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The Diversity and Commonality of Cells Cells come in an amazing variety of sizes and shapes Figure 1-1. Some move rapidly and have fast-changing structures as we can see in movies of amoebae and rotifers. Others are largely stationary and structurally stable. Oxygen kills some cells but is an absolute requirement for others. Most cells in multicellular organisms are intimately involved with other cells. Although some unicellular organisms live in isolation others form colonies or live in close association with other types of organisms such as the bacteria that help plants to ex- tract nitrogen from the air or the bacteria that live in our in- testines and help us digest food. Despite these and numerous 1.1 1 A single 200 micrometer m cell the human egg with sperm which are also single cells. From the union of an egg and sperm will arise the 10 trillion cells of a human body. Photo Researchers Inc. LIFE BEGINS WITH CELLS L ike ourselves the individual cells that form our bodies can grow reproduce process information respond to stimuli and carry out an amazing array of chemical re- actions. These abilities define life. We and other multicellular organisms contain billions or trillions of cells organized into complex structures but many organisms consist of a single cell. Even simple unicellular organisms exhibit all the hall- mark properties of life indicating that the cell is the funda- mental unit of life. As the twenty-first century opens we face an explosion of new data about the components of cells what structures they contain how they touch and influence each other. Still an immense amount remains to be learned particularly about how information flows through cells and how they decide on the most appropriate ways to respond. Molecular cell biology is a rich integrative science that brings together biochemistry biophysics molecular biology microscopy genetics physiology computer science and de- velopmental biology. Each of these fields has its own em- phasis and style of experimentation. In the following chapters we will describe insights and experimental ap- proaches drawn from all of these fields gradually weaving the multifaceted story of the birth life and death of cells. We start in this prologue chapter by introducing the diversity of cells their basic constituents and critical functions and what we can learn from the various ways of studying cells. 1 OUTLINE 1.1 The Diversity and Commonality of Cells 1.2 The Molecules of a Cell 1.3 The Work of Cells 1.4 Investigating Cells and Their Parts 1.5 A Genome Perspective on Evolution

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other differences all cells share certain structural features and carry out many complicated processes in basically the same way. As the story of cells unfolds throughout this book we will focus on the molecular basis of both the differences and similarities in the structure and function of various cells. All Cells Are Prokaryotic or Eukaryotic The biological universe consists of two types of cells— prokaryotic and eukaryotic. Prokaryotic cells consist of a sin- gle closed compartment that is surrounded by the plasma membrane lacks a defined nucleus and has a relatively simple internal organization Figure 1-2a. All prokaryotes have cells of this type. Bacteria the most numerous prokaryotes are single-celled organisms the cyanobacteria or blue-green algae can be unicellular or filamentous chains of cells. Although bac- terial cells do not have membrane-bounded compartments many proteins are precisely localized in their aqueous interior or cytosol indicating the presence of internal organization. A single Escherichia coli bacterium has a dry weight of about 2 CHAPTER 1 • Life Begins with Cells ▲ FIGURE 1-1 Cells come in an astounding assortment of shapes and sizes. Some of the morphological variety of cells is illustrated in these photographs. In addition to morphology cells differ in their ability to move internal organization prokaryotic versus eukaryotic cells and metabolic activities. a Eubacteria note dividing cells. These are Lactococcus lactis which are used to produce cheese such as Roquefort Brie and Camembert. b A mass of archaebacteria Methanosarcina that produce their energy by converting carbon dioxide and hydrogen gas to methane. Some species that live in the rumen of cattle give rise to 150 liters of methane gas/day. c Blood cells shown in false color. The red blood cells are oxygen-bearing erythrocytes the white blood cells leukocytes are part of the immune system and fight infection and the green cells are platelets that provide substances to make blood clot at a wound. d Large single cells: fossilized dinosaur eggs. e A colonial single-celled green alga Volvox aureus. The large spheres are made up of many individual cells visible as blue or green dots. The yellow masses inside are daughter colonies each made up of many cells. f A single Purkinje neuron of the cerebellum which can form more than a hundred thousand connections with other cells through the branched network of dendrites. The cell was made visible by introduction of a fluorescent protein the cell body is the bulb at the bottom. g Cells can form an epithelial sheet as in the slice through intestine shown here. Each finger-like tower of cells a villus contains many cells in a continuous sheet. Nutrients are transferred from digested food through the epithelial sheet to the blood for transport to other parts of the body. New cells form continuously near the bases of the villi and old cells are shed from the top. h Plant cells are fixed firmly in place in vascular plants supported by a rigid cellulose skeleton. Spaces between the cells are joined into tubes for transport of water and food. Part a Gary Gaugler/ Photo Researchers Inc. Part b Ralph Robinson/ Visuals Inlimited Inc. Part c NIH/Photo Researchers Inc. Part d John D. Cunningham/Visuals Unlimited Inc. Part e Carolina Biological/Visuals Unlimited Inc. Part f Helen M. Blau Stanford University. Part g Jeff Gordon Washington University School of Medicine. Part h Richard Kessel and C. Shih/Visuals Unlimited Inc. e f g h a b c d

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25 10 14 g. Bacteria account for an estimated 1–1.5 kg of the average human’s weight. The estimated number of bacte- ria on earth is 5 10 30 weighing a total of about 10 12 kg. Prokaryotic cells have been found 7 miles deep in the ocean and 40 miles up in the atmosphere they are quite adaptable The carbon stored in bacteria is nearly as much as the carbon stored in plants. Eukaryotic cells unlike prokaryotic cells contain a de- fined membrane-bound nucleus and extensive internal mem- branes that enclose other compartments the organelles Fig- ure 1-2b. The region of the cell lying between the plasma membrane and the nucleus is the cytoplasm comprising the cytosol aqueous phase and the organelles. Eukaryotes com- prise all members of the plant and animal kingdoms includ- ing the fungi which exist in both multicellular forms molds and unicellular forms yeasts and the protozoans proto primitive zoan animal which are exclusively unicellular. Eukaryotic cells are commonly about 10–100 m across 1.1 • The Diversity and Commonality of Cells 3 Inner plasma membrane a Prokaryotic cell b Eukaryotic cell Cell wall Periplasmic space Outer membrane Nucleus Nuclear membrane Plasma membrane Golgi vesicles Lysosome Secretory vesicle Peroxisome Mitochondrion Rough endoplasmic reticulum Periplasmic space and cell wall Outer membrane Inner plasma membrane Nucleoid 0.5 m 1 m Nucleus Golgi vesicles Lysosome Mitochondrion Endoplasmic reticulum Nucleoid ▲ FIGURE 1-2 Prokaryotic cells have a simpler internal organization than eukaryotic cells. a Electron micrograph of a thin section of Escherichia coli a common intestinal bacterium. The nucleoid consisting of the bacterial DNA is not enclosed within a membrane. E. coli and some other bacteria are surrounded by two membranes separated by the periplasmic space. The thin cell wall is adjacent to the inner membrane. b Electron micrograph of a plasma cell a type of white blood cell that secretes antibodies. Only a single membrane the plasma membrane surrounds the cell but the interior contains many membrane-limited compartments or organelles. The defining characteristic of eukaryotic cells is segregation of the cellular DNA within a defined nucleus which is bounded by a double membrane. The outer nuclear membrane is continuous with the rough endoplasmic reticulum a factory for assembling proteins. Golgi vesicles process and modify proteins mitochondria generate energy lysosomes digest cell materials to recycle them peroxisomes process molecules using oxygen and secretory vesicles carry cell materials to the surface to release them. Part a courtesy of I. D. J. Burdett and R. G. E. Murray. Part b from P . C. Cross and K. L. Mercer 1993 Cell and Tissue Ultrastructure: A Functional Perspective W. H. Freeman and Company.

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generally much larger than bacteria. A typical human fi- broblast a connective tissue cell might be about 15 m across with a volume and dry weight some thousands of times those of an E. coli bacterial cell. An amoeba a single- celled protozoan can be more than 0.5 mm long. An ostrich egg begins as a single cell that is even larger and easily visi- ble to the naked eye. All cells are thought to have evolved from a common pro- genitor because the structures and molecules in all cells have so many similarities. In recent years detailed analysis of the DNA sequences from a variety of prokaryotic organisms has revealed two distinct types: the so-called “true” bacteria or eu- bacteria and archaea also called archaebacteria or archaeans. Working on the assumption that organisms with more similar genes evolved from a common progenitor more recently than those with more dissimilar genes researchers have developed the evolutionary lineage tree shown in Figure 1-3. According to this tree the archaea and the eukaryotes diverged from the true bacteria before they diverged from each other. Many archaeans grow in unusual often extreme envi- ronments that may resemble ancient conditions when life first appeared on earth. For instance halophiles “salt lov- ing” require high concentrations of salt to survive and thermoacidophiles “heat and acid loving” grow in hot 80 C sulfur springs where a pH of less than 2 is common. Still other archaeans live in oxygen-free milieus and generate methane CH 4 by combining water with carbon dioxide. Unicellular Organisms Help and Hurt Us Bacteria and archaebacteria the most abundant single-celled organisms are commonly 1–2 m in size. Despite their small size and simple architecture they are remarkable biochemi- cal factories converting simple chemicals into complex bio- logical molecules. Bacteria are critical to the earth’s ecology but some cause major diseases: bubonic plague Black Death from Yersinia pestis strep throat from Streptomyces tuber- culosis from Mycobacterium tuberculosis anthrax from Bacillus anthracis cholera from Vibrio cholerae food poi- soning from certain types of E. coli and Salmonella. Humans are walking repositories of bacteria as are all plants and animals. We provide food and shelter for a stag- gering number of “bugs” with the greatest concentration in our intestines. Bacteria help us digest our food and in turn are able to reproduce. A common gut bacterium E. coli is also a favorite experimental organism. In response to signals from bacteria such as E. coli the intestinal cells form appro- priate shapes to provide a niche where bacteria can live thus facilitating proper digestion by the combined efforts of the bacterial and the intestinal cells. Conversely exposure to in- testinal cells changes the properties of the bacteria so that they participate more effectively in digestion. Such commu- nication and response is a common feature of cells. The normal peaceful mutualism of humans and bacteria is sometimes violated by one or both parties. When bacteria begin to grow where they are dangerous to us e.g. in the blood- stream or in a wound the cells of our immune system fight back neutralizing or devouring the intruders. Powerful antibi- otic medicines which selectively poison prokaryotic cells provide rapid assistance to our relatively slow-developing immune response. Understanding the molecular biology of bac- terial cells leads to an understanding of how bacteria are nor- mally poisoned by antibiotics how they become resistant to antibiotics and what processes or structures present in bacter- ial but not human cells might be usefully targeted by new drugs. 4 CHAPTER 1 • Life Begins with Cells Plants Fungi EUKARYOTA EUBACTERIA ARCHAEA Animals Microsporidia Euglena Sulfolobus Thermococcus Methanobacterium Halococcus Halobacterium Methanococcus jannaschii Borrelia burgdorferi E. coli B. subtilus Diplomonads Giardia lamblia Ciliates Slime molds Thermotoga Flavobacteria Green sulfur bacteria Presumed common progenitor of all extant organisms Presumed common progenitor of archaebacteria and eukaryotes ▲ FIGURE 1-3 All organisms from simple bacteria to complex mammals probably evolved from a common single- celled progenitor. This family tree depicts the evolutionary relations among the three major lineages of organisms. The structure of the tree was initially ascertained from morphological criteria: Creatures that look alike were put close together. More recently the sequences of DNA and proteins have been examined as a more information-rich criterion for assigning relationships. The greater the similarities in these macromolecular sequences the more closely related organisms are thought to be. The trees based on morphological comparisons and the fossil record generally agree well with those those based on molecular data. Although all organisms in the eubacterial and archaean lineages are prokaryotes archaea are more similar to eukaryotes than to eubacteria “true” bacteria in some respects. For instance archaean and eukaryotic genomes encode homologous histone proteins which associate with DNA in contrast bacteria lack histones. Likewise the RNA and protein components of archaean ribosomes are more like those in eukaryotes than those in bacteria.

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Like bacteria protozoa are usually beneficial members of the food chain. They play key roles in the fertility of soil con- trolling bacterial populations and excreting nitrogenous and phosphate compounds and are key players in waste treat- ment systems—both natural and man-made. These unicellu- lar eukaryotes are also critical parts of marine ecosystems consuming large quantities of phytoplankton and harboring photosynthetic algae which use sunlight to produce biologi- cally useful energy forms and small fuel molecules. However some protozoa do give us grief: Entamoeba histolytica causes dysentery Trichomonas vaginalis vagini- tis and Trypanosoma brucei sleeping sickness. Each year the worst of the protozoa Plasmodium falciparum and related species is the cause of more than 300 million new cases of malaria a disease that kills 1.5 to 3 million people annually. These protozoans inhabit mammals and mosquitoes alter- nately changing their morphology and behavior in response to signals in each of these environments. They also recog- nize receptors on the surfaces of the cells they infect. The complex life cycle of Plasmodium dramatically illustrates how a single cell can adapt to each new challenge it encoun- ters Figure 1-4. All of the transformations in cell type that 1.1 • The Diversity and Commonality of Cells 5 a Red blood cell Merozoites Liver Sporozoites Oocyst Mosquito Human Gametocytes Sporulation Merozoites Sperm Egg Zygote 2 1 8 7 6 5 4 3 ▲ FIGURE 1-4 Plasmodium organisms the parasites that cause malaria are single-celled protozoans with a remarkable life cycle. Many Plasmodium species are known and they can infect a variety of animals cycling between insect and vertebrate hosts. The four species that cause malaria in humans undergo several dramatic transformations within their human and mosquito hosts. a Diagram of the life cycle. Sporozoites enter a human host when an infected Anopheles mosquito bites a person . They migrate to the liver where they develop into merozoites which are released into the blood . Merozoites differ substantially from sporozoites so this transformation is a metamorphosis Greek “to transform” or “many shapes”. Circulating merozoites invade red blood cells RBCs and reproduce within them . Proteins produced by some Plasmodium species move to the surface of infected RBCs causing the cells to adhere to the walls of blood vessels. This prevents infected RBCs cells from circulating to the spleen where cells of the immune system would destroy the RBCs and the Plasmodium organisms they harbor. After growing and reproducing in RBCs for a period of time characteristic of each Plasmodium species the merozoites suddenly burst forth in synchrony from large numbers of infected cells . It is this 4 3 2 1 event that brings on the fevers and shaking chills that are the well-known symptoms of malaria. Some of the released merozoites infect additional RBCs creating a cycle of production and infection. Eventually some merozoites develop into male and female gametocytes another metamorphosis. These cells which contain half the usual number of chromosomes cannot survive for long unless they are transferred in blood to an Anopheles mosquito. In the mosquito’s stomach the gametocytes are transformed into sperm or eggs gametes yet another metamorphosis marked by development of long hairlike flagella on the sperm . Fusion of sperm and eggs generates zygotes which implant into the cells of the stomach wall and grow into oocysts essentially factories for producing sporozoites. Rupture of an oocyst releases thousands of sporozoites these migrate to the salivary glands setting the stage for infection of another human host. b Scanning electron micrograph of mature oocysts and emerging sporozoites. Oocysts abut the external surface of stomach wall cells and are encased within a membrane that protects them from the host immune system. Part b courtesy of R. E. Sinden. 8 7 6 5 b MEDIA CONNECTIONS Video: Plasmodium Sporozoite Entering and Exiting a Liver Cell

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occur during the Plasmodium life cycle are governed by in- structions encoded in the genetic material of this parasite and triggered by environmental inputs. The other group of single-celled eukaryotes the yeasts also have their good and bad points as do their multicellular cousins the molds. Yeasts and molds which collectively con- stitute the fungi have an important ecological role in break- ing down plant and animal remains for reuse. They also make numerous antibiotics and are used in the manufacture of bread beer wine and cheese. Not so pleasant are fungal diseases which range from relatively innocuous skin infec- tions such as jock itch and athlete’s foot to life-threatening Pneumocystis carinii pneumonia a common cause of death among AIDS patients. Even Single Cells Can Have Sex The common yeast used to make bread and beer Saccha- romyces cerevisiae appears fairly frequently in this book be- cause it has proven to be a great experimental organism. Like many other unicellular organisms yeasts have two mating types that are conceptually like the male and female gametes eggs and sperm of higher organisms. Two yeast cells of op- posite mating type can fuse or mate to produce a third cell type containing the genetic material from each cell Figure 1-5. Such sexual life cycles allow more rapid changes in ge- netic inheritance than would be possible without sex result- ing in valuable adaptations while quickly eliminating detrimental mutations. That and not just Hollywood is probably why sex is so ubiquitous. Viruses Are the Ultimate Parasites Virus-caused diseases are numerous and all too familiar: chicken pox influenza some types of pneumonia polio measles rabies hepatitis the common cold and many oth- ers. Smallpox once a worldwide scourge was eradicated by a decade-long global immunization effort beginning in the mid-1960s. Viral infections in plants e.g. dwarf mosaic virus in corn have a major economic impact on crop pro- duction. Planting of virus-resistant varieties developed by traditional breeding methods and more recently by genetic engineering techniques can reduce crop losses significantly. Most viruses have a rather limited host range infecting cer- tain bacteria plants or animals Figure 1-6. Because viruses cannot grow or reproduce on their own they are not considered to be alive. To survive a virus must infect a host cell and take over its internal machinery to syn- thesize viral proteins and in some cases to replicate the viral genetic material. When newly made viruses are released the cycle starts anew. Viruses are much smaller than cells on the order of 100 nanometer nm in diameter in comparison bacterial cells are usually 1000 nm 1 nm 10 9 meters. A virus is typically composed of a protein coat that encloses a core containing the genetic material which carries the infor- mation for producing more viruses Chapter 4. The coat protects a virus from the environment and allows it to stick to or enter specific host cells. In some viruses the protein coat is surrounded by an outer membrane-like envelope. The ability of viruses to transport genetic material into cells and tissues represents a medical menace and a medical opportunity. Viral infections can be devastatingly destructive causing cells to break open and tissues to fall apart. However many methods for manipulating cells depend upon using 6 CHAPTER 1 • Life Begins with Cells ▲ FIGURE 1-5 The yeast Saccharomyces cerevisiae reproduces sexually and asexually. a Two cells that differ in mating type called a and can mate to form an a/ cell . The a and cells are haploid meaning they contain a single copy of each yeast chromosome half the usual number. Mating yields a diploid a/ cell containing two copies of each chromosome. During vegetative growth diploid cells multiply by mitotic budding an asexual process . Under starvation conditions diploid cells undergo meiosis a special type of cell division to form haploid ascospores . Rupture of an ascus releases four haploid spores which can germinate into haploid cells . These also can multiply asexually . b Scanning electron micrograph of budding yeast cells. After each bud breaks free a scar is left at the budding site so the number of previous buds can be counted. The orange cells are bacteria. Part b M. Abbey/Visuals Unlimited Inc. 5 4 3 2 1 Vegetative growth of diploid cells Bud Starvation causes ascus formation meiosis Four haploid ascospores within ascus Vegetative growth of haploid cells Ascus ruptures spores germinate a Mating between haploid cells of opposite mating type Diploid cells a/ α α b 1 2 3 4 5 a Budding S. cerevisiae

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viruses to convey genetic material into cells. To do this the portion of the viral genetic material that is potentially harm- ful is replaced with other genetic material including human genes. The altered viruses or vectors still can enter cells tot- ing the introduced genes with them Chapter 9. One day dis- eases caused by defective genes may be treated by using viral vectors to introduce a normal copy of a defective gene into patients. Current research is dedicated to overcoming the con- siderable obstacles to this approach such as getting the in- troduced genes to work at the right places and times. We Develop from a Single Cell In 1827 German physician Karl von Baer discovered that mammals grow from eggs that come from the mother’s ovary. Fertilization of an egg by a sperm cell yields a zygote a visually unimpressive cell 200 m in diameter. Every human being begins as a zygote which houses all the neces- sary instructions for building the human body containing about 100 trillion 10 14 cells an amazing feat. Development begins with the fertilized egg cell dividing into two four then eight cells forming the very early embryo Figure 1-7. Con- tinued cell proliferation and then differentiation into distinct cell types gives rise to every tissue in the body. One initial cell the fertilized egg zygote generates hundreds of differ- ent kinds of cells that differ in contents shape size color mobility and surface composition. We will see how genes and signals control cell diversification in Chapters 15 and 22. Making different kinds of cells—muscle skin bone neu- ron blood cells—is not enough to produce the human body. The cells must be properly arranged and organized into tis- sues organs and appendages. Our two hands have the same kinds of cells yet their different arrangements—in a mirror image—are critical for function. In addition many cells ex- hibit distinct functional and/or structural asymmetries a property often called polarity. From such polarized cells arise 1.1 • The Diversity and Commonality of Cells 7 a T4 bacteriophage b Tobacco mosaic virus c Adenovirus 100 nm 50 nm 50 nm ▲ FIGURE 1-6 Viruses must infect a host cell to grow and reproduce. These electron micrographs illustrate some of the structural variety exhibited by viruses. a T4 bacteriophage bracket attaches to a bacterial cell via a tail structure. Viruses that infect bacteria are called bacteriophages or simply phages. b Tobacco mosaic virus causes a mottling of the leaves of infected tobacco plants and stunts their growth. c Adenovirus causes eye and respiratory tract infections in humans. This virus has an outer membranous envelope from which long glycoprotein spikes protrude. Part a from A. Levine 1991 Viruses Scientific American Library p. 20. Part b courtesy of R. C. Valentine. Part c courtesy of Robley C. Williams University of California. a b c FIGURE 1-7 The first few cell divisions of a fertilized egg set the stage for all subsequent development. A developing mouse embryo is shown at a the two-cell b four-cell and c eight-cell stages. The embryo is surrounded by supporting membranes. The corresponding steps in human development occur during the first few days after fertilization. Claude Edelmann/Photo Researchers Inc. MEDIA CONNECTIONS Video: Early Embryonic Development

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asymmetric polarized tissues such as the lining of the intes- tines and structures like hands and hearts. The features that make some cells polarized and how they arise also are cov- ered in later chapters. Stem Cells Cloning and Related Techniques Offer Exciting Possibilities but Raise Some Concerns Identical twins occur naturally when the mass of cells com- posing an early embryo divides into two parts each of which develops and grows into an individual animal. Each cell in an eight-cell-stage mouse embryo has the potential to give rise to any part of the entire animal. Cells with this capabil- ity are referred to as embryonic stem ES cells. As we learn in Chapter 22 ES cells can be grown in the laboratory cul- tured and will develop into various types of differentiated cells under appropriate conditions. The ability to make and manipulate mammalian embryos in the laboratory has led to new medical opportunities as well as various social and ethical concerns. In vitro fertiliza- tion for instance has allowed many otherwise infertile cou- ples to have children. A new technique involves extraction of nuclei from defective sperm incapable of normally fertiliz- ing an egg injection of the nuclei into eggs and implantation of the resulting fertilized eggs into the mother. In recent years nuclei taken from cells of adult animals have been used to produce new animals. In this procedure the nucleus is removed from a body cell e.g. skin or blood cell of a donor animal and introduced into an unfertilized mammalian egg that has been deprived of its own nucleus. This manipulated egg which is equivalent to a fertilized egg is then implanted into a foster mother. The ability of such a donor nucleus to direct the development of an entire animal suggests that all the information required for life is retained in the nuclei of some adult cells. Since all the cells in an ani- mal produced in this way have the genes of the single origi- nal donor cell the new animal is a clone of the donor Figure 1-8. Repeating the process can give rise to many clones. So far however the majority of embryos produced by this tech- nique of nuclear-transfer cloning do not survive due to birth defects. Even those animals that are born live have shown abnormalities including accelerated aging. The “rooting” of plants in contrast is a type of cloning that is readily ac- complished by gardeners farmers and laboratory technicians. The technical difficulties and possible hazards of nuclear- transfer cloning have not deterred some individuals from pur- suing the goal of human cloning. However cloning of humans per se has very limited scientific interest and is op- posed by most scientists because of its high risk. Of greater scientific and medical interest is the ability to generate specific cell types starting from embryonic or adult stem cells. The sci- entific interest comes from learning the signals that can un- leash the potential of the genes to form a certain cell type. The medical interest comes from the possibility of treating the nu- merous diseases in which particular cell types are damaged or missing and of repairing wounds more completely. The Molecules of a Cell Molecular cell biologists explore how all the remarkable properties of the cell arise from underlying molecular events: the assembly of large molecules binding of large molecules to each other catalytic effects that promote particular chem- ical reactions and the deployment of information carried by giant molecules. Here we review the most important kinds of molecules that form the chemical foundations of cell struc- ture and function. Small Molecules Carry Energy Transmit Signals and Are Linked into Macromolecules Much of the cell’s contents is a watery soup flavored with small molecules e.g. simple sugars amino acids vitamins and ions e.g. sodium chloride calcium ions. The locations and concentrations of small molecules and ions within the cell are controlled by numerous proteins inserted in cellular membranes. These pumps transporters and ion channels move nearly all small molecules and ions into or out of the cell and its organelles Chapter 7. 1.2 8 CHAPTER 1 • Life Begins with Cells ▲ FIGURE 1-8 Five genetically identical cloned sheep. An early sheep embryo was divided into five groups of cells and each was separately implanted into a surrogate mother much like the natural process of twinning. At an early stage the cells are able to adjust and form an entire animal later in development the cells become progressively restricted and can no longer do so. An alternative way to clone animals is to replace the nuclei of multiple single-celled embryos with donor nuclei from cells of an adult sheep. Each embryo will be genetically identical to the adult from which the nucleus was obtained. Low percentages of embryos survive these procedures to give healthy animals and the full impact of the techniques on the animals is not yet known. Geoff Tompkinson/Science Photo Library/Photo Researchers Inc.

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One of the best-known small molecules is adenosine triphosphate ATP which stores readily available chemical energy in two of its chemical bonds see Figure 2-24. When cells split apart these energy-rich bonds in ATP the released energy can be harnessed to power an energy-requiring process like muscle contraction or protein biosynthesis. To obtain energy for making ATP cells break down food mole- cules. For instance when sugar is degraded to carbon diox- ide and water the energy stored in the original chemical bonds is released and much of it can be “captured” in ATP Chapter 8. Bacterial plant and animal cells can all make ATP by this process. In addition plants and a few other or- ganisms can harvest energy from sunlight to form ATP in photosynthesis. Other small molecules act as signals both within and be- tween cells such signals direct numerous cellular activities Chapters 13–15. The powerful effect on our bodies of a frightening event comes from the instantaneous flooding of the body with epinephrine a small-molecule hormone that mobilizes the “fight or flight” response. The movements needed to fight or flee are triggered by nerve impulses that flow from the brain to our muscles with the aid of neuro- transmitters another type of small-molecule signal that we discuss in Chapter 7. Certain small molecules monomers in the cellular soup can be joined to form polymers through repetition of a single type of chemical-linkage reaction see Figure 2-11. Cells produce three types of large polymers commonly called macromolecules: polysaccharides proteins and nucleic acids. Sugars for example are the monomers used to form polysaccharides. These macromolecules are critical structural components of plant cell walls and insect skeletons. A typical polysaccharide is a linear or branched chain of repeating identical sugar units. Such a chain carries information: the number of units. However if the units are not identical then the order and type of units carry additional information. As we see in Chapter 6 some polysaccharides exhibit the greater informational complexity associated with a linear code made up of different units assembled in a particular order. This property however is most typical of the two other types of biological macromolecules—proteins and nucleic acids. Proteins Give Cells Structure and Perform Most Cellular Tasks The varied intricate structures of proteins enable them to carry out numerous functions. Cells string together 20 dif- ferent amino acids in a linear chain to form a protein see Figure 2-13. Proteins commonly range in length from 100 to 1000 amino acids but some are much shorter and others longer. We obtain amino acids either by synthesizing them from other molecules or by breaking down proteins that we eat. The “essential” amino acids from a dietary standpoint are the eight that we cannot synthesize and must obtain from food. Beans and corn together have all eight making their combination particularly nutritious. Once a chain of amino acids is formed it folds into a complex shape conferring a distinctive three-dimensional structure and function on each protein Figure 1-9. 1.2 • The Molecules of a Cell 9 Glutamine synthetase Insulin Hemoglobin Immunoglobulin Adenylate kinase DNA molecule Lipid bilayer ▲ FIGURE 1-9 Proteins vary greatly in size shape and function. These models of the water-accessible surface of some representative proteins are drawn to a common scale and reveal the numerous projections and crevices on the surface. Each protein has a defined three-dimensional shape conformation that is stabilized by numerous chemical interactions discussed in Chapters 2 and 3. The illustrated proteins include enzymes glutamine synthetase and adenylate kinase an antibody immunoglobulin a hormone insulin and the blood’s oxygen carrier hemoglobin. Models of a segment of the nucleic acid DNA and a small region of the lipid bilayer that forms cellular membranes see Section 1.3 demonstrate the relative width of these structures compared with typical proteins. Courtesy of Gareth White.

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Some proteins are similar to one another and therefore can be considered members of a protein family. A few hun- dred such families have been identified. Most proteins are de- signed to work in particular places within a cell or to be released into the extracellular extra “outside” space. Elab- orate cellular pathways ensure that proteins are transported to their proper intracellular intra within locations or se- creted Chapters 16 and 17. Proteins can serve as structural components of a cell for example by forming an internal skeleton Chapters 5 19 and 20. They can be sensors that change shape as temperature ion concentrations or other properties of the cell change. They can import and export substances across the plasma mem- brane Chapter 7. They can be enzymes causing chemical re- actions to occur much more rapidly than they would without the aid of these protein catalysts Chapter 3. They can bind to a specific gene turning it on or off Chapter 11. They can be extracellular signals released from one cell to communicate with other cells or intracellular signals carrying information within the cell Chapters 13–15. They can be motors that move other molecules around burning chemical energy ATP to do so Chapters 19 and 20. How can 20 amino acids form all the different proteins needed to perform these varied tasks Seems impossible at first glance. But if a “typical” protein is about 400 amino acids long there are 20 400 possible different protein se- quences. Even assuming that many of these would be func- tionally equivalent unstable or otherwise discountable the number of possible proteins is well along toward infinity. Next we might ask how many protein molecules a cell needs to operate and maintain itself. To estimate this num- ber let’s take a typical eukaryotic cell such as a hepatocyte liver cell. This cell roughly a cube 15 m 0.0015 cm on a side has a volume of 3.4 10 9 cm 3 or milliliters. As- suming a cell density of 1.03 g/ml the cell would weigh 3.5 10 9 g. Since protein accounts for approximately 20 percent of a cell’s weight the total weight of cellular pro- tein is 7 10 10 g. The average yeast protein has a mo- lecular weight of 52700 g/mol. Assuming this value is typical of eukaryotic proteins we can calculate the total number of protein molecules per liver cell as about 7.9 10 9 from the total protein weight and Avogadro’s number the number of molecules per mole of any chemical com- pound 6.02 10 23 . To carry this calculation one step further consider that a liver cell contains about 10000 different proteins thus a cell contains close to a million molecules of each type of protein on average. In actuality the abundance of different proteins varies widely from the quite rare insulin-binding receptor protein 20000 mole- cules to the abundant structural protein actin 5 10 8 molecules. Nucleic Acids Carry Coded Information for Making Proteins at the Right Time and Place The information about how when and where to produce each kind of protein is carried in the genetic material a polymer called deoxyribonucleic acid DNA. The three-dimensional structure of DNA consists of two long helical strands that are coiled around a common axis forming a double helix. DNA strands are composed of monomers called nucleotides these often are referred to as bases because their structures contain cyclic organic bases Chapter 4. Four different nucleotides abbreviated A T C and G are joined end to end in a DNA strand with the base parts projecting out from the helical backbone of the strand. Each DNA double helix has a simple construction: wherever there is an A in one strand there is a T in the other and each C is matched with a G Figure 1-10. This complementary match- ing of the two strands is so strong that if complementary strands are separated they will spontaneously zip back to- gether in the right salt and temperature conditions. Such hybridization is extremely useful for detecting one strand using the other. For example if one strand is purified and attached to a piece of paper soaking the paper in a solution contain- ing the other complementary strand will lead to zippering 10 CHAPTER 1 • Life Begins with Cells Parental strands A G TC Daughter strands ▲ FIGURE 1-10 DNA consists of two complementary strands wound around each other to form a double helix. Left The double helix is stabilized by weak hydrogen bonds between the A and T bases and between the C and G bases. Right During replication the two strands are unwound and used as templates to produce complementary strands. The outcome is two copies of the original double helix each containing one of the original strands and one new daughter complementary strand.

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even if the solution also contains many other DNA strands that do not match. The genetic information carried by DNA resides in its se- quence the linear order of nucleotides along a strand. The information-bearing portion of DNA is divided into discrete functional units the genes which typically are 5000 to 100000 nucleotides long. Most bacteria have a few thou- sand genes humans about 40000. The genes that carry in- structions for making proteins commonly contain two parts: a coding region that specifies the amino acid sequence of a protein and a regulatory region that controls when and in which cells the protein is made. Cells use two processes in series to convert the coded in- formation in DNA into proteins Figure 1-11. In the first called transcription the coding region of a gene is copied into a single-stranded ribonucleic acid RNA version of the double-stranded DNA. A large enzyme RNA polymerase catalyzes the linkage of nucleotides into a RNA chain using DNA as a template. In eukaryotic cells the initial RNA product is processed into a smaller messenger RNA mRNA molecule which moves to the cytoplasm. Here the ribosome an enormously complex molecular machine composed of both RNA and protein carries out the second process called translation. During translation the ribosome assembles and links together amino acids in the precise order dictated by the mRNA sequence according to the nearly universal genetic code. We examine the cell components that carry out tran- scription and translation in detail in Chapter 4. All organisms have ways to control when and where their genes can be transcribed. For instance nearly all the cells in our bodies contain the full set of human genes but in each cell type only some of these genes are active or turned on and used to make proteins. That’s why liver cells produce some proteins that are not produced by kidney cells and vice versa. Moreover many cells can respond to external signals or changes in external conditions by turning specific genes on or off thereby adapting their repertoire of proteins to meet current needs. Such control of gene activity depends on DNA-binding proteins called transcription factors which bind to DNA and act as switches either activating or re- pressing transcription of particular genes Chapter 11. Transcription factors are shaped so precisely that they are able to bind preferentially to the regulatory regions of just a few genes out of the thousands present in a cell’s DNA. Typ- ically a DNA-binding protein will recognize short DNA se- quences about 6–12 base pairs long. A segment of DNA containing 10 base pairs can have 4 10 possible sequences 1048576 since each position can be any of four nu- cleotides. Only a few copies of each such sequence will occur in the DNA of a cell assuring the specificity of gene activation and repression. Multiple copies of one type of transcription factor can coordinately regulate a set of genes if binding sites for that factor exist near each gene in the set. Transcription factors often work as multiprotein complexes with more than one protein contributing its own DNA-binding speci- ficity to selecting the regulated genes. In complex organisms hundreds of different transcription factors are employed to form an exquisite control system that activates the right genes in the right cells at the right times. The Genome Is Packaged into Chromosomes and Replicated During Cell Division Most of the DNA in eukaryotic cells is located in the nucleus extensively folded into the familiar structures we know as chromosomes Chapter 10. Each chromosome contains a sin- gle linear DNA molecule associated with certain proteins. In prokaryotic cells most or all of the genetic information resides 1.2 • The Molecules of a Cell 11 Nucleus Cytosol Transcription factor DNA pre-mRNA mRNA Ribosome RNA polymerase Transcribed region of DNA Nontranscribed region of DNA Protein-coding region of RNA Noncoding region of RNA Protein Start Activation Transcription Processing Translation 1 2 3 4 Amino acid chain ▲ FIGURE 1-11 The coded information in DNA is converted into the amino acid sequences of proteins by a multistep process. Step : Transcription factors bind to the regulatory regions of the specific genes they control and activate them. Step : Following assembly of a multiprotein initiation complex bound to the DNA RNA polymerase begins transcription of an activated gene at a specific location the start site. The polymerase moves along the DNA linking nucleotides into a single-stranded pre-mRNA transcript using one of the DNA strands as a template. Step : The transcript is processed to remove noncoding sequences. Step : In a eukaryotic cell the mature messenger RNA mRNA moves to the cytoplasm where it is bound by ribosomes that read its sequence and assemble a protein by chemically linking amino acids into a linear chain. 4 3 2 1

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in a single circular DNA molecule about a millimeter in length this molecule lies folded back on itself many times in the cen- tral region of the cell see Figure 1-2a. The genome of an or- ganism comprises its entire complement of DNA. With the exception of eggs and sperm every normal human cell has 46 chromosomes Figure 1-12. Half of these and thus half of the genes can be traced back to Mom the other half to Dad. Every time a cell divides a large multiprotein replication machine the replisome separates the two strands of double- helical DNA in the chromosomes and uses each strand as a template to assemble nucleotides into a new complementary strand see Figure 1-10. The outcome is a pair of double he- lices each identical to the original. DNA polymerase which is responsible for linking nucleotides into a DNA strand and the many other components of the replisome are described in Chapter 4. The molecular design of DNA and the remarkable properties of the replisome assure rapid highly accurate copy- ing. Many DNA polymerase molecules work in concert each one copying part of a chromosome. The entire genome of fruit flies about 1.2 10 8 nucleotides long can be copied in three minutes Because of the accuracy of DNA replication nearly all the cells in our bodies carry the same genetic instructions and we can inherit Mom’s brown hair and Dad’s blue eyes. A rather dramatic example of gene control involves in- activation of an entire chromosome in human females. Women have two X chromosomes whereas men have one X chromosome and one Y chromosome which has differ- ent genes than the X chromosome. Yet the genes on the X chromosome must for the most part be equally active in fe- male cells XX and male cells XY. To achieve this balance one of the X chromosomes in female cells is chemically mod- ified and condensed into a very small mass called a Barr body which is inactive and never transcribed. Surprisingly we inherit a small amount of genetic mate- rial entirely and uniquely from our mothers. This is the cir- cular DNA present in mitochondria the organelles in eukaryotic cells that synthesize ATP using the energy released by the breakdown of nutrients. Mitochondria contain mul- tiple copies of their own DNA genomes which code for some of the mitochondrial proteins Chapter 10. Because each human inherits mitochondrial DNA only from his or her mother it comes with the egg but not the sperm the dis- tinctive features of a particular mitochondrial DNA can be used to trace maternal history. Chloroplasts the organelles that carry out photosynthesis in plants also have their own circular genomes. Mutations May Be Good Bad or Indifferent Mistakes occasionally do occur spontaneously during DNA replication causing changes in the sequence of nucleotides. Such changes or mutations also can arise from radiation 12 CHAPTER 1 • Life Begins with Cells ▲ FIGURE 1-12 Chromosomes can be “painted” for easy identification. A normal human has 23 pairs of morphologically distinct chromosomes one member of each pair is inherited from the mother and the other member from the father. Left A chromosome spread from a human body cell midway through mitosis when the chromosomes are fully condensed. This preparation was treated with fluorescent-labeled staining reagents that allow each of the 22 pairs and the X and Y chromosomes to appear in a different color when viewed in a fluorescence microscope. This technique of multiplex fluorescence in situ hybridization M-FISH sometimes is called chromosome painting Chapter 10. Right Chromosomes from the preparation on the left arranged in pairs in descending order of size an array called a karyotype. The presence of X and Y chromosomes identifies the sex of the individual as male. Courtesy of M. R. Speicher.

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that causes damage to the nucleotide chain or from chemi- cal poisons such as those in cigarette smoke that lead to er- rors during the DNA-copying process Chapter 23. Mutations come in various forms: a simple swap of one nu- cleotide for another the deletion insertion or inversion of one to millions of nucleotides in the DNA of one chromo- some and translocation of a stretch of DNA from one chro- mosome to another. In sexually reproducing animals like ourselves mutations can be inherited only if they are present in cells that poten- tially contribute to the formation of offspring. Such germ-line cells include eggs sperm and their precursor cells. Body cells that do not contribute to offspring are called somatic cells. Mutations that occur in these cells never are inherited al- though they may contribute to the onset of cancer . Plants have a less distinct division between somatic and germ-line cells since many plant cells can function in both capacities. Mutated genes that encode altered proteins or that can- not be controlled properly cause numerous inherited dis- eases. For example sickle cell disease is attributable to a single nucleotide substitution in the hemoglobin gene which encodes the protein that carries oxygen in red blood cells. The single amino acid change caused by the sickle cell mu- tation reduces the ability of red blood cells to carry oxygen from the lungs to the tissues. Recent advances in detecting disease-causing mutations and in understanding how they af- fect cell functions offer exciting possibilities for reducing their often devastating effects. Sequencing of the human genome has shown that a very large proportion of our DNA does not code for any RNA or have any discernible regulatory function a quite unexpected finding. Mutations in these regions usually produce no im- mediate effects—good or bad. However such “indifferent” mutations in nonfunctional DNA may have been a major player in evolution leading to creation of new genes or new regulatory sequences for controlling already existing genes. For instance since binding sites for transcription factors typ- ically are only 10–12 nucleotides long a few single-nucleotide mutations might convert a nonfunctional bit of DNA into a functional protein-binding regulatory site. Much of the nonessential DNA in both eukaryotes and prokaryotes consists of highly repeated sequences that can move from one place in the genome to another. These mobile DNA elements can jump transpose into genes most com- monly damaging but sometimes activating them. Jumping generally occurs rarely enough to avoid endangering the host organism. Mobile elements which were discovered first in plants are responsible for leaf color variegation and the diverse beautiful color patterns of Indian corn kernels. By jumping in and out of genes that control pigmentation as plant development progresses the mobile elements give rise to elaborate colored patterns. Mobile elements were later found in bacteria in which they often carry and unfortu- nately disseminate genes for antibiotic resistance. Now we understand that mobile elements have multi- plied and slowly accumulated in genomes over evolutionary time becoming a universal property of genomes in present- day organisms. They account for an astounding 45 percent of the human genome. Some of our own mobile DNA ele- ments are copies—often highly mutated and damaged—of genomes from viruses that spend part of their life cycle as DNA segments inserted into host-cell DNA. Thus we carry in our chromosomes the genetic residues of infections ac- quired by our ancestors. Once viewed only as molecular par- asites mobile DNA elements are now thought to have contributed significantly to the evolution of higher organ- isms Chapter 10. The Work of Cells In essence any cell is simply a compartment with a watery interior that is separated from the external environment by a surface membrane the plasma membrane that prevents the free flow of molecules in and out of cells. In addition as we’ve noted eukaryotic cells have extensive internal mem- branes that further subdivide the cell into various compart- ments the organelles. The plasma membrane and other cellular membranes are composed primarily of two layers of phospholipid molecules. These bipartite molecules have a “water-loving” hydrophilic end and a “water-hating” hy- drophobic end. The two phospholipid layers of a mem- brane are oriented with all the hydrophilic ends directed to- ward the inner and outer surfaces and the hydrophobic ends buried within the interior Figure 1-13. Smaller amounts of 1.3 1.3 • The Work of Cells 13 Water Cholesterol Fatty chains Water-seeking head group ▲ FIGURE 1-13 The watery interior of cells is surrounded by the plasma membrane a two-layered shell of phospholipids. The phospholipid molecules are oriented with their fatty acyl chains black squiggly lines facing inward and their water-seeking head groups white spheres facing outward. Thus both sides of the membrane are lined by head groups mainly charged phosphates adjacent to the watery spaces inside and outside the cell. All biological membranes have the same basic phospholipid bilayer structure. Cholesterol red and various proteins not shown are embedded in the bilayer. In actuality the interior space is much larger relative to the volume of the plasma membrane depicted here.

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other lipids such as cholesterol and many kinds of proteins are inserted into the phospholipid framework. The lipid mol- ecules and some proteins can float sidewise in the plane of the membrane giving membranes a fluid character. This flu- idity allows cells to change shape and even move. However the attachment of some membrane proteins to other mole- cules inside or outside the cell restricts their lateral move- ment. We learn more about membranes and how molecules cross them in Chapters 5 and 7. The cytosol and the internal spaces of organelles differ from each other and from the cell exterior in terms of acidity ionic composition and protein contents. For example the composition of salts inside the cell is often drastically differ- ent from what is outside. Because of these different “micro- climates” each cell compartment has its own assigned tasks in the overall work of the cell Chapter 5. The unique func- tions and micro-climates of the various cell compartments are due largely to the proteins that reside in their membranes or interior. We can think of the entire cell compartment as a factory dedicated to sustaining the well-being of the cell. Much cel- lular work is performed by molecular machines some housed in the cytosol and some in various organelles. Here we quickly review the major tasks that cells carry out in their pursuit of the good life. Cells Build and Degrade Numerous Molecules and Structures As chemical factories cells produce an enormous number of complex molecules from simple chemical building blocks. All of this synthetic work is powered by chemical energy ex- tracted primarily from sugars and fats or sunlight in the case of plant cells and stored primarily in ATP the universal “currency” of chemical energy Figure 1-14. In animal and plant cells most ATP is produced by large molecular ma- chines located in two organelles mitochondria and chloro- plasts. Similar machines for generating ATP are located in the plasma membrane of bacterial cells. Both mitochondria and chloroplasts are thought to have originated as bacteria that took up residence inside eukaryotic cells and then be- came welcome collaborators Chapter 8. Directly or indi- rectly all of our food is created by plant cells using sunlight to build complex macromolecules during photosynthesis. Even underground oil supplies are derived from the decay of plant material. Cells need to break down worn-out or obsolete parts into small molecules that can be discarded or recycled. This housekeeping task is assigned largely to lysosomes or- ganelles crammed with degradative enzymes. The interior of lysosomes has a pH of about 5.0 roughly 100 times more acidic than that of the surrounding cytosol. This aids in the breakdown of materials by lysosomal enzymes which are specially designed to function at such a low pH. To create the low pH environment proteins located in the lysosomal mem- brane pump hydrogen ions into the lysosome using energy supplied from ATP Chapter 7. Lysosomes are assisted in the cell’s cleanup work by peroxisomes. These small organelles are specialized for breaking down the lipid components of membranes and rendering various toxins harmless. Most of the structural and functional properties of cells depend on proteins. Thus for cells to work properly the nu- 14 CHAPTER 1 • Life Begins with Cells Energy ATP Light photosynthesis or compounds with high potential energy respiration Synthesis of cellular macro- molecules DNA RNA proteins polysaccharides Synthesis of other cellular constituents such as membrane phospholipids and certain required metabolites Cellular movements including muscle con- traction crawling move- ments of entire cells and movement of chromosomes during mitosis Transport of molecules against a concentration gradient Generation of an electric potential across a membrane important for nerve function Heat ADP + P i ▲ FIGURE 1-14 ATP is the most common molecule used by cells to capture and transfer energy. ATP is formed from ADP and inorganic phosphate P i by photosynthesis in plants and by the breakdown of sugars and fats in most cells. The energy released by the splitting hydrolysis of P i from ATP drives many cellular processes. MEDIA CONNECTIONS Overview Animation: Biological Energy Interconversions

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merous proteins composing the various working compart- ments must be transported from where they are made to their proper locations Chapters 16 and 17. Some proteins are made on ribosomes that are free in the cytosol. Proteins secreted from the cell and most membrane proteins however are made on ribosomes associated with the endoplasmic reticulum ER. This organelle produces processes and ships out both proteins and lipids. Protein chains produced on the ER move to the Golgi apparatus where they are further modified before being forwarded to their final destinations. Proteins that travel in this way contain short sequences of amino acids or attached sugar chains oligosaccharides that serve as addresses for directing them to their correct desti- nations. These addresses work because they are recognized and bound by other proteins that do the sorting and shipping in various cell compartments. Animal Cells Produce Their Own External Environment and Glues The simplest multicellular animals are single cells embedded in a jelly of proteins and polysaccharides called the extracel- lular matrix. Cells themselves produce and secrete these ma- terials thus creating their own immediate environment Chapter 6. Collagen the single most abundant protein in the animal kingdom is a major component of the extracel- lular matrix in most tissues. In animals the extracellular ma- trix cushions and lubricates cells. A specialized especially tough matrix the basal lamina forms a supporting layer un- derlying sheetlike cell layers and helps prevent the cells from ripping apart. The cells in animal tissues are “glued” together by cell- adhesion molecules CAMs embedded in their surface membranes. Some CAMs bind cells to one another other types bind cells to the extracellular matrix forming a cohe- sive unit. The cells of higher plants contain relatively few such molecules instead plants cells are rigidly tied together by extensive interlocking of the cell walls of neighboring cells. The cytosols of adjacent animal or plant cells often are connected by functionally similar but structurally different “bridges” called gap junctions in animals and plasmodes- mata in plants. These structures allow cells to exchange small molecules including nutrients and signals facilitating coor- dinated functioning of the cells in a tissue. Cells Change Shape and Move Although cells sometimes are spherical they more commonly have more elaborate shapes due to their internal skeletons and external attachments. Three types of protein filaments organized into networks and bundles form the cytoskeleton within animal cells Figure 1-15. The cytoskeleton prevents the plasma membrane of animal cells from relaxing into a sphere Chapter 5 it also functions in cell locomotion and the intracellular transport of vesicles chromosomes and macromolecules Chapters 19 and 20. The cytoskeleton can be linked through the cell surface to the extracellular matrix or to the cytoskeleton of other cells thus helping to form tis- sues Chapter 6. All cytoskeletal filaments are long polymers of protein subunits. Elaborate systems regulate the assembly and disas- sembly of the cytoskeleton thereby controlling cell shape. In some cells the cytoskeleton is relatively stable but in others it changes shape continuously. Shrinkage of the cytoskeleton in some parts of the cell and its growth in other parts can pro- duce coordinated changes in shape that result in cell locomo- tion. For instance a cell can send out an extension that attaches to a surface or to other cells and then retract the cell body from the other end. As this process continues due to co- ordinated changes in the cytoskeleton the cell moves for- ward. Cells can move at rates on the order of 20 m/second. Cell locomotion is used during embryonic development of multicellular animals to shape tissues and during adulthood to defend against infection to transport nutrients and to heal wounds. This process does not play a role in the growth and development of multicellular plants because new plant cells 1.3 • The Work of Cells 15 Intermediate filaments Microtubules Microfilaments ▲ FIGURE 1-15 The three types of cytoskeletal filaments have characteristic distributions within cells. Three views of the same cell. A cultured fibroblast was treated with three different antibody preparations. Each antibody binds specifically to the protein monomers forming one type of filament and is chemically linked to a differently colored fluorescent dye green blue or red. Visualization of the stained cell in a fluorescence microscope reveals the location of filaments bound to a particular dye-antibody preparation. In this case intermediate filaments are stained green microtubules blue and microfilaments red. All three fiber systems contribute to the shape and movements of cells. Courtesy of V. Small.

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are generated by the division of existing cells that share cell walls. As a result plant development involves cell enlarge- ment but not movement of cells from one position to another . Cells Sense and Send Information A living cell continuously monitors its surroundings and ad- justs its own activities and composition accordingly. Cells also communicate by deliberately sending signals that can be received and interpreted by other cells. Such signals are common not only within an individual organism but also between organisms. For instance the odor of a pear detected by us and other animals signals a food source consumption of the pear by an animal aids in distributing the pear’s seeds. Everyone benefits The signals employed by cells include sim- ple small chemicals gases proteins light and mechanical movements. Cells possess numerous receptor proteins for de- tecting signals and elaborate pathways for transmitting them within the cell to evoke a response. At any time a cell may be able to sense only some of the signals around it and how a cell responds to a signal may change with time. In some cases receiving one signal primes a cell to respond to a sub- sequent different signal in a particular way. Both changes in the environment e.g. an increase or de- crease in a particular nutrient or the light level and signals received from other cells represent external information that cells must process. The most rapid responses to such signals generally involve changes in the location or activity of pre- existing proteins. For instance soon after you eat a carbo- hydrate-rich meal glucose pours into your bloodstream. The rise in blood glucose is sensed by cells in the pancreas which respond by releasing their stored supply of the protein hormone insulin. The circulating insulin signal causes glu- cose transporters in the cytoplasm of fat and muscle cells to move to the cell surface where they begin importing glucose. Meanwhile liver cells also are furiously taking in glucose via a different glucose transporter. In both liver and muscle cells an intracellular signaling pathway triggered by binding of in- sulin to cell-surface receptors activates a key enzyme needed to make glycogen a large glucose polymer Figure 1-16a. The net result of these cell responses is that your blood glu- cose level falls and extra glucose is stored as glycogen which your cells can use as a glucose source when you skip a meal to cram for a test. The ability of cells to send and respond to signals is cru- cial to development. Many developmentally important sig- nals are secreted proteins produced by specific cells at specific times and places in a developing organism. Often a receiving cell integrates multiple signals in deciding how to behave for example to differentiate into a particular tissue type to extend a process to die to send back a confirming signal yes I’m here or to migrate. The functions of about half the proteins in humans roundworms yeast and several other eukaryotic organisms have been predicted based on analyses of genomic sequences Chapter 9. Such analyses have revealed that at least 10–15 percent of the proteins in eukaryotes function as secreted ex- tracellular signals signal receptors or intracellular signal- transduction proteins which pass along a signal through a series of steps culminating in a particular cellular response e.g. increased glycogen synthesis. Clearly signaling and signal transduction are major activities of cells. Cells Regulate Their Gene Expression to Meet Changing Needs In addition to modulating the activities of existing proteins cells often respond to changing circumstances and to signals from other cells by altering the amount or types of proteins they contain. Gene expression the overall process of selectively reading and using genetic information is commonly controlled at the level of transcription the first step in the production of proteins. In this way cells can produce a particular mRNA only when the encoded protein is needed thus minimizing wasted energy . Producing a mRNA is however only the first in a chain of regulated events that together determine whether an active protein product is produced from a particular gene. 16 CHAPTER 1 • Life Begins with Cells mRNA Cytosolic receptor Increased transcription of specific genes a Surface receptors b Nucleus Receptor-hormone complex Bound signal Active enzyme Inactive enzyme mRNA Protein ▲ FIGURE 1-16 External signals commonly cause a change in the activity of preexisting proteins or in the amounts and types of proteins that cells produce. a Binding of a hormone or other signaling molecule to its specific receptors can trigger an intracellular pathway that increases or decreases the activity of a preexisting protein. For example binding of insulin to receptors in the plasma membrane of liver and muscle cells leads to activation of glycogen synthase a key enzyme in the synthesis of glycogen from glucose. b The receptors for steroid hormones are located within cells not on the cell surface. The hormone-receptor complexes activate transcription of specific target genes leading to increased production of the encoded proteins. Many signals that bind to receptors on the cell surface also act by more complex pathways to modulate gene expression.

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Transcriptional control of gene expression was first de- cisively demonstrated in the response of the gut bacterium E. coli to different sugar sources. E. coli cells prefer glucose as a sugar source but they can survive on lactose in a pinch. These bacteria use both a DNA-binding repressor protein and a DNA-binding activator protein to change the rate of transcription of three genes needed to metabolize lactose de- pending on the relative amounts of glucose and lactose pres- ent Chapter 4. Such dual positive/negative control of gene expression fine tunes the bacterial cell’s enzymatic equipment for the job at hand. Like bacterial cells unicellular eukaryotes may be sub- jected to widely varying environmental conditions that re- quire extensive changes in cellular structures and function. For instance in starvation conditions yeast cells stop grow- ing and form dormant spores see Figure 1-4. In multicellu- lar organisms however the environment around most cells is relatively constant. The major purpose of gene control in us and in other complex organisms is to tailor the properties of various cell types to the benefit of the entire animal or plant. Control of gene activity in eukaryotic cells usually in- volves a balance between the actions of transcriptional acti- vators and repressors. Binding of activators to specific DNA regulatory sequences called enhancers turns on transcription and binding of repressors to other regulatory sequences called silencers turns off transcription. In Chapters 11 and 12 we take a close look at transcriptional activators and re- pressors and how they operate as well as other mechanisms for controlling gene expression. In an extreme case expres- sion of a particular gene could occur only in part of the brain only during evening hours only during a certain stage of development only after a large meal and so forth. Many external signals modify the activity of transcrip- tional activators and repressors that control specific genes. For example lipid-soluble steroid hormones such as estro- gen and testosterone can diffuse across the plasma mem- brane and bind to their specific receptors located in the cytoplasm or nucleus Figure 1-16b. Hormone binding changes the shape of the receptor so that it can bind to spe- cific enhancer sequences in the DNA thus turning the recep- tor into a transcriptional activator. By this rather simple signal-transduction pathway steroid hormones cause cells to change which genes they transcribe Chapter 11. Since steroid hormones can circulate in the bloodstream they can affect the properties of many or all cells in a temporally co- ordinated manner. Binding of many other hormones and of growth factors to receptors on the cell surface triggers dif- ferent signal-transduction pathways that also lead to changes in the transcription of specific genes Chapters 13–15. Al- though these pathways involve multiple components and are more complicated than those transducing steroid hormone signals the general idea is the same. Cells Grow and Divide The most remarkable feature of cells and entire organisms is their ability to reproduce. Biological reproduction combined with continuing evolutionary selection for a highly functional body plan is why today’s horseshoe crabs look much as they did 300 million years ago a time span during which entire mountain ranges have risen or fallen. The Teton Mountains in Wyoming now about 14000 feet high and still growing did not exist a mere 10 million years ago. Yet horseshoe crabs with a life span of about 19 years have faithfully reproduced their ancient selves more than half a million times during that period. The common impression that biological structure is transient and geological structure is stable is the exact oppo- site of the truth. Despite the limited duration of our individ- ual lives reproduction gives us a potential for immortality that a mountain or a rock does not have. The simplest type of reproduction entails the division of a “parent” cell into two “daughter” cells. This occurs as part of the cell cycle a series of events that prepares a cell to divide followed by the actual division process called mitosis. The eukaryotic cell cycle commonly is represented as four stages Figure 1-17. The chromosomes and the DNA they carry are copied during the S synthesis phase. The replicated chro- mosomes separate during the M mitotic phase with each daughter cell getting a copy of each chromosome during cell division. The M and S phases are separated by two gap stages the G 1 phase and G 2 phase during which mRNAs and pro- teins are made. In single-celled organisms both daughter cells 1.3 • The Work of Cells 17 G 0 Nondividing cells Resting cells M G 2 S RNA and protein synthesis DNA replication RNA and protein synthesis Cell division G 1 ▲ FIGURE 1-17 During growth eukaryotic cells continually progress through the four stages of the cell cycle generating new daughter cells. In most proliferating cells the four phases of the cell cycle proceed successively taking from 10–20 hours depending on cell type and developmental state. During interphase which consists of the G 1 S and G 2 phases the cell roughly doubles its mass. Replication of DNA during S leaves the cell with four copies of each type of chromosome. In the mitotic M phase the chromosomes are evenly partitioned to two daughter cells and the cytoplasm divides roughly in half in most cases. Under certain conditions such as starvation or when a tissue has reached its final size cells will stop cycling and remain in a waiting state called G 0 . Most cells in G 0 can reenter the cycle if conditions change. MEDIA CONNECTIONS Overview Animation: Life Cycle of a Cell

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often though not always resemble the parent cell. In multi- cellular organisms stem cells can give rise to two different cells one that resembles the parent cell and one that does not. Such asymmetric cell division is critical to the generation of different cell types in the body Chapter 22. During growth the cell cycle operates continuously with newly formed daughter cells immediately embarking on their own path to mitosis. Under optimal conditions bacteria can di- vide to form two daughter cells once every 30 minutes. At this rate in an hour one cell becomes four in a day one cell be- comes more than 10 14 which if dried would weigh about 25 grams. Under normal circumstances however growth cannot continue at this rate because the food supply becomes limiting. Most eukaryotic cells take considerably longer than bac- terial cells to grow and divide. Moreover the cell cycle in adult plants and animals normally is highly regulated Chapter 21. This tight control prevents imbalanced excessive growth of tissues while assuring that worn-out or damaged cells are re- placed and that additional cells are formed in response to new circumstances or developmental needs. For instance the pro- liferation of red blood cells increases substantially when a per- son ascends to a higher altitude and needs more capacity to capture oxygen. Some highly specialized cells in adult animals such as nerve cells and striated muscle cells rarely divide if at all. The fundamental defect in cancer is loss of the ability to control the growth and division of cells. In Chapter 23 we examine the molecular and cellular events that lead to inap- propriate uncontrolled proliferation of cells. Mitosis is an asexual process since the daughter cells carry the exact same genetic information as the parental cell. In sexual reproduction fusion of two cells produces a third cell that contains genetic information from each parental cell. Since such fusions would cause an ever-increasing num- ber of chromosomes sexual reproductive cycles employ a special type of cell division called meiosis that reduces the number of chromosomes in preparation for fusion see Fig- ure 9-3. Cells with a full set of chromosomes are called diploid cells. During meiosis a diploid cell replicates its chro- mosomes as usual for mitosis but then divides twice without copying the chromosomes in-between. Each of the resulting four daughter cells which has only half the full number of chromosomes is said to be haploid. Sexual reproduction occurs in animals and plants and even in unicellular organisms such as yeasts see Figure 1-5. Animals spend considerable time and energy generating eggs and sperm the haploid cells called gametes that are used for sexual reproduction. A human female will produce about half a million eggs in a lifetime all these cells form before she is born a young human male about 100 million sperm each day. Gametes are formed from diploid precursor germ-line cells which in humans contain 46 chromosomes. In humans the X and Y chromosomes are called sex chromosomes because they determine whether an individual is male or female. In human diploid cells the 44 remaining chromosomes called auto- somes occur as pairs of 22 different kinds. Through meiosis a man produces sperm that have 22 chromosomes plus either an X or a Y and a woman produces ova unfertilized eggs with 22 chromosomes plus an X. Fusion of an egg and sperm fer- tilization yields a fertilized egg the zygote with 46 chromo- somes one pair of each of the 22 kinds and a pair of X’s in females or an X and a Y in males Figure 1-18. Errors during meiosis can lead to disorders resulting from an abnormal num- ber of chromosomes. These include Down’s syndrome caused by an extra chromosome 21 and Klinefelter’s syndrome caused by an extra X chromosome. Cells Die from Aggravated Assault or an Internal Program When cells in multicellular organisms are badly damaged or infected with a virus they die. Cell death resulting from such a traumatic event is messy and often releases potentially toxic cell constituents that can damage surrounding cells. Cells also may die when they fail to receive a life-maintaining signal or when they receive a death signal. In this type of pro- grammed cell death called apoptosis a dying cell actually produces proteins necessary for self-destruction. Death by apoptosis avoids the release of potentially toxic cell con- stituents Figure 1-19. Programmed cell death is critical to the proper develop- ment and functioning of our bodies Chapter 22. During fetal life for instance our hands initially develop with “web- bing” between the fingers the cells in the webbing subse- quently die in an orderly and precise pattern that leaves the 18 CHAPTER 1 • Life Begins with Cells 44 A XX 44 A XX 22 A X 22 A X 44 A XY 44 A XY 22 A Y 22 A X FEMALE MALE Meiosis Fertilization One type of female gamete Two types of male gamete Female zygote Male zygote Diploid 2n Haploid 1n Haploid 1n Diploid 2n ▲ FIGURE 1-18 Dad made you a boy or girl. In animals meiosis of diploid precursor cells forms eggs and sperm gametes. The male parent produces two types of sperm and determines the sex of the zygote. In humans as shown here X and Y are the sex chromosomes the zygote must receive a Y chromosome from the male parent to develop into a male. A autosomes non-sex chromosomes.

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fingers and thumb free to play the piano. Nerve cells in the brain soon die if they do not make proper or useful electri- cal connections with other cells. Some developing lympho- cytes the immune-system cells intended to recognize foreign proteins and polysaccharides have the ability to react against our own tissues. Such self-reactive lymphocytes be- come programmed to die before they fully mature. If these cells are not weeded out before reaching maturity they can cause autoimmune diseases in which our immune system de- stroys the very tissues it is meant to protect. Investigating Cells and Their Parts To build an integrated understanding of how the various mo- lecular components that underlie cellular functions work to- gether in a living cell we must draw on various perspectives. Here we look at how five disciplines—cell biology biochem- istry genetics genomics and developmental biology—can contribute to our knowledge of cell structure and function. The experimental approaches of each field probe the cell’s inner workings in different ways allowing us to ask differ- ent types of questions about cells and what they do. Cell di- vision provides a good example to illustrate the role of different perspectives in analyzing a complex cellular process. The realm of biology ranges in scale more than a billion- fold Figure 1-20. Beyond that it’s ecology and earth science 1.4 1.4 • Investigating Cells and Their Parts 19 ▲ FIGURE 1-19 Apoptotic cells break apart without spewing forth cell constituents that might harm neighboring cells. White blood cells normally look like the cell on the left. Cells undergoing programmed cell death apoptosis like the cell on the right form numerous surface blebs that eventually are released. The cell is dying because it lacks certain growth signals. Apoptosis is important to eliminate virus-infected cells remove cells where they are not needed like the webbing that disappears as fingers develop and to destroy immune system cells that would react with our own bodies. Gopal Murti/Visuals Unlimited Inc. 10 -10 m10 -9 m10 -8 m10 -7 m10 -6 m10 -5 m10 -4 m10 -3 m10 -2 m10 -1 m10 0 m C _ C bond Glucose Hemoglobin Ribosome Mitochondrion Bacterium Red blood cell Atoms Small molecules Macro- molecules Cells 1 µ m 1 nm 1 mm 1 m 0.1 nm 10 nm 100 nm 10 µ m 100 µ m 10 mm 100 mm Bumblebee Multicellular organisms C. elegans Newborn human Assemblies Nanometers Micrometers Millimeters Meters PHOTO DNA base pairs a b c d ▲ FIGURE 1-20 Biologists are interested in objects ranging in size from small molecules to the tallest trees. A sampling of biological objects aligned on a logarithmic scale. a The DNA double helix has a diameter of about 2 nm. b Eight-cell-stage human embryo three days after fertilization about 200 m across. c A wolf spider about 15 mm across. d Emperor penguins are about 1 m tall. Part a Will and Deni McIntyre. Part b Yorgas Nikas/Photo Researchers Inc. Part c Gary Gaugler/Visuals Unlimited Inc. Part d Hugh S. Rose/Visuals Unlimited Inc.

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at the “macro” end chemistry and physics at the “micro” end. The visible plants and animals that surround us are measured in meters 10 0 –10 2 m. By looking closely we can see a bio- logical world of millimeters 1 mm 10 3 m and even tenths of millimeters 10 4 m. Setting aside oddities like chicken eggs most cells are 1–100 micrometers 1 m 10 6 m long and thus clearly visible only when magnified. To see the struc- tures within cells we must go farther down the size scale to 10–100 nanometers 1 nm 10 9 m. Cell Biology Reveals the Size Shape and Location of Cell Components Actual observation of cells awaited development of the first crude microscopes in the early 1600s. A compound micro- scope the most useful type of light microscope has two lenses. The total magnifying power is the product of the magnification by each lens. As better lenses were invented the magnifying power and the ability to distinguish closely spaced objects the resolution increased greatly. Modern compound microscopes magnify the view about a thousand- fold so that a bacterium 1 micrometer 1 m long looks like it’s a millimeter long. Objects about 0.2 m apart can be dis- cerned in these instruments. Microscopy is most powerful when particular compo- nents of the cell are stained or labeled specifically enabling them to be easily seen and located within the cell. A simple example is staining with dyes that bind specifically to DNA to visualize the chromosomes. Specific proteins can be de- tected by harnessing the binding specificity of antibodies the proteins whose normal task is to help defend animals against infection and foreign substances. In general each type of antibody binds to one protein or large polysaccha- ride and no other Chapter 3. Purified antibodies can be chemically linked to a fluorescent molecule which permits their detection in a special fluorescence microscope Chap- ter 5. If a cell or tissue is treated with a detergent that par- tially dissolves cell membranes fluorescent antibodies can drift in and bind to the specific protein they recognize. When the sample is viewed in the microscope the bound fluorescent antibodies identify the location of the target pro- tein see Figure 1-15. Better still is pinpointing proteins in living cells with in- tact membranes. One way of doing this is to introduce an engineered gene that codes for a hybrid protein: part of the hybrid protein is the cellular protein of interest the other part is a protein that fluoresces when struck by ultraviolet light. A common fluorescent protein used for this purpose is green fluorescent protein GFP a natural protein that makes some jellyfish colorful and fluorescent. GFP “tag- ging” could reveal for instance that a particular protein is first made on the endoplasmic reticulum and then is moved by the cell into the lysosomes. In this case first the endoplasmic reticulum and later the lysosomes would glow in the dark. Chromosomes are visible in the light microscope only during mitosis when they become highly condensed. The ex- traordinary behavior of chromosomes during mitosis first was discovered using the improved compound microscopes of the late 1800s. About halfway through mitosis the repli- cated chromosomes begin to move apart. Microtubules one of the three types of cytoskeletal filaments participate in this movement of chromosomes during mitosis. Fluorescent tag- ging of tubulin the protein subunit that polymerizes to form microtubules reveals structural details of cell division that otherwise could not be seen and allows observation of chro- mosome movement Figure 1-21. Electron microscopes use a focused beam of electrons in- stead of a beam of light. In transmission electron microscopy specimens are cut into very thin sections and placed under a high vacuum precluding examination of living cells. The res- olution of transmission electron microscopes about 0.1 nm permits fine structural details to be distinguished and their powerful magnification would make a 1- m-long bacterial cell look like a soccer ball. Most of the organelles in eukary- otic cells and the double-layered structure of the plasma membrane were first observed with electron microscopes Chapter 5. With new specialized electron microscopy tech- niques three-dimensional models of organelles and large protein complexes can be constructed from multiple images. But to obtain a more detailed look at the individual macro- molecules within cells we must turn to techniques within the purview of biochemistry. 20 CHAPTER 1 • Life Begins with Cells ▲ FIGURE 1-21 During the later stages of mitosis microtubules red pull the replicated chromosomes black toward the ends of a dividing cell. This plant cell is stained with a DNA-binding dye ethidium to reveal chromosomes and with fluorescent-tagged antibodies specific for tubulin to reveal microtubules. At this stage in mitosis the two copies of each replicated chromosome called chromatids have separated and are moving away from each other. Courtesy of Andrew Bajer.

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Biochemistry Reveals the Molecular Structure and Chemistry of Purified Cell Constituents Biochemists extract the contents of cells and separate the constituents based on differences in their chemical or phys- ical properties a process called fractionation. Of particular interest are proteins the workhorses of many cellular processes. A typical fractionation scheme involves use of various separation techniques in a sequential fashion. These separation techniques commonly are based on dif- ferences in the size of molecules or the electrical charge on their surface Chapter 3. To purify a particular protein of interest a purification scheme is designed so that each step yields a preparation with fewer and fewer contaminating proteins until finally only the protein of interest remains Figure 1-22. The initial purification of a protein of interest from a cell extract often is a tedious time-consuming task. Once a small amount of purified protein is obtained antibodies to it can be produced by methods discussed in Chapter 6. For a bio- chemist antibodies are near-perfect tools for isolating larger amounts of a protein of interest for further analysis. In effect antibodies can “pluck out” the protein they specifically rec- ognize and bind from a semipure sample containing numer- ous different proteins. An increasingly common alternative is to engineer a gene that encodes a protein of interest with a small attached protein “tag” which can be used to pull out the protein from whole cell extracts. Purification of a protein is a necessary prelude to studies on how it catalyzes a chemical reaction or carries out other functions and how its activity is regulated. Some enzymes are made of multiple protein chains subunits with one chain catalyzing a chemical reaction and other chains regulating when and where that reaction occurs. The molecular ma- chines that perform many critical cell processes constitute even larger assemblies of proteins. By separating the individ- ual proteins composing such assemblies their individual cat- alytic or other activities can be assessed. For example purification and study of the activity of the individual pro- teins composing the DNA replication machine provided clues about how they work together to replicate DNA during cell division Chapter 4. The folded three-dimensional structure or conforma- tion of a protein is vital to its function. To understand the re- lation between the function of a protein and its form we need to know both what it does and its detailed structure. The most widely used method for determining the complex structures of proteins DNA and RNA is x-ray crystallogra- phy. Computer-assisted analysis of the data often permits the location of every atom in a large complex molecule to be de- termined. The double-helix structure of DNA which is key to its role in heredity was first proposed based on x-ray crys- tallographic studies. Throughout this book you will en- counter numerous examples of protein structures as we zero in on how proteins work. Genetics Reveals the Consequences of Damaged Genes Biochemical and crystallographic studies can tell us much about an individual protein but they cannot prove that it is required for cell division or any other cell process. The im- portance of a protein is demonstrated most firmly if a mu- 1.4 • Investigating Cells and Their Parts 21 Homogenate Salt fractionation Ion exchange chromatography Gel filtration chromatography Affinity chromatography 12345 ▲ FIGURE 1-22 Biochemical purification of a protein from a cell extract often requires several separation techniques. The purification can be followed by gel electrophoresis of the starting protein mixture and the fractions obtained from each purification step. In this procedure a sample is applied to wells in the top of a gelatin-like slab and an electric field is applied. In the presence of appropriate salt and detergent concentrations the proteins move through the fibers of the gel toward the anode with larger proteins moving more slowly through the gel than smaller ones see Figure 3-32. When the gel is stained separated proteins are visible as distinct bands whose intensities are roughly proportional to the protein concentration. Shown here are schematic depictions of gels for the starting mixture of proteins lane 1 and samples taken after each of several purification steps. In the first step salt fractionation proteins that precipitated with a certain amount of salt were re-dissolved electrophoresis of this sample lane 2 shows that it contains fewer proteins than the original mixture. The sample then was subjected in succession to three types of column chromatography that separate proteins by electrical charge size or binding affinity for a particular small molecule see Figure 3-34. The final preparation is quite pure as can be seen from the appearance of just one protein band in lane 5. After J. Berg et al. 2002 Biochemistry W. H. Freeman and Company p. 87 .

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tation that prevents its synthesis or makes it nonfunctional adversely affects the process under study. We define the genotype of an organism as its composition of genes the term also is commonly used in reference to dif- ferent versions of a single gene or a small number of genes of interest in an individual organism. A diploid organism generally carries two versions alleles of each gene one de- rived from each parent. There are important exceptions such as the genes on the X and Y chromosomes in males of some species including our own. The phenotype is the visible out- come of a gene’s action like blue eyes versus brown eyes or the shapes of peas. In the early days of genetics the location and chemical identity of genes were unknown all that could be followed were the observable characteristics the pheno- types. The concept that genes are like “beads” on a long “string” the chromosome was proposed early in the 1900s based on genetic work with the fruit fly Drosophila. In the classical genetics approach mutants are isolated that lack the ability to do something a normal organism can do. Often large genetic “screens” are done looking for many different mutant individuals e.g. fruit flies yeast cells that are unable to complete a certain process such as cell division or muscle formation. In experimental organisms or cultured cells mutations usually are produced by treatment with a mutagen a chemical or physical agent that promotes muta- tions in a largely random fashion. But how can we isolate and maintain mutant organisms or cells that are defective in some process such as cell division that is necessary for sur- vival One way is to look for temperature-sensitive mutants. These mutants are able to grow at one temperature the per- missive temperature but not at another usually higher tem- perature the nonpermissive temperature. Normal cells can grow at either temperature. In most cases a temperature- sensitive mutant produces an altered protein that works at the permissive temperature but unfolds and is nonfunctional at the nonpermissive temperature. Temperature-sensitive screens are readily done with viruses bacteria yeast round- worms and fruit flies. By analyzing the effects of numerous different temperature- sensitive mutations that altered cell division geneticists discov- ered all the genes necessary for cell division without knowing anything initially about which proteins they encode or how these proteins participate in the process. The great power of ge- netics is to reveal the existence and relevance of proteins with- out prior knowledge of their biochemical identity or molecular function. Eventually these “mutation-defined” genes were iso- lated and replicated cloned with recombinant DNA tech- niques discussed in Chapter 9. With the isolated genes in hand the encoded proteins could be produced in the test tube or in engineered bacteria or cultured cells. Then the biochemists could investigate whether the proteins associate with other pro- teins or DNA or catalyze particular chemical reactions during cell division Chapter 21. The analysis of genome sequences from various organ- isms during the past decade has identified many previously unknown DNA regions that are likely to encode proteins i.e. protein-coding genes. The general function of the pro- tein encoded by a sequence-identified gene may be deduced by analogy with known proteins of similar sequence. Rather than randomly isolating mutations in novel genes several techniques are now available for inactivating specific genes by engineering mutations into them Chapter 9. The effects of such deliberate gene-specific mutations provide informa- tion about the role of the encoded proteins in living organ- isms. This application of genetic techniques starts with a gene/protein sequence and ends up with a mutant phenotype traditional genetics starts with a mutant phenotype and ends up with a gene/protein sequence. Genomics Reveals Differences in the Structure and Expression of Entire Genomes Biochemistry and genetics generally focus on one gene and its encoded protein at a time. While powerful these traditional approaches do not give a comprehensive view of the struc- ture and activity of an organism’s genome its entire set of genes. The field of genomics does just that encompassing the molecular characterization of whole genomes and the deter- mination of global patterns of gene expression. The recent completion of the genome sequences for more than 80 species of bacteria and several eukaryotes now permits com- parisons of entire genomes from different species. The results provide overwhelming evidence of the molecular unity of life and the evolutionary processes that made us what we are see Section 1.5. Genomics-based methods for comparing thou- sands of pieces of DNA from different individuals all at the same time are proving useful in tracing the history and mi- grations of plants and animals and in following the inheri- tance of diseases in human families. New methods using DNA microarrays can simultane- ously detect all the mRNAs present in a cell thereby indi- cating which genes are being transcribed. Such global patterns of gene expression clearly show that liver cells tran- scribe a quite different set of genes than do white blood cells or skin cells. Changes in gene expression also can be moni- tored during a disease process in response to drugs or other external signals and during development. For instance the recent identification of all the mRNAs present in cultured fi- broblasts before during and after they divide has given us an overall view of transcriptional changes that occur during cell division Figure 1-23. Cancer diagnosis is being trans- formed because previously indistinguishable cancer cells have distinct gene expression patterns and prognoses Chap- ter 23. Similar studies with different organisms and cell types are revealing what is universal about the genes involved in cell division and what is specific to particular organisms. The entire complement of proteins in a cell its proteome is controlled in part by changes in gene transcription. The regulated synthesis processing localization and degradation of specific proteins also play roles in determining the pro- teome of a particular cell and the association of certain pro- teins with one another is critical to the functional abilities 22 CHAPTER 1 • Life Begins with Cells

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of cells. New techniques for monitoring the presence and in- teractions of numerous proteins simultaneously called pro- teomics are one way of assembling a comprehensive view of the proteins and molecular machines important for cell functioning. The field of proteomics will advance dramati- cally once high-throughput x-ray crystallography currently under development permits researchers to rapidly determine the structures of hundreds or thousands of proteins. Developmental Biology Reveals Changes in the Properties of Cells as They Specialize Another approach to viewing cells comes from studying how they change during development of a complex organism. Bacteria algae and unicellular eukaryotes protozoans yeasts often but by no means always can work solo. The concerted actions of the trillions of cells that compose our bodies require an enormous amount of communication and division of labor. During the development of multicellular or- ganisms differentiation processes form hundreds of cell types each specialized for a particular task: transmission of electrical signals by neurons transport of oxygen by red blood cells destruction of infecting bacteria by macro- phages contraction by muscle cells chemical processing by liver cells. Many of the differences among differentiated cells are due to production of specific sets of proteins needed to carry out the unique functions of each cell type. That is only a subset of an organism’s genes is transcribed at any given time or in any given cell. Such differential gene expression at dif- ferent times or in different cell types occurs in bacteria fungi plants animals and even viruses. Differential gene expres- sion is readily apparent in an early fly embryo in which all the cells look alike until they are stained to detect the pro- teins encoded by particular genes Figure 1-24. Transcrip- tion can change within one cell type in response to an external signal or in accordance with a biological clock some genes for instance undergo a daily cycle between low and high transcription rates. 1.4 • Investigating Cells and Their Parts 23 ▲ FIGURE 1-23 DNA microarray analysis gives a global view of changes in transcription following addition of serum to cultured human cells. Serum contains growth factors that stimulate nondividing cells to begin growing and dividing. DNA microarray analysis can detect the relative transcription of genes in two different cell populations see Figure 9-35. The microarray consists of tiny spots of DNA attached to a microscope slide. Each spot contains many copies of a DNA sequence from a single human gene. One preparation of RNA containing all the different types of RNA being made in nongrowing cells cultured without serum is labeled with green fluorescent molecules. Another RNA population from growing serum-treated cells is labeled with red. The two are mixed and hybridized to the slide where they "zipper up" with their corresponding genes. Green spots e.g. spot 3 therefore indicate genes that are transcribed in nondividing serum-deprived cells red spots e.g. spot 4 indicate genes that are transcribed in dividing cells and yellow spots e.g. spots 1 and 2 indicate genes that are transcribed equally in dividing and nondividing cells. From V. R. Iyer et al. 1999 Science 283:83. ▲ FIGURE 1-24 Differential gene expression can be detected in early fly embryos before cells are morphologically different. An early Drosophila embryo has about 6000 cells covering its surface most of which are indistinguishable by simple light microscopy. If the embryo is made permeable to antibodies with a detergent that partially dissolves membranes the antibodies can find and bind to the proteins they recognize. In this embryo we see antibodies tagged with a fluorescent label bound to proteins that are in the nuclei each small sphere corresponds to one nucleus. Three different antibodies were used each specific for a different protein and each giving a distinct color yellow green or blue in a fluorescence microscope. The red color is added to highlight overlaps between the yellow and blue stains. The locations of the different proteins show that the cells are in fact different at this early stage with particular genes turned on in specific stripes of cells. These genes control the subdivision of the body into repeating segments like the black and yellow stripes of a hornet. Courtesy of Sean Carroll University of Wisconsin.

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Producing different kinds of cells is not enough to make an organism any more than collecting all the parts of a truck in one pile gives you a truck. The various cell types must be or- ganized and assembled into all the tissues and organs. Even more remarkable these body parts must work almost imme- diately after their formation and continue working during the growth process. For instance the human heart begins to beat when it is less than 3 mm long when we are mere 23-day-old embryos and continues beating as it grows into a fist-size muscle. From a few hundred cells to billions and still ticking. In the developing organism cells grow and divide at some times and not others they assemble and communicate they prevent or repair errors in the developmental process and they coordinate each tissue with others. In the adult or- ganism cell division largely stops in most organs. If part of an organ such as the liver is damaged or removed cell divi- sion resumes until the organ is regenerated. The legend goes that Zeus punished Prometheus for giving humans fire by chaining him to a rock and having an eagle eat his liver. The punishment was eternal because as the Greeks evidently knew the liver regenerates. Developmental studies involve watching where when and how different kinds of cells form discovering which sig- nals trigger and coordinate developmental events and un- derstanding the differential gene action that underlies differentiation Chapters 15 and 22. During development we can see cells change in their normal context of other cells. Cell biology biochemistry cell biology genetics and ge- nomics approaches are all employed in studying cells during development. Choosing the Right Experimental Organism for the Job Our current understanding of the molecular functioning of cells rests on studies with viruses bacteria yeast protozoa slime molds plants frogs sea urchins worms insects fish chickens mice and humans. For various reasons some or- ganisms are more appropriate than others for answering par- ticular questions. Because of the evolutionary conservation of genes proteins organelles cell types and so forth dis- coveries about biological structures and functions obtained with one experimental organism often apply to others. Thus researchers generally conduct studies with the organism that is most suitable for rapidly and completely answering the question being posed knowing that the results obtained in one organism are likely to be broadly applicable. Figure 1-25 summarizes the typical experimental uses of various organ- isms whose genomes have been sequenced completely or nearly so. The availability of the genome sequences for these organisms makes them particularly useful for genetics and genomics studies. Bacteria have several advantages as experimental organ- isms: They grow rapidly possess elegant mechanisms for controlling gene activity and have powerful genetics. This 24 CHAPTER 1 • Life Begins with Cells FIGURE 1-25 Each experimental organism used in cell biology has advantages for certain types of studies. Viruses and bacteria have small genomes amenable to genetic dissection. Many insights into gene control initially came from studies with these organisms. The yeast Saccharomyces cerevisiae has the cellular organization of a eukaryote but is a relatively simple single-celled organism that is easy to grow and to manipulate genetically. In the nematode worm Caenorhabditis elegans which has a small number of cells arranged in a nearly identical way in every worm the formation of each individual cell can be traced. The fruit fly Drosophila melanogaster first used to discover the properties of chromosomes has been especially valuable in identifying genes that control embryonic development. Many of these genes are evolutionarily conserved in humans. The zebrafish Danio rerio is used for rapid genetic screens to identify genes that control development and organogenesis. Of the experimental animal systems mice Mus musculus are evolutionarily the closest to humans and have provided models for studying numerous human genetic and infectious diseases. The mustard-family weed Arabidopsis thaliana sometimes described as the Drosophila of the plant kingdom has been used for genetic screens to identify genes involved in nearly every aspect of plant life. Genome sequencing is completed for many viruses and bacterial species the yeast Saccharomyces cerevisiae the roundworm C. elegans the fruit fly D. melanogaster humans and the plant Arabidopsis thaliana. It is mostly completed for mice and in progress for zebrafish. Other organisms particularly frogs sea urchins chickens and slime molds continue to be immensely valuable for cell biology research. Increasingly a wide variety of other species are used especially for studies of evolution of cells and mechanisms. Part a Visuals Unlimited Inc. Part b Kari Lountmaa/Science Photo Library/ Photo Researchers Inc. Part c Scimat/Photo Researchers Inc. Part d Photo Researchers Inc. Part e Darwin Dale/Photo Researchers Inc. Part f Inge Spence/Visuals Unlimited Inc. Part g J. M. Labat/Jancana/Visuals Unlimited Inc. Part h Darwin Dale/Photo Researchers Inc. latter property relates to the small size of bacterial genomes the ease of obtaining mutants the availability of techniques for transferring genes into bacteria an enormous wealth of knowledge about bacterial gene control and protein func- tions and the relative simplicity of mapping genes relative to one another in the genome. Single-celled yeasts not only have some of the same advantages as bacteria but also pos- sess the cell organization marked by the presence of a nu- cleus and organelles that is characteristic of all eukaryotes. Studies of cells in specialized tissues make use of animal and plant “models” that is experimental organisms with at- tributes typical of many others. Nerve cells and muscle cells for instance traditionally were studied in mammals or in creatures with especially large or accessible cells such as the giant neural cells of the squid and sea hare or the flight mus- cles of birds. More recently muscle and nerve development have been extensively studied in fruit flies Drosophila melanogaster roundworms Caenorhabditis elegans and zebrafish in which mutants can be readily isolated. Organ- isms with large-celled embryos that develop outside the

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1.4 • Investigating Cells and Their Parts 25 Plant Arabidopsis thaliana Development and patterning of tissues Genetics of cell biology Agricultural applications Physiology Gene regulation Immunity Infectious disease Roundworm Caenorhabditis elegans Development of the body plan Cell lineage Formation and function of the nervous system Control of programmed cell death Cell proliferation and cancer genes Aging Behavior Gene regulation and chromosome structure Viruses Proteins involved in DNA RNA protein synthesis Gene regulation Cancer and control of cell proliferation Transport of proteins and organelles inside cells Infection and immunity Possible gene therapy approaches Bacteria Proteins involved in DNA RNA protein synthesis metabolism Gene regulation Targets for new antibiotics Cell cycle Signaling Yeast Saccharomyces cerevisiae Control of cell cycle and cell division Protein secretion and membrane biogenesis Function of the cytoskeleton Cell differentiation Aging Gene regulation and chromosome structure Fruit fly Drosophila melanogaster Development of the body plan Generation of differentiated cell lineages Formation of the nervous system heart and musculature Programmed cell death Genetic control of behavior Cancer genes and control of cell proliferation Control of cell polarization Effects of drugs alcohol pesticides Zebrafish Development of vertebrate body tissues Formation and function of brain and nervous system Birth defects Cancer Mice including cultured cells Development of body tissues Function of mammalian immune system Formation and function of brain and nervous system Models of cancers and other human diseases Gene regulation and inheritance Infectious disease a b c d e f g h mother e.g. frogs sea urchins fish and chickens are ex- tremely useful for tracing the fates of cells as they form differ- ent tissues and for making extracts for biochemical studies. For instance a key protein in regulating mitosis was first identified in studies with frog and sea urchin embryos and subsequently purified from extracts Chapter 21.

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Using recombinant DNA techniques researchers can engineer specific genes to contain mutations that inactivate or increase production of their encoded proteins. Such genes can be introduced into the embryos of worms flies frogs sea urchins chickens mice a variety of plants and other organisms permitting the effects of activating a gene abnormally or inhibiting a normal gene function to be as- sessed. This approach is being used extensively to produce mouse versions of human genetic diseases. New techniques specifically for inactivating particular genes by injecting short pieces of RNA are making quick tests of gene func- tions possible in many organisms. Mice have one enormous advantage over other experi- mental organisms: they are the closest to humans of any an- imal for which powerful genetic approaches are feasible. Engineered mouse genes carrying mutations similar to those associated with a particular inherited disease in humans can be introduced into mouse embryonic stem ES cells. These cells can be injected into an early embryo which is then im- planted into a pseudopregnant female mouse Chapter 9. If the mice that develop from the injected ES cells exhibit dis- eases similar to the human disease then the link between the disease and mutations in a particular gene or genes is sup- ported. Once mouse models of a human disease are avail- able further studies on the molecular defects causing the disease can be done and new treatments can be tested thereby minimizing human exposure to untested treatments. A continuous unplanned genetic screen has been per- formed on human populations for millennia. Thousands of inherited traits have been identified and more recently mapped to locations on the chromosomes. Some of these traits are inherited propensities to get a disease others are eye color or other minor characteristics. Genetic variations in virtually every aspect of cell biology can be found in human populations allowing studies of normal and disease states and of variant cells in culture. Less-common experimental organisms offer possibilities for exploring unique or exotic properties of cells and for studying standard properties of cells that are exaggerated in a useful fashion in a particular animal. For example the ends of chromosomes the telomeres are extremely dilute in most cells. Human cells typically contain 92 telomeres 46 chro- mosomes 2 ends per chromosome. In contrast some pro- tozoa with unusual “fragmented” chromosomes contain millions of telomeres per cell. Recent discoveries about telomere structure have benefited greatly from using this nat- ural variation for experimental advantage. A Genome Perspective on Evolution Comprehensive studies of genes and proteins from many or- ganisms are giving us an extraordinary documentation of the history of life. We share with other eukaryotes thousands of 1.5 individual proteins hundreds of macromolecular machines and most of our organelles all as a result of our shared evo- lutionary history. New insights into molecular cell biology arising from genomics are leading to a fuller appreciation of the elegant molecular machines that arose during billions of years of genetic tinkering and evolutionary selection for the most efficient precise designs. Despite all that we currently know about cells many new proteins new macromolecular assemblies and new activities of known ones remain to be discovered. Once a more complete description of cells is in hand we will be ready to fully investigate the rippling flow- ing dynamics of living systems. Metabolic Proteins the Genetic Code and Organelle Structures Are Nearly Universal Even organisms that look incredibly different share many bio- chemical properties. For instance the enzymes that catalyze degradation of sugars and many other simple chemical reac- tions in cells have similar structures and mechanisms in most living things. The genetic code whereby the nucleotide se- quences of mRNA specifies the amino acid sequences of pro- teins can be read equally well by a bacterial cell and a human cell. Because of the universal nature of the genetic code bac- terial “factories” can be designed to manufacture growth fac- tors insulin clotting factors and other human proteins with therapeutic uses. The biochemical similarities among organ- isms also extend to the organelles found in eukaryotic cells. The basic structures and functions of these subcellular com- ponents are largely conserved in all eukaryotes. Computer analysis of DNA sequence data now available for numerous bacterial species and several eukaryotes can locate protein-coding genes within genomes. With the aid of the genetic code the amino acid sequences of proteins can be deduced from the corresponding gene sequences. Although simple conceptually “finding” genes and deducing the amino acid sequences of their encoded proteins is complicated in practice because of the many noncoding regions in eukary- otic DNA Chapter 9. Despite the difficulties and occasional ambiguities in analyzing DNA sequences comparisons of the genomes from a wide range of organisms provide stunning compelling evidence for the conservation of the molecular mechanisms that build and change organisms and for the common evolutionary history of all species. Many Genes Controlling Development Are Remarkably Similar in Humans and Other Animals As humans we probably have a biased and somewhat exag- gerated view of our status in the animal kingdom. Pride in our swollen forebrain and its associated mental capabilities may blind us to the remarkably sophisticated abilities of other species: navigation by birds the sonar system of bats homing by salmon or the flight of a fly. 26 CHAPTER 1 • Life Begins with Cells

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Despite all the evidence for evolutionary unity at the cel- lular and physiological levels everyone expected that genes regulating animal development would differ greatly from one phylum to the next. After all insects and sea urchins and mammals look so different. We must have many unique proteins to create a brain like ours . . . or must we The fruits of research in developmental genetics during the past two decades reveal that insects and mammals which have a common ancestor about half a billion years ago possess many similar development-regulating genes Figure 1-26. Indeed a large number of these genes appear to be con- served in many and perhaps all animals. Remarkably the developmental functions of the proteins encoded by these genes are also often preserved. For instance certain proteins involved in eye development in insects are related to pro- tein regulators of eye development in mammals. Same for development of the heart gut lungs and capillaries and for placement of body parts along the head-to-tail and back- to-front body axes Chapter 15. This is not to say that all genes or proteins are evolution- arily conserved. Many striking examples exist of proteins that as far as we can tell are utterly absent from certain lin- eages of animals. Plants not surprisingly exhibit many such differences from animals after a billion-year separation in their evolution. Yet certain DNA-binding proteins differ be- tween peas and cows at only two amino acids out of 102 Darwin’s Ideas About the Evolution of Whole Animals Are Relevant to Genes Darwin did not know that genes exist or how they change but we do: the DNA replication machine makes an error or a mutagen causes replacement of one nucleotide with an- other or breakage of a chromosome. Some changes in the genome are innocuous some mildly harmful some deadly a very few are beneficial. Mutations can change the sequence of a gene in a way that modifies the activity of the encoded protein or alters when where and in what amounts the pro- tein is produced in the body. Gene-sequence changes that are harmful will be lost from a population of organisms because the affected individuals cannot survive as well as their relatives. This selection process is exactly what Darwin described without knowing the underlying mechanisms that cause organisms to vary. Thus the selection of whole organisms for survival is really a selection of genes or more accurately sets of genes. A pop- ulation of organisms often contains many variants that are 1.5 • A Genome Perspective on Evolution 27 Mammal Genes Fly a b c FIGURE 1-26 Similar genes conserved during evolution regulate many developmental processes in diverse animals. Insects and mammals are estimated to have had a common ancestor about half a billion years ago. They share genes that control similar processes such as growth of heart and eyes and organization of the body plan indicating conservation of function from ancient times. a Hox genes are found in clusters on the chromosomes of most or all animals. Hox genes encode related proteins that control the activities of other genes. Hox genes direct the development of different segments along the head-to- tail axis of many animals as indicated by corresponding colors. Each gene is activated transcriptually in a specific region along the head-to-toe axis and controls the growth of tissues there. For example in mice the Hox genes are responsible for the distinctive shapes of vertebrae. Mutations affecting Hox genes in flies cause body parts to form in the wrong locations such as legs in lieu of antennae on the head. These genes provide a head-to-tail address and serve to direct formation of the right structures in the right places. b Development of the large compound eyes in fruit flies requires a gene called eyeless named for the mutant phenotype. c Flies with inactivated eyeless genes lack eyes. d Normal human eyes require the human gene called Pax6 that corresponds to eyeless. e People lacking adequate Pax6 function have the genetic disease aniridia a lack of irises in the eyes. Pax6 and eyeless encode highly related proteins that regulate the activities of other genes and are descended from the same ancestral gene. Parts a and b Andreas Hefti Interdepartmental Electron Microscopy IEM Biocenter University of Basel. Part d © Simon Fraser/Photo Researchers Inc. d e

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all roughly equally well-suited to the prevailing conditions. When conditions change—a fire a flood loss of preferred food supply climate shift—variants that are better able to adapt will survive and those less suited to the new condi- tions will begin to die out. In this way the genetic composi- tion of a population of organisms can change over time. Human Medicine Is Informed by Research on Other Organisms Mutations that occur in certain genes during the course of our lives contribute to formation of various human cancers. The normal wild-type forms of such “cancer-causing” genes generally encode proteins that help regulate cell proliferation or death Chapter 23. We also can inherit from our parents mutant copies of genes that cause all manner of genetic dis- eases such as cystic fibrosis muscular dystrophy sickle cell anemia and Huntington’s disease. Happily we can also in- herit genes that make us robustly resist disease. A remarkable number of genes associated with cancer and other human diseases are present in evolutionarily distant animals. For ex- ample a recent study shows that more than three-quarters of the known human disease genes are related to genes found in the fruit fly Drosophila. With the identification of human disease genes in other organisms experimental studies in experimentally tractable organisms should lead to rapid progress in understanding the normal functions of the disease-related genes and what occurs when things go awry. Conversely the disease states themselves constitute a genetic analysis with well-studied phenotypes. All the genes that can be altered to cause a cer- tain disease may encode a group of functionally related proteins. Thus clues about the normal cellular functions of proteins come from human diseases and can be used to guide initial research into mechanism. For instance genes initially identified because of their link to cancer in humans can be studied in the context of normal development in var- ious model organisms providing further insight about the functions of their protein products. 28 CHAPTER 1 • Life Begins with Cells

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T he life of a cell depends on thousands of chemical in- teractions and reactions exquisitely coordinated with one another in time and space and under the influence of the cell’s genetic instructions and its environment. How does a cell extract critical nutrients and information from its environment How does a cell convert the energy stored in nutrients into work movement synthesis of critical com- ponents How does a cell transform nutrients into the fun- damental structures required for its survival cell wall nucleus nucleic acids proteins cytoskeleton How does a cell link itself to other cells to form a tissue How do cells communicate with one another so that the organism as a whole can function One of the goals of molecular cell bi- ology is to answer such questions about the structure and function of cells and organisms in terms of the properties of individual molecules and ions. Life first arose in a watery environment and the proper- ties of this ubiquitous substance have a profound influence on the chemistry of life. Constituting 70–80 percent by weight of most cells water is the most abundant molecule in biological systems. About 7 percent of the weight of liv- ing matter is composed of inorganic ions and small molecules such as amino acids the building blocks of proteins nu- cleotides the building blocks of DNA and RNA lipids the building blocks of biomembranes and sugars the building blocks of starches and cellulose the remainder being the macromolecules and macromolecular aggregates composed of these building blocks. Many biomolecules e.g. sugars readily dissolve in water these water-liking molecules are described as hy- drophilic. Other biomolecules e.g. fats like triacylglycerols shun water these are said to be hydrophobic water-fearing. Still other biomolecules e.g. phospholipids referred to as amphipathic are a bit schizophrenic containing both hy- drophilic and hydrophobic regions. These are used to build the membranes that surround cells and their internal or- ganelles Chapter 5. The smooth functioning of cells tis- sues and organisms depends on all these molecules from the smallest to the largest. Indeed the chemistry of the simple proton H with a mass of 1 dalton Da can be as impor- tant to the survival of a human cell as that of each gigantic DNA molecule with a mass as large as 8.6 10 10 Da sin- gle strand of DNA from human chromosome 1. A relatively small number of principles and facts of chem- istry are essential for understanding cellular processes at the molecular level Figure 2-1. In this chapter we review some of these key principles and facts beginning with the cova- lent bonds that connect atoms into a molecule and the non- covalent forces that stabilize groups of atoms within and between molecules. We then consider the key properties of the basic building blocks of cellular structures. After review- ing those aspects of chemical equilibrium that are most rele- vant to biological systems we end the chapter with basic 29 OUTLINE 2.1 Atomic Bonds and Molecular Interactions 2.2 Chemical Building Blocks of Cells 2.3 Chemical Equilibrium 2.4 Biochemical Energetics 2 CHEMICAL FOUNDATIONS Polysaccharide chains on the surface of cellulose visualized by atomic force microscopy. Courtesy of M. Miles from A. A. Baker et al. 2000 Biophys J. 79:1139–1145.

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principles of biochemical energetics including the central role of ATP adenosine triphosphate in capturing and trans- ferring energy in cellular metabolism. Atomic Bonds and Molecular Interactions Strong and weak attractive forces between atoms are the glue that holds them together in individual molecules and permits interactions between different biological molecules. Strong forces form a covalent bond when two atoms share one pair of electrons “single” bond or multiple pairs of electrons “double” bond “triple” bond etc.. The weak attractive forces of noncovalent interactions are equally important in 2.1 determining the properties and functions of biomolecules such as proteins nucleic acids carbohydrates and lipids. There are four major types of noncovalent interactions: ionic interactions hydrogen bonds van der Waals interactions and the hydrophobic effect. Each Atom Has a Defined Number and Geometry of Covalent Bonds Hydrogen oxygen carbon nitrogen phosphorus and sulfur are the most abundant elements found in biological mole- cules. These atoms which rarely exist as isolated entities readily form covalent bonds with other atoms using elec- trons that reside in the outermost electron orbitals sur- rounding their nuclei. As a rule each type of atom forms a 30 CHAPTER 2 • Chemical Foundations Protein B Protein A CH 3 CH 3 + − O C O C + + _ _ O C H O N H H O a CH 3 CH 3 CH 3 CH 3 b c d Noncovalent interactions γ β α Adenosine triphosphate ATP "High-energy" phosphoanhydride bonds Small molecule subunits k f k r k eq k f k r Macromolecule ▲ FIGURE 2-1 Chemistry of life: key concepts. a Covalent and noncovalent interactions lie at the heart of all biomolecules as when two proteins with complementary shapes and chemical properties come together to form a tightly bound complex. In addition to the covalent bonds that hold the atoms of an amino acid together and link amino acids together noncovlent interactions help define the structure of each individual protein and serve to help hold the complementary structures together. b Small molecules serve as building blocks for larger structures. For example to generate the information-carrying macromolecule DNA the four small nucleotide building blocks deoxyadenylate A deoxythymidylate T deoxyguanylate G and deoxycytidylate C are covalently linked together into long strings polymers which then dimerize into the double helix. c Chemical reactions are reversible and the distribution of the chemicals between starting compounds left and the products of the reactions right depends on the rate constants of the forward k f upper arrow and reverse k r lower arrow reactions. In the reaction shown the forward reaction rate constant is faster than the reverse reaction indicated by the thickness of the arrows. The ratio of these K eq provides an informative measure of the relative amounts of products and reactants that will be present at equilibrium. d In many cases the source of energy for chemical reactions in cells is the hydrolysis of the molecule ATP. This energy is released when a high-energy phosphoanhydride bond linking the α and β or the β and γ phosphates in the ATP molecule yellow is broken by the addition of a water molecule. Proteins can efficiently transfer the energy of ATP hydrolysis to other chemicals thus fueling other chemical reactions or to other biomolecules for physical work.

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characteristic number of covalent bonds with other atoms with a well-defined geometry determined by the atom’s size and by both the distribution of electrons around the nucleus and the number of electrons that it can share. In some cases e.g. carbon the number of stable covalent bonds formed is fixed in other cases e.g. sulfur different numbers of sta- ble covalent bonds are possible. All the biological building blocks are organized around the carbon atom which normally forms four covalent bonds with two to four other atoms. As illustrated by the methane CH 4 molecule when carbon is bonded to four other atoms the angle between any two bonds is 109.5º and the positions of bonded atoms define the four points of a tetrahedron Figure 2-2a. This geometry helps define the structures of many biomolecules. A carbon or any other atom bonded to four dissimilar atoms or groups in a nonplanar configuration is said to be asymmetric. The tetrahedral orientation of bonds formed by an asymmetric carbon atom can be arranged in three-dimensional space in two different ways producing molecules that are mirror images of each other a property called chirality. Such molecules are called optical isomers or stereoisomers. Many molecules in cells contain at least one asymmetric carbon atom often called a chiral carbon atom. The different stereoisomers of a molecule usually have com- pletely different biological activities because the arrangement of atoms within their structures differs yielding their unique abilities to interact and chemically react with other molecules. Carbon can also bond to three other atoms in which all atoms are in a common plane. In this case the carbon atom forms two typical single bonds with two atoms and a dou- ble bond two shared electron pairs with the third atom Figure 2-2b. In the absence of other constraints atoms joined by a single bond generally can rotate freely about the bond axis while those connected by a double bond cannot. The rigid planarity imposed by double bonds has enormous significance for the shapes and flexibility of large biological molecules such as proteins and nucleic acids. The number of covalent bonds formed by other common atoms is shown in Table 2-1. A hydrogen atom forms only one bond. An atom of oxygen usually forms only two cova- lent bonds but has two additional pairs of electrons that can participate in noncovalent interactions. Sulfur forms two co- valent bonds in hydrogen sulfide H 2 S but also can accom- modate six covalent bonds as in sulfuric acid H 2 SO 4 and its sulfate derivatives. Nitrogen and phosphorus each have five electrons to share. In ammonia NH 3 the nitrogen atom forms three covalent bonds the pair of electrons around the atom not involved in a covalent bond can take part in non- covalent interactions. In the ammonium ion NH 4 nitro- gen forms four covalent bonds which have a tetrahedral geometry. Phosphorus commonly forms five covalent bonds as in phosphoric acid H 3 PO 4 and its phosphate derivatives which form the backbone of nucleic acids. Phosphate groups attached to proteins play a key role in regulating the activ- ity of many proteins Chapter 3 and the central molecule in cellular energetics ATP contains three phosphate groups see Section 2.4. 2.1 • Atomic Bonds and Molecular Interactions 31 C H H H H Chemical structure Ball-and-stick model Space-filling model a Methane H H O C b Formaldehyde 109.5° H H H H C O C H H 120° ▲ FIGURE 2-2 Geometry of bonds when carbon is covalently linked to four or three other atoms. a If a carbon atom forms four single bonds as in methane CH 4 the bonded atoms all H in this case are oriented in space in the form of a tetrahedron. The letter representation on the left clearly indicates the atomic composition of the molecule and the bonding pattern. The ball-and- stick model in the center illustrates the geometric arrangement of the atoms and bonds but the diameters of the balls representing the atoms and their nonbonding electrons are unrealistically small compared with the bond lengths. The sizes of the electron clouds in the space-filling model on the right more accurately represent the structure in three dimensions. b A carbon atom also can be bonded to three rather than four other atoms as in formaldehyde CH 2 O. In this case the carbon bonding electrons participate in two single bonds and one double bond which all lie in the same plane. Unlike atoms connected by a single bond which usually can rotate freely about the bond axis those connected by a double bond cannot. TABLE 2-1 Bonding Properties of Atoms Most Abundant in Biomolecules Atom and Outer Usual Number Electrons of Covalent Bonds Bond Geometry 1 2 2 4 or 6 3 or 4 5 4 H O S N P C H O N P C S

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Covalent Bonds Are Much Stronger and More Stable Than Noncovalent Interactions Covalent bonds are very stable because the energies required to break them are much greater than the thermal energy available at room temperature 25 ºC or body temperature 37 ºC. For example the thermal energy at 25 ºC is ap- proximately 0.6 kilocalorie per mole kcal/mol whereas the energy required to break the carbon-carbon single bond COC in ethane is about 140 times larger Figure 2-4. Con- sequently at room temperature 25 ºC fewer than 1 in 10 12 ethane molecules is broken into a pair of ·CH 3 radicals each containing an unpaired nonbonding electron. Covalent single bonds in biological molecules have ener- gies similar to that of the COC bond in ethane. Because more electrons are shared between atoms in double bonds they require more energy to break than single bonds. For in- stance it takes 84 kcal/mol to break a single COO bond but 170 kcal/mol to break a CUO double bond. The most com- mon double bonds in biological molecules are CUO CUN CUC and PUO. The energy required to break noncovalent interactions is only 1–5 kcal/mol much less than the bond energies of covalent bonds see Figure 2-4. Indeed noncovalent inter- actions are weak enough that they are constantly being 32 CHAPTER 2 • Chemical Foundations 104.5° HH δ + δ + δ − δ − − + Dipole moment O ▲ FIGURE 2-3 The dipole nature of a water molecule. The symbol represents a partial charge a weaker charge than the one on an electron or a proton. Because of the difference in the electronegativities of H and O each of the polar HOO bonds in water has a dipole moment. The sizes and directions of the dipole moments of each of the bonds determine the net dipole moment of the molecule. 0.24 × 10 0 0.24 × 10 1 0.24 × 10 2 0.24 × 10 3 Thermal energy van der Waals Electrostatic Hydrogen bonds Hydrolysis of ATP phosphoanhydride bond C−CCC Covalent bonds Noncovalent interactions kcal/mol FIGURE 2-4 Relative energies of covalent bonds and noncovalent interactions. Bond energies are determined as the energy required to break a particular type of linkage. Covalent bonds are one to two powers of 10 stronger than noncovalent interactions. The latter are somewhat greater than the thermal energy of the environment at normal room temperature 25 ˚C. Many biological processes are coupled to the energy released during hydrolysis of a phosphoanhydride bond in ATP . Electrons Are Shared Unequally in Polar Covalent Bonds In many molecules the bonded atoms exert different attrac- tions for the electrons of the covalent bond resulting in un- equal sharing of the electrons. The extent of an atom’s ability to attract an electron is called its electronegativity. A bond between atoms with identical or similar electronegativities is said to be nonpolar. In a nonpolar bond the bonding elec- trons are essentially shared equally between the two atoms as is the case for most COC and COH bonds. However if two atoms differ in their electronegativities the bond be- tween them is said to be polar. One end of a polar bond has a partial negative charge and the other end has a partial positive charge . In an OOH bond for example the greater electronegativity of the oxygen atom relative to hydrogen results in the electrons spending more time around the oxygen atom than the hydro- gen. Thus the OOH bond possesses an electric dipole a pos- itive charge separated from an equal but opposite negative charge. We can think of the oxygen atom of the OOH bond as having on average a charge of 25 percent of an electron with the H atom having an equivalent positive charge. Be- cause of its two OOH bonds water molecules H 2 O are dipoles that form electrostatic noncovalent interactions with one another and with other molecules Figure 2-3. These interactions play a critical role in almost every biochemical interaction and are thus fundamental to cell biology. The polarity of the OUP double bond in H 3 PO 4 results in a “resonance hybrid” a structure between the two forms shown below in which nonbonding electrons are shown as pairs of dots: In the resonance hybrid on the right one of the electrons from the PUO double bond has accumulated around the O atom giving it a negative charge and leaving the P atom with a positive charge. These charges are important in noncova- lent interactions. HH P H HH P H O O O O O O O O ▲

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formed and broken at room temperature. Although these interactions are weak and have a transient existence at physiological temperatures 25–37 ºC multiple noncova- lent interactions can act together to produce highly stable and specific associations between different parts of a large molecule or between different macromolecules. We first re- view the four main types of noncovalent interactions and then consider their role in the binding of biomolecules to one another and to other molecules. Ionic Interactions Are Attractions Between Oppositely Charged Ions Ionic interactions result from the attraction of a positively charged ion—a cation—for a negatively charged ion—an anion. In sodium chloride NaCl for example the bonding electron contributed by the sodium atom is completely trans- ferred to the chlorine atom. Unlike covalent bonds ionic interactions do not have fixed or specific geometric orientations because the electrostatic field around an ion— its attraction for an opposite charge—is uniform in all directions. In aqueous solutions simple ions of biological signifi- cance such as Na K Ca 2 Mg 2 and Cl do not exist as free isolated entities. Instead each is hydrated sur- rounded by a stable shell of water molecules which are held in place by ionic interactions between the central ion and the oppositely charged end of the water dipole Figure 2-5. Most ionic compounds dissolve readily in water because the energy of hydration the energy released when ions tightly bind water molecules is greater than the lattice energy that stabilizes the crystal structure. Parts or all of the aqueous hy- dration shell must be removed from ions when they directly interact with proteins. For example water of hydration is lost when ions pass through protein pores in the cell mem- brane during nerve conduction Chapter 7. The relative strength of the interaction between two ions A and C depends on the concentration of other ions in a solution. The higher the concentration of other ions e.g. Na and Cl the more opportunities A and C have to interact ionically with these other ions and thus the lower the energy required to break the interaction between A and C . As a result increasing the concentrations of salts such as NaCl in a solution of biological molecules can weaken and even dis- rupt the ionic interactions holding the biomolecules together. Hydrogen Bonds Determine Water Solubility of Uncharged Molecules A hydrogen bond is the interaction of a partially positively charged hydrogen atom in a molecular dipole e.g. water with unpaired electrons from another atom either in the same intramolecular or in a different intermolecular mol- ecule. Normally a hydrogen atom forms a covalent bond with only one other atom. However a hydrogen atom cova- lently bonded to an electronegative donor atom D may form an additional weak association the hydrogen bond with an acceptor atom A which must have a nonbonding pair of electrons available for the interaction: The length of the covalent DOH bond is a bit longer than it would be if there were no hydrogen bond because the ac- ceptor “pulls” the hydrogen away from the donor. An im- portant feature of all hydrogen bonds is directionality. In the strongest hydrogen bonds the donor atom the hydrogen atom and the acceptor atom all lie in a straight line. Non- linear hydrogen bonds are weaker than linear ones still mul- tiple nonlinear hydrogen bonds help to stabilize the three-dimensional structures of many proteins. Hydrogen bonds are both longer and weaker than cova- lent bonds between the same atoms. In water for example the distance between the nuclei of the hydrogen and oxygen atoms of adjacent hydrogen-bonded molecules is about 0.27 nm about twice the length of the covalent OOH bonds within a single water molecule Figure 2-6a. The strength of a hydrogen bond between water molecules approxi- mately 5 kcal/mol is much weaker than a covalent OOH bond roughly 110 kcal/mol although it is greater than that for many other hydrogen bonds in biological molecules 1–2 kcal/mol. The extensive hydrogen bonding between water molecules accounts for many of the key properties of this compound including its unusually high melting and boiling points and its ability to interact with many other molecules. The solubility of uncharged substances in an aqueous en- vironment depends largely on their ability to form hydrogen bonds with water. For instance the hydroxyl group OOH in methanol CH 3 OH and the amino group ONH 2 in methylamine CH 3 NH 2 can form several hydrogen bonds with water enabling these molecules to dissolve in water to Hydrogen bond A A H D H D 2.1 • Atomic Bonds and Molecular Interactions 33 Mg 2 O H H ▲ FIGURE 2-5 Electrostatic interaction between water and a magnesium ion Mg 2 . Water molecules are held in place by electrostatic interactions between the two positive charges on the ion and the partial negative charge on the oxygen of each water molecule. In aqueous solutions all ions are surrounded by a similar hydration shell.

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high concentrations Figure 2-6b. In general molecules with polar bonds that easily form hydrogen bonds with water can readily dissolve in water that is they are hydrophilic. Many biological molecules contain in addition to hydroxyl and amino groups peptide and ester groups which form hydro- gen bonds with water Figure 2-6c. X-ray crystallography combined with computational analysis permits an accurate depiction of the distribution of electrons in covalent bonds and the outermost unbonded electrons of atoms as illus- trated in Figure 2-7. These unbonded electrons can form hy- drogen bonds with donor hydrogens. Van der Waals Interactions Are Caused by Transient Dipoles When any two atoms approach each other closely they cre- ate a weak nonspecific attractive force called a van der Waals interaction. These nonspecific interactions result from the momentary random fluctuations in the distribution of the electrons of any atom which give rise to a transient unequal distribution of electrons. If two noncovalently bonded atoms are close enough together electrons of one atom will perturb the electrons of the other. This perturbation generates a tran- sient dipole in the second atom and the two dipoles will at- tract each other weakly Figure 2-8. Similarly a polar covalent bond in one molecule will attract an oppositely ori- ented dipole in another. Van der Waals interactions involving either transiently induced or permanent electric dipoles occur in all types of molecules both polar and nonpolar. In particular van der Waals interactions are responsible for the cohesion between molecules of nonpolar liquids and solids such as heptane CH 3 OCH 2 5 OCH 3 that cannot form hydrogen bonds or ionic interactions with other molecules. The strength of van der Waals interactions decreases rapidly with increasing dis- tance thus these noncovalent bonds can form only when 34 CHAPTER 2 • Chemical Foundations Water-water a OHO H O O H H HH H H H O H b Methanol-water O H O H H H O H CH 3 Methylamine-water N H H O H H H O H CH 3 c Peptide group−water Ester group−water O C N H O H H O H H O C O O H H ▲ FIGURE 2-6 Hydrogen bonding of water with itself and with other compounds. Each pair of nonbonding outer electrons in an oxygen or nitrogen atom can accept a hydrogen atom in a hydrogen bond. The hydroxyl and the amino groups can also form hydrogen bonds with water. a In liquid water each water molecule apparently forms transient hydrogen bonds with several others creating a dynamic network of hydrogen-bonded molecules. b Water also can form hydrogen bonds with methanol and methylamine accounting for the high solubility of these compounds. c The peptide group and ester group which are present in many biomolecules commonly participate in hydrogen bonds with water or polar groups in other molecules. N H C O C α ▲ FIGURE 2-7 Distribution of bonding and outer non- bonding electrons in the peptide group. Shown here is one amino acid within a protein called crambin. The black lines diagrammatically represent the covalent bonds between atoms. The red negative and blue positive lines represent contours of charge. The greater the number of contour lines the higher the charge. The high density of red contour lines between atoms represents the covalent bonds shared electron pairs. The two sets of red contour lines emanating from the oxygen O and not falling on a covalent bond black line represent the two pairs of nonbonded electrons on the oxygen that are available to participate in hydrogen bonding. The high density of blue contour lines near the hydrogen H bonded to nitrogen N represents a partial positive charge indicating that this H can act as a donor in hydrogen bonding. From C. Jelsch et al. 2000 Proc. Nat’l. Acad. Sci. USA 97:3171. Courtesy of M. M. Teeter.

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atoms are quite close to one another. However if atoms get too close together they become repelled by the negative charges of their electrons. When the van der Waals attraction between two atoms exactly balances the repulsion between their two electron clouds the atoms are said to be in van der Waals contact. The strength of the van der Waals interaction is about 1 kcal/mol weaker than typical hydrogen bonds and only slightly higher than the average thermal energy of mol- ecules at 25 ºC. Thus multiple van der Waals interactions a van der Waals interaction in conjunction with other nonco- valent interactions or both are required to significantly in- fluence intermolecular contacts. The Hydrophobic Effect Causes Nonpolar Molecules to Adhere to One Another Because nonpolar molecules do not contain charged groups possess a dipole moment or become hydrated they are in- soluble or almost insoluble in water that is they are hy- drophobic. The covalent bonds between two carbon atoms and between carbon and hydrogen atoms are the most com- mon nonpolar bonds in biological systems. Hydrocarbons— molecules made up only of carbon and hydrogen—are virtually insoluble in water. Large triacylglycerols or triglyc- erides which comprise animal fats and vegetable oils also are insoluble in water. As we see later the major portion of these molecules consists of long hydrocarbon chains. After being shaken in water triacylglycerols form a separate phase. A familiar example is the separation of oil from the water- based vinegar in an oil-and-vinegar salad dressing. Nonpolar molecules or nonpolar portions of molecules tend to aggregate in water owing to a phenomenon called the hydrophobic effect. Because water molecules cannot form hydrogen bonds with nonpolar substances they tend to form “cages” of relatively rigid hydrogen-bonded pentagons and hexagons around nonpolar molecules Figure 2-9 left. This state is energetically unfavorable because it decreases the randomness entropy of the population of water molecules. The role of entropy in chemical systems is discussed in a later section. If nonpolar molecules in an aqueous environ- ment aggregate with their hydrophobic surfaces facing each other there is a reduction in the hydrophobic surface area exposed to water Figure 2-9 right. As a consequence less water is needed to form the cages surrounding the nonpolar molecules and entropy increases an energetically more fa- vorable state relative to the unaggregated state. In a sense then water squeezes the nonpolar molecules into sponta- neously forming aggregates. Rather than constituting an at- tractive force such as in hydrogen bonds the hydrophobic effect results from an avoidance of an unstable state exten- sive water cages around individual nonpolar molecules. Nonpolar molecules can also associate albeit weakly through van der Waals interactions. The net result of the hy- drophobic and van der Waals interactions is a very power- ful tendency for hydrophobic molecules to interact with one another not with water. Simply put like dissolves like. Polar molecules dissolve in polar solvents such as water nonpolar molecules dissolve in nonpolar solvents such as hexane. 2.1 • Atomic Bonds and Molecular Interactions 35 Covalent radius 0.062 nm van der Waals radius 0.14 nm δ δ δ δ ▲ FIGURE 2-8 Two oxygen molecules in van der Waals contact. In this space-filling model red indicates negative charge and blue indicates positive charge. Transient dipoles in the electron clouds of all atoms give rise to weak attractive forces called van der Waals interactions. Each type of atom has a characteristic van der Waals radius at which van der Waals interactions with other atoms are optimal. Because atoms repel one another if they are close enough together for their outer electrons to overlap the van der Waals radius is a measure of the size of the electron cloud surrounding an atom. The covalent radius indicated here is for the double bond of OUO the single- bond covalent radius of oxygen is slightly longer. Highly ordered water molecules Hydrophobic aggregation Nonpolar substance Unaggregated state: Water population highly ordered Lower entropy energetically unfavorable Aggregated state: Water population less ordered Higher entropy energetically more favorable Waters released into bulk solution ▲ FIGURE 2-9 Schematic depiction of the hydrophobic effect. Cages of water molecules that form around nonpolar molecules in solution are more ordered than water molecules in the surrounding bulk liquid. Aggregation of nonpolar molecules reduces the number of water molecules involved in highly ordered cages resulting in a higher-entropy more energetically favorable state right compared with the unaggregated state left.

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■ In an aqueous environment nonpolar molecules or non- polar portions of larger molecules are driven together by the hydrophobic effect thereby reducing the extent of their direct contact with water molecules see Figure 2-9. ■ Molecular complementarity is the lock-and-key fit be- tween molecules whose shapes charges and other physi- cal properties are complementary. Multiple noncovalent in- teractions can form between complementary molecules causing them to bind tightly see Figure 2-10 but not be- tween molecules that are not complementary. ■ The high degree of binding specificity that results from molecular complementarity is one of the features that dis- tinguish biochemistry from typical solution chemistry. Chemical Building Blocks of Cells The three most abundant biological macromolecules— proteins nucleic acids and polysaccharides—are all poly- mers composed of multiple covalently linked identical or nearly identical small molecules or monomers Figure 2-11. The covalent bonds between monomer molecules usually are formed by dehydration reactions in which a water molecule is lost: HOX 1 OOH HOX 2 OOH n HOX 1 OX 2 OOH H 2 O Proteins are linear polymers containing ten to several thousand amino acids linked by peptide bonds. Nucleic acids 2.2 2.2 • Chemical Building Blocks of Cells 37 MONOMERS Amino acid HO H 2 NOH C C R Nucleotide Monosaccharide Polar group Glycerol O C O C Phosphate Hydrophilic head group Hydrophobic fatty acyl tails Glycerophospholipid Phospholipid bilayer O HO 4 1 OH OH HO OH Base O HO P O O 5 3 1 Sugar HO POLYMERS OH H 2 O H H C N R 5 C O C Polypeptide N H H C N H R 1 C OH C H C O N H H O N H H O C C C C COH R 2 R 3 R 4 H 2 O O 4 1 OH OH HO HO Polysaccharide O O 4 4 1 1 OH OH OH OH HO HO O O OH H 2 O 5 3 B 4 OH O P O O HO Nucleic acid 5 3 B 1 3 B 2 3 B 3 5 5 OH O O P O O O O P O O O HO P O O OH ▲ FIGURE 2-11 Covalent and noncovalent linkage of monomers to form biopolymers and membranes. Overview of the cell’s chemical building blocks and the macrostructures formed from them. Top The three major types of biological macromolecules are each assembled by the polymerization of multiple small molecules monomers of a particular type: proteins from amino acids Chapter 3 nucleic acids from nucleotides Chapter 4 and polysaccharides from monosaccharides sugars. The monomers are covalently linked into polymers by coupled reactions whose net result is condensation through the dehydration reaction shown. Bottom In contrast phospholipid monomers noncovalently assemble into bilayer structure which forms the basis of all cellular membranes Chapter 5.

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are linear polymers containing hundreds to millions of nu- cleotides linked by phosphodiester bonds. Polysaccharides are linear or branched polymers of monosaccharides sugars such as glucose linked by glycosidic bonds. A similar approach is used to form various large struc- tures in which the repeating components associate by non- covalent interactions. For instance the fibers of the cytoskeleton are composed of many repeating protein mole- cules. And as we discuss below phospholipids assemble noncovalently to form a two-layered bilayer structure that is the basis of all cellular membranes see Figure 2-11. Thus a repeating theme in biology is the construction of large mol- ecules and structures by the covalent or noncovalent associ- ation of many similar or identical smaller molecules. Amino Acids Differing Only in Their Side Chains Compose Proteins The monomeric building blocks of proteins are 20 amino acids all of which have a characteristic structure consisting of a central carbon atom C bonded to four different chemical groups: an amino NH 2 group a carboxyl COOH group a hydrogen H atom and one variable group called a side chain or R group. Because the carbon in all amino acids except glycine is asymmetric these mole- cules can exist in two mirror-image forms called by conven- tion the D dextro and the L levo isomers Figure 2-12. The two isomers cannot be interconverted one made iden- tical with the other without breaking and then re-forming a chemical bond in one of them. With rare exceptions only the L forms of amino acids are found in proteins. We discuss the properties of the covalent peptide bond that links amino acids into long chains in Chapter 3. To understand the structures and functions of proteins you must be familiar with some of the distinctive properties of the amino acids which are determined by their side chains. The side chains of different amino acids vary in size shape charge hydrophobicity and reactivity. Amino acids can be classified into several broad categories based prima- rily on their solubility in water which is influenced by the polarity of their side chains Figure 2-13. Amino acids with polar side chains are hydrophilic and tend to be on the sur- faces of proteins by interacting with water they make pro- teins soluble in aqueous solutions and can form noncovalent interactions with other water-soluble molecules. In contrast amino acids with nonpolar side chains are hydrophobic they avoid water and often aggregate to help form the water- insoluble cores of many proteins. The polarity of amino acid side chains thus is responsible for shaping the final three- dimensional structure of proteins. A subset of the hydrophilic amino acids are charged ion- ized at the pH ≈7 typical of physiological conditions see Section 2.3. Arginine and lysine are positively charged as- partic acid and glutamic acid are negatively charged their charged forms are called aspartate and glutamate. These four amino acids are the prime contributors to the overall charge of a protein. A fifth amino acid histidine has an im- idazole side chain which can shift from being positively charged to uncharged with small changes in the acidity of its environment: The activities of many proteins are modulated by shifts in environmental acidity through protonation of histidine side chains. Asparagine and glutamine are uncharged but have polar side chains containing amide groups with extensive hydrogen-bonding capacities. Similarly serine and threonine are uncharged but have polar hydroxyl groups which also participate in hydrogen bonds with other polar molecules. The side chains of hydrophobic amino acids are insoluble or only slightly soluble in water. The noncyclic side chains of alanine valine leucine isoleucine and methionine consist entirely of hydrocarbons except for the one sulfur atom in methionine and all are nonpolar. Phenylalanine tyrosine and tryptophan have large bulky aromatic side chains. In later chapters we will see in detail how hydrophobic residues line the surface of proteins that are embedded within biomembranes. Lastly cysteine glycine and proline exhibit special roles in proteins because of the unique properties of their side chains. The side chain of cysteine contains a reactive sulfhydryl group OSH which can oxidize to form a cova- lent disulfide bond OSOSO to a second cysteine: Regions within a protein chain or in separate chains sometimes are cross-linked through disulfide bonds. Disulfide bonds are commonly found in extracellular proteins where they help stabilize the folded structure. The smallest amino acid glycine has a single hydrogen atom as its R group. Its small size allows it to fit into tight spaces. Unlike the other common amino acids the side chain of proline bends around to form a ring by covalently bonding to the nitrogen atom amino group attached to the C . As a result proline is very rigid and creates a fixed kink in a protein chain limiting how a protein can fold in the region of proline residues. H OC N H H S CH 2 C O C C H S CH 2 H N H O C N H H C O C H SH CH 2 C CH 2 H HS N C C C N N H H H H CH 2 C C C N N H H H CH 2 pH 7.8 pH 5.8 38 CHAPTER 2 • Chemical Foundations

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2.2 • Chemical Building Blocks of Cells 39 D isomer L isomer COO − C α NH 3 + H C α H NH 3 + COO − R R ▲ FIGURE 2-12 Common structure of amino acids. The carbon atom C of each amino acid is bonded to four chemical groups. The side chain or R group is unique to each type of amino acid see Figure 2-13. Because the C in all amino acids except glycine is asymmetric these molecules have two mirror- image forms designated L and D. Although the chemical properties of such optical isomers are identical their biological activities are distinct. Only L amino acids are found in proteins. CH Lysine Lys or K Arginine Arg or R Histidine His or H H C CH 2 COO H CH 2 CH 2 CH 2 NH 3 H C CH 2 COO CNH N C CH HH C CH 2 COO CH 2 CH 2 NH C NH 2 NH 2 Basic amino acids H 3 C Serine Ser or S H C CH 2 COO OH Threonine Thr or T H C COO OH HC CH 3 Asparagine Asn or N H C CH 2 COO C O H 2 N CH 2 Glutamine Gln or Q H C COO Polar amino acids with uncharged R groups CH 2 C O H 2 N H C COO CH 2 CH 2 HC Alanine Ala or A Tryptophan Trp or W Tyrosine Tyr or Y Phenylalanine Phe or F Methionine Met or M Leucine Leu or L Isoleucine Ile or I Valine Val or V H H 3 NC CH 3 CH 3 H 3 N H 3 N H 3 N H 2 N H 3 N H 3 N H 3 N H 3 N H 3 N H 3 N H 3 N H 3 N H 3 N H 3 N H 3 N H 3 N H 3 N H 3 N H 3 N COO H C CH CH COO HYDROPHILIC AMINO ACIDS HYDROPHOBIC AMINO ACIDS H 3 C CH 3 H C COO CH 3 CH 3 CH 2 H C COO CH 2 S CH 3 CH 2 H C COO CH 2 H C OH COO CH 2 H C C NH COO SPECIAL AMINO ACIDS Cysteine Cys or C Glycine Gly or G Proline Pro or P H 2 C H C CH 2 CH 2 CH 2 SH COO H C COO H C H COO Acidic amino acids Aspartate Asp or D H C CH 2 COO COO Glutamate Glu or E H C CH 2 COO CH 2 COO ▲ FIGURE 2-13 The 20 common amino acids used to build proteins. The side chain R group red determines the characteristic properties of each amino acid and is the basis for grouping amino acids into three main categories: hydrophobic hydrophilic and special. Shown are the ionized forms that exist at the pH ≈7 of the cytosol. In parentheses are the three-letter and one-letter abbreviations for each amino acid. ▲

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Some amino acids are more abundant in proteins than other amino acids. Cysteine tryptophan and methionine are rare amino acids together they constitute approximately 5 percent of the amino acids in a protein. Four amino acids— leucine serine lysine and glutamic acid—are the most abun- dant amino acids totaling 32 percent of all the amino acid residues in a typical protein. However the amino acid com- position of proteins can vary widely from these values. Five Different Nucleotides Are Used to Build Nucleic Acids Two types of chemically similar nucleic acids DNA deoxyri- bonucleic acid and RNA ribonucleic acid are the principal information-carrying molecules of the cell. The monomers from which DNA and RNA are built called nucleotides all have a common structure: a phosphate group linked by a phosphoester bond to a pentose a five-carbon sugar molecule that in turn is linked to a nitrogen- and carbon-containing ring structure commonly referred to as a “base” Figure 2-14a. In RNA the pentose is ribose in DNA it is deoxyribose Figure 2-14b. The bases adenine guanine and cytosine are found in both DNA and RNA thymine is found only in DNA and uracil is found only in RNA. Adenine and guanine are purines which contain a pair of fused rings cytosine thymine and uracil are pyrimidines which contain a single ring Figure 2-15. The bases are often abbreviated A G C T and U respectively these same single- letter abbreviations are also commonly used to denote the entire nucleotides in nucleic acid polymers. In nucleotides the 1 carbon atom of the sugar ribose or deoxyribose is attached to the nitrogen at position 9 of a purine N 9 or at position 1 of a pyrimidine N 1 . The acidic character of nu- cleotides is due to the phosphate group which under normal intracellular conditions releases a hydrogen ion H leav- ing the phosphate negatively charged see Figure 2-14a. Most nucleic acids in cells are associated with proteins which form ionic interactions with the negatively charged phosphates. Cells and extracellular fluids in organisms contain small concentrations of nucleosides combinations of a base and a sugar without a phosphate. Nucleotides are nucleosides that have one two or three phosphate groups esterified at the 5 hydroxyl. Nucleoside monophosphates have a single esteri- fied phosphate see Figure 2-14a diphosphates contain a pyrophosphate group: and triphosphates have a third phosphate. Table 2-2 lists the names of the nucleosides and nucleotides in nucleic acids and the various forms of nucleoside phosphates. The nucleoside triphosphates are used in the synthesis of nucleic acids which we cover in Chapter 4. Among their other functions in the cell GTP participates in intracellular signaling and acts as an energy reservoir particularly in protein synthesis and ATP discussed later in this chapter is the most widely used biological energy carrier. O OO O O OP P O Pyrophosphate 40 CHAPTER 2 • Chemical Foundations H H H H O N N N N NH 2 O OH O CH 2 CH HC C C C OH P O O Adenosine 5 -monophosphate AMP a 1 2 2 1 4 5 3 4 5 6 7 8 9 Adenine Phosphate Ribose 3 b H H H H O HOCH 2 H OH OH 2 1 4 5 3 2-Deoxyribose H H H H O HOCH 2 OH OH OH 2 1 4 5 3 Ribose ▲ FIGURE 2-14 Common structure of nucleotides. a Adenosine 5-monophosphate AMP a nucleotide present in RNA. By convention the carbon atoms of the pentose sugar in nucleotides are numbered with primes. In natural nucleotides the 1 carbon is joined by a linkage to the base in this case adenine both the base blue and the phosphate on the 5 hydroxyl red extend above the plane of the furanose ring. b Ribose and deoxyribose the pentoses in RNA and DNA respectively. N N N N NH 2 CH HC C C C H Adenine A 1 2 4 5 6 3 7 8 9 HN N C C CH CH H O Uracil U 3 2 6 5 4 1 O HN N C C CCH 3 CH H O Thymine T 3 2 6 5 4 1 O N HN N N O CH C C C C H H 2 N Guanine G 1 2 4 5 6 3 7 8 9 N NH 2 C 3 2 6 5 4 1 CH CH N H C O Cytosine C PURINES PYRIMIDINES ▲ FIGURE 2-15 Chemical structures of the principal bases in nucleic acids. In nucleic acids and nucleotides nitrogen 9 of purines and nitrogen 1 of pyrimidines red are bonded to the 1 carbon of ribose or deoxyribose. U is only in RNA and T is only in DNA. Both RNA and DNA contain A G and C.

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Monosaccharides Joined by Glycosidic Bonds Form Linear and Branched Polysaccharides The building blocks of the polysaccharides are the simple sugars or monosaccharides. Monosaccharides are carbohy- drates which are literally covalently bonded combinations of carbon and water in a one-to-one ratio CH 2 O n where n equals 3 4 5 6 or 7. Hexoses n 6 and pentoses n 5 are the most common monosaccharides. All monosaccha- rides contain hydroxyl OOH groups and either an alde- hyde or a keto group: D-Glucose C 6 H 12 O 6 is the principal external source of energy for most cells in higher organisms and can exist in three different forms: a linear structure and two different hemiacetal ring structures Figure 2-16a. If the aldehyde group on carbon 1 reacts with the hydroxyl group on carbon 5 the resulting hemiacetal D-glucopyranose contains a six- member ring. In the anomer of D-glucopyranose the hy- droxyl group attached to carbon 1 points “downward” from the ring as shown in Figure 2-16a in the anomer this hy- droxyl points “upward.” In aqueous solution the and anomers readily interconvert spontaneously at equilibrium there is about one-third anomer and two-thirds with very little of the open-chain form. Because enzymes can dis- tinguish between the and anomers of D-glucose these forms have distinct biological roles. Condensation of the hy- droxyl group on carbon 4 of the linear glucose with its alde- H CC O Aldehyde CC C O Keto 2.2 • Chemical Building Blocks of Cells 41 TABLE 2-2 Terminology of Nucleosides and Nucleotides Bases Purines Pyrimidines Uracil U Adenine A Guanine G Cytosine C Thymine T Nucleosides in RNA Adenosine Guanosine Cytidine Uridine in DNA Deoxyadenosine Deoxyguanosine Deoxycytidine Deoxythymidine Nucleotides in RNA Adenylate Guanylate Cytidylate Uridylate in DNA Deoxyadenylate Deoxyguanylate Deoxycytidylate Deoxythymidylate Nucleoside monophosphates AMP GMP CMP UMP Nucleoside diphosphates ADP GDP CDP UDP Nucleoside triphosphates ATP GTP CTP UTP Deoxynucleoside mono- di- and triphosphates dAMP etc. H O C 2 3 1 COH H 4 COH H 5 6 C CH 2 OH OH H CH HO D-Glucose 6 2 1 4 3 5 CH 2 OH HCOH O H H H H OH OH OH D-Glucofuranose rare 6 1 4 32 CH 2 OH O H H H H H OH OH HO OH D-Glucopyranose common 5 a b H O C 2 3 1 CH HO 4 COH H 5 6 C CH 2 OH OH H CH HO D-Mannose H O C 2 3 1 COH H 4 CH HO 5 6 C CH 2 OH OH H CH HO D-Galactose ▲ FIGURE 2-16 Chemical structures of hexoses. All hexoses have the same chemical formula C 6 H 12 O 6 and contain an aldehyde or keto group. a The ring forms of D-glucose are generated from the linear molecule by reaction of the aldehyde at carbon 1 with the hydroxyl on carbon 5 or carbon 4. The three forms are readily interconvertible although the pyranose form right predominates in biological systems. b In D-mannose and D-galactose the configuration of the H green and OH blue bound to one carbon atom differs from that in glucose. These sugars like glucose exist primarily as pyranoses.

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hyde group results in the formation of D-glucofuranose a hemiacetal containing a five-member ring. Although all three forms of D-glucose exist in biological systems the pyranose form is by far the most abundant. Many biologically important sugars are six-carbon sug- ars that are structurally related to D-glucose Figure 2-16b. Mannose is identical with glucose except that the orientation of the groups bonded to carbon 2 is reversed. Similarly galactose another hexose differs from glucose only in the orientation of the groups attached to carbon 4. Interconver- sion of glucose and mannose or galactose requires the break- ing and making of covalent bonds such reactions are carried out by enzymes called epimerases. The pyranose ring in Figure 2-16a is depicted as planar . In fact because of the tetrahedral geometry around carbon atoms the most stable conformation of a pyranose ring has a nonplanar chairlike shape. In this conformation each bond from a ring carbon to a nonring atom e.g. H or O is either nearly perpendicular to the ring referred to as axial a or nearly in the plane of the ring referred to as equatorial e: The enzymes that make the glycosidic bonds linking monosaccharides into polysaccharides are specific for the or anomer of one sugar and a particular hydroxyl group on the other. In principle any two sugar molecules can be linked in a variety of ways because each monosaccharide has mul- tiple hydroxyl groups that can participate in the formation of glycosidic bonds. Furthermore any one monosaccharide has the potential of being linked to more than two other mono- saccharides thus generating a branch point and nonlinear e e e e e a a a a a 5 4 6 3 1 2 CH 2 OH O Pyranoses HO HO HO O H H H OH H H -D-Glucopyranose polymers. Glycosidic bonds are usually formed between a co- valently modified sugar and the growing polymer chain. Such modifications include a phosphate e.g. glucose 6- phosphate or a nucleotide e.g. UDP-galactose: The epimerase enzymes that interconvert different monosac- charides often do so using the nucleotide sugars rather than the unsubstituted sugars. Disaccharides formed from two monosaccharides are the simplest polysaccharides. The disaccharide lactose com- posed of galactose and glucose is the major sugar in milk the disaccharide sucrose composed of glucose and fructose is a principal product of plant photosynthesis and is refined into common table sugar Figure 2-17. Larger polysaccharides containing dozens to hundreds of monosaccharide units can function as reservoirs for glu- cose as structural components or as adhesives that help hold cells together in tissues. The most common storage car- bohydrate in animal cells is glycogen a very long highly branched polymer of glucose. As much as 10 percent by weight of the liver can be glycogen. The primary storage car- bohydrate in plant cells starch also is a glucose polymer. It occurs in an unbranched form amylose and lightly branched form amylopectin. Both glycogen and starch are composed of the anomer of glucose. In contrast cellulose the major constituent of plant cell walls is an unbranched polymer of the anomer of glucose. Human digestive en- zymes can hydrolyze the glycosidic bonds in starch but not the glycosidic bonds in cellulose. Many species of plants bacteria and molds produce cellulose-degrading enzymes. 42 CHAPTER 2 • Chemical Foundations 6 1 CH 2 OPO 3 2 O H H H OH H OH OH HO Glucose 6-phosphate CH 2 OH H 6 1 O H H H OH H O OH OH UDP-galactose H O OO O O P P O Uridine 1 CH 2 OH O H H H H H OH HO OH HO OH Galactose 12 CH 2 OH CH 2 OH CH 2 OH O O H H H H H H H OH H OH OH HO HO OH HO Glucose Fructose 4 CH 2 OH H 2 O O H H H H H OH OH OH Glucose HO 1 CH 2 OH O O H H H H H OH OH Lactose H 4 CH 2 OH O H H H H OH OH OH H 2 O HO 1 CH 2 OH O O H H H H H OH OH Sucrose 2 CH 2 OH CH 2 OH O H H H OH HO ▲ FIGURE 2-17 Formation of the disaccharides lactose and sucrose. In any glycosidic linkage the anomeric carbon of one sugar molecule in either the or conformation is linked to a hydroxyl oxygen on another sugar molecule. The linkages are named accordingly: thus lactose contains a 1n 4 bond and sucrose contains an 1n 2 bond. ▲

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Cows and termites can break down cellulose because they harbor cellulose-degrading bacteria in their gut. Many complex polysaccharides contain modified sugars that are covalently linked to various small groups particu- larly amino sulfate and acetyl groups. Such modifications are abundant in glycosaminoglycans major polysaccharide components of the extracellular matrix that we describe in Chapter 6. Fatty Acids Are Precursors for Many Cellular Lipids Before considering phospholipids and their role in the struc- ture of biomembranes we briefly review the properties of fatty acids. Like glucose fatty acids are an important energy source for many cells and are stored in the form of triacyl- glycerols within adipose tissue Chapter 8. Fatty acids also are precursors for phospholipids and many other lipids with a variety of functions Chapter 18. Fatty acids consist of a hydrocarbon chain attached to a carboxyl group OCOOH. They differ in length although the predominant fatty acids in cells have an even number of carbon atoms usually 14 16 18 or 20. The major fatty acids in phospholipids are listed in Table 2-3. Fatty acids often are designated by the abbreviation Cx:y where x is the number of carbons in the chain and y is the number of dou- ble bonds. Fatty acids containing 12 or more carbon atoms are nearly insoluble in aqueous solutions because of their long hydrophobic hydrocarbon chains. Fatty acids with no carbon-carbon double bonds are said to be saturated those with at least one double bond are un- saturated. Unsaturated fatty acids with more than one carbon- carbon double bond are referred to as polyunsaturated. Two “essential” polyunsaturated fatty acids linoleic acid C18:2 and linolenic acid C18:3 cannot be synthesized by mammals and must be supplied in their diet. Mammals can synthesize other common fatty acids. Two stereoisomeric configurations cis and trans are possible around each carbon-carbon double bond: A cis double bond introduces a rigid kink in the otherwise flexible straight chain of a fatty acid Figure 2-18. In gen- eral the fatty acids in biological systems contain only cis double bonds. Fatty acids can be covalently attached to another molecule by a type of dehydration reaction called esterification in which the OH from the carboxyl group of the fatty acid and a H from a hydroxyl group on the other molecule are lost. In the com- bined molecule formed by this reaction the portion derived from the fatty acid is called an acyl group or fatty acyl group. This is illustrated by triacylglycerols which contain three acyl groups esterfied to glycerol: C O O CH 2 n H 3 C CH 2 CH 2 C O O CH 2 n H 3 C CH C O O CH 2 n H 3 C Triacylglycerol C C H H CH 2 Trans C C H H CH 2 H 2 C H 2 C Cis 2.2 • Chemical Building Blocks of Cells 43 TABLE 2-3 Fatty Acids That Predominate in Phospholipids Common Name of Acid Ionized Form in Parentheses Abbreviation Chemical Formula SATURATED FATTY ACIDS Myristic myristate C14:0 CH 3 CH 2 12 COOH Palmitic palmitate C16:0 CH 3 CH 2 14 COOH Stearic stearate C18:0 CH 3 CH 2 16 COOH UNSATURATED FATTY ACIDS Oleic oleate C18:1 CH 3 CH 2 7 CHUCHCH 2 7 COOH Linoleic linoleate C18:2 CH 3 CH 2 4 CHUCHCH 2 CHUCHCH 2 7 COOH Arachidonic arachidonate C20:4 CH 3 CH 2 4 CHUCHCH 2 3 CHUCHCH 2 3 COOH

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If the acyl groups are long enough these molecules are insoluble in water even though they contain three polar ester bonds. Fatty acyl groups also form the hydrophobic portion of phospholipids which we discuss next. Phospholipids Associate Noncovalently to Form the Basic Bilayer Structure of Biomembranes Biomembranes are large flexible sheets that serve as the boundaries of cells and their intracellular organelles and form the outer surfaces of some viruses. Membranes liter- ally define what is a cell the outer membrane and the con- tents within the membrane and what is not the extracellular space outside the membrane. Unlike the proteins nucleic acids and polysaccharides membranes are assembled by the noncovalent association of their component building blocks. The primary building blocks of all biomembranes are phos- pholipids whose physical properties are responsible for the formation of the sheetlike structure of membranes. Phospholipids consist of two long-chain nonpolar fatty acyl groups linked usually by an ester bond to small highly polar groups including a phosphate. In phosphoglycerides the major class of phospholipids fatty acyl side chains are es- terified to two of the three hydroxyl groups in glycerol. The third hydroxyl group is esterified to phosphate. The simplest phospholipid phosphatidic acid contains only these compo- nents. In most phospholipids found in membranes the phos- phate group is esterified to a hydroxyl group on another hydrophilic compound. In phosphatidylcholine for example choline is attached to the phosphate Figure 2-19. The neg- ative charge on the phosphate as well as the charged or polar groups esterified to it can interact strongly with water. The 44 CHAPTER 2 • Chemical Foundations H 3 C Palmitate ionized form of palmitic acid H H C H H C H H C H H C H H C H H C H H C H H C H H C H H C H H C H H C H H C H H C C O O H 3 C Oleate ionized form of oleic acid H H C H H C H H C H H C H H C H H C H H C H C H C H H C H H C H H C H H C H H C H H C H H C C O O ▲ FIGURE 2-18 The effect of a double bond on the shape of fatty acids. Shown are space-filling models and chemical structures of the ionized form of palmitic acid a saturated fatty acid with 16 C atoms and oleic acid an unsaturated one with 18 C atoms. In saturated fatty acids the hydrocarbon chain is often linear the cis double bond in oleate creates a rigid kink in the hydrocarbon chain. After L. Stryer 1994 Biochemistry 4th ed. W. H. Freeman and Company p. 265. Choline PHOSPHATIDYLCHOLINE Hydrophobic tail Hydrophilic head Phosphate Glycerol Fatty acid chains CH 3 C H 2 H 2 C N + CH 3 CH 3 O C O O C O O P O O O − H 2 C CH CH 2 ▲ FIGURE 2-19 Phosphatidylcholine a typical phospho- glyceride. All phosphoglycerides are amphipathic having a hydrophobic tail yellow and a hydrophilic head blue in which glycerol is linked via a phosphate group to an alcohol. Either of or both the fatty acyl side chains in a phosphoglyceride may be saturated or unsaturated. In phosphatidic acid red the simplest phospholipid the phosphate is not linked to an alcohol.

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phosphate and its associated esterified group the “head” group of a phospholipid is hydrophilic whereas the fatty acyl chains the “tails” are hydrophobic. The amphipathic nature of phospholipids which governs their interactions is critical to the structure of biomem- branes. When a suspension of phospholipids is mechanically dispersed in aqueous solution the phospholipids aggregate into one of three forms: spherical micelles and liposomes and sheetlike two-molecule-thick phospholipid bilayers Figure 2-20. The type of structure formed by a pure phospholipid or a mixture of phospholipids depends on several factors in- cluding the length of the fatty acyl chains their degree of saturation and temperature. In all three structures the hy- drophobic effect causes the fatty acyl chains to aggregate and exclude water molecules from the “core.” Micelles are rarely formed from natural phosphoglycerides whose fatty acyl chains generally are too bulky to fit into the interior of a micelle. If one of the two fatty acyl chains is removed by hydrolysis forming a lysophospholipid the predominant type of aggregate that forms is the micelle. Common deter- gents and soaps form micelles in aqueous solution that be- have as tiny ball bearings thus giving soap solutions their slippery feel and lubricating properties. Under suitable conditions phospholipids of the compo- sition present in cells spontaneously form symmetric phos- pholipid bilayers. Each phospholipid layer in this lamellar structure is called a leaflet. The fatty acyl chains in each leaflet minimize contact with water by aligning themselves tightly together in the center of the bilayer forming a hydrophobic core that is about 3 nm thick see Figure 2-20. The close packing of these nonpolar tails is stabilized by the hydrophobic effect and van der Waals interactions between them. Ionic and hydrogen bonds stabilize the interaction of the phospholipid polar head groups with one another and with water. A phospholipid bilayer can be of almost unlimited size— from micrometers m to millimeters mm in length or width—and can contain tens of millions of phospholipid molecules. Because of their hydrophobic core bilayers are virtually impermeable to salts sugars and most other small hydrophilic molecules. The phospholipid bilayer is the basic structural unit of nearly all biological membranes thus al- though they contain other molecules e.g. cholesterol gly- colipids proteins biomembranes have a hydrophobic core that separates two aqueous solutions and acts as a perme- ability barrier. The structural organization of biomembranes and the general properties of membrane proteins are de- scribed in Chapter 5. KEY CONCEPTS OF SECTION 2.2 Chemical Building Blocks of Cells ■ Three major biopolymers are present in cells: proteins composed of amino acids linked by peptide bonds nucleic acids composed of nucleotides linked by phosphodiester bonds and polysaccharides composed of monosaccharides sugars linked by glycosidic bonds see Figure 2-11. ■ Many molecules in cells contain at least one asymmet- ric carbon atom which is bonded to four dissimilar atoms. Such molecules can exist as optical isomers mirror im- ages designated D and L which have different biological activities. In biological systems nearly all sugars are D iso- mers while nearly all amino acids are L isomers. ■ Differences in the size shape charge hydrophobicity and reactivity of the side chains of amino acids determine the chemical and structural properties of proteins see Fig- ure 2-13. ■ Amino acids with hydrophobic side chains tend to clus- ter in the interior of proteins away from the surrounding aqueous environment those with hydrophilic side chains usually are toward the surface. ■ The bases in the nucleotides composing DNA and RNA are heterocyclic rings attached to a pentose sugar. They form two groups: the purines—adenine A and guanine G—and the pyrimidines—cytosine C thymine T and uracil U see Figure 2-15. A G T and C are in DNA and A G U and C are in RNA. ■ Glucose and other hexoses can exist in three forms: an open-chain linear structure a six-member pyranose ring and 2.2 • Chemical Building Blocks of Cells 45 Phospholipid bilayer Liposome Micelle ▲ FIGURE 2-20 Cross-sectional views of the three struc- tures formed by phospholipids in aqueous solutions. The white spheres depict the hydrophilic heads of the phospholipids and the squiggly black lines in the yellow regions represent the hydrophobic tails. Shown are a spherical micelle with a hydrophobic interior composed entirely of fatty acyl chains a spherical liposome which has two phospholipid layers and an aqueous center and a two-molecule-thick sheet of phospholipids or bilayer the basic structural unit of biomembranes.

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a five-member furanose ring see Figure 2-16. In biological systems the pyranose form of D-glucose predominates. ■ Glycosidic bonds are formed between either the or anomer of one sugar and a hydroxyl group on another sugar leading to formation of disaccharides and other poly- saccharides see Figure 2-17. ■ The long hydrocarbon chain of a fatty acid may contain no carbon-carbon double bond saturated or one or more double bonds unsaturated which bends the chain. ■ Phospholipids are amphipathic molecules with a hydro- phobic tail often two fatty acyl chains and a hydrophilic head see Figure 2-19. ■ In aqueous solution the hydrophobic effect and van der Waals interactions organize and stabilize phospholipids into one of three structures: a micelle liposome or sheet- like bilayer see Figure 2-20. ■ In a phospholipid bilayer which constitutes the basic structure of all biomembranes fatty acyl chains in each leaflet are oriented toward one another forming a hy- drophobic core and the polar head groups line both sur- faces and directly interact with the aqueous solution. Chemical Equilibrium We now shift our discussion to chemical reactions in which bonds primarily covalent bonds in reactant chemicals are broken and new bonds are formed to generate reaction prod- ucts. At any one time several hundred different kinds of chemical reactions are occurring simultaneously in every cell and many chemicals can in principle undergo multiple chemical reactions. Both the extent to which reactions can proceed and the rate at which they take place determine the chemical composition of cells. When reactants first mix together—before any products have been formed—their rate of reaction is determined in part by their initial concentrations. As the reaction products accumulate the concentration of each reactant decreases and so does the reaction rate. Meanwhile some of the product molecules begin to participate in the reverse reaction which re-forms the reactants microscopic reversibility. This re- verse reaction is slow at first but speeds up as the concentra- tion of product increases. Eventually the rates of the forward and reverse reactions become equal so that the concentra- tions of reactants and products stop changing. The system is then said to be in chemical equilibrium. At equilibrium the ratio of products to reactants called the equilibrium constant is a fixed value that is independ- ent of the rate at which the reaction occurs. The rate of a chemical reaction can be increased by a catalyst which brings reactants together and accelerates their interactions but is not permanently changed during a reaction. In this sec- tion we discuss several aspects of chemical equilibria in the 2.3 next section we examine energy changes during reactions and their relationship to equilibria. Equilibrium Constants Reflect the Extent of a Chemical Reaction The equilibrium constant K eq depends on the nature of the reactants and products the temperature and the pressure particularly in reactions involving gases. Under standard physical conditions 25 ºC and 1 atm pressure for biologi- cal systems the K eq is always the same for a given reaction whether or not a catalyst is present. For the general reaction aA bB cC zZ yY xX 2-1 where capital letters represent particular molecules or atoms and lowercase letters represent the number of each in the re- action formula the equilibrium constant is given by 2-2 where brackets denote the concentrations of the molecules. The rate of the forward reaction left to right in Equation 2-1 is Rate forward k f A a B b C c where k f is the rate constant for the forward reaction. Simi- larly the rate of the reverse reaction right to left in Equation 2-1 is Rate reverse k r X x Y y Z z where k r is the rate constant for the reverse reaction. At equilibrium the forward and reverse rates are equal so Rate forward /Rate reverse 1. By rearranging these equations we can express the equilibrium constant as the ratio of the rate constants 2-3 Chemical Reactions in Cells Are at Steady State Under appropriate conditions and given sufficient time indi- vidual biochemical reactions carried out in a test tube even- tually will reach equilibrium. Within cells however many reactions are linked in pathways in which a product of one reaction serves as a reactant in another or is pumped out of the cell. In this more complex situation when the rate of for- mation of a substance is equal to the rate of its consumption the concentration of the substance remains constant and the system of linked reactions for producing and consuming that substance is said to be in a steady state Figure 2-21. One consequence of such linked reactions is that they prevent the accumulation of excess intermediates protecting cells from the harmful effects of intermediates that have the potential of being toxic at high concentrations. K eq k f k r K eq X x Y y Z z A a B b C c 46 CHAPTER 2 • Chemical Foundations

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Dissociation Constants for Binding Reactions Reflect the Affinity of Interacting Molecules The concept of chemical equilibrium also applies to the bind- ing of one molecule to another. Many important cellular processes depend on such binding “reactions” which involve the making and breaking of various noncovalent interactions rather than covalent bonds as discussed above. A common example is the binding of a ligand e.g. the hormone insulin or adrenaline to its receptor on the surface of a cell trigger- ing a biological response. Another example is the binding of a protein to a specific sequence of base pairs in a molecule of DNA which frequently causes the expression of a nearby gene to increase or decrease Chapter 11. If the equilibrium constant for a binding reaction is known the intracellular stability of the resulting complex can be predicted. To illus- trate the general approach for determining the concentration of noncovalently associated complexes we will calculate the extent to which a protein is bound to DNA in a cell. Most commonly binding reactions are described in terms of the dissociation constant K d which is the reciprocal of the equilibrium constant. For the binding reaction P D PD where PD is the specific complex of a protein P and DNA D the dissociation constant is given by 2-4 Typical reactions in which a protein binds to a specific DNA sequence have a K d of 10 10 M where M symbolizes molar- ity or moles per liter mol/L. To relate the magnitude of this dissociation constant to the intracellular ratio of bound to unbound DNA let’s consider the simple example of a bac- terial cell having a volume of 1.5 10 15 L and containing K d PD PD 1 molecule of DNA and 10 molecules of the DNA-binding protein P. In this case given a K d of 10 10 M 99 percent of the time this specific DNA sequence will have a molecule of protein bound to it and 1 percent of the time it will not even though the cell contains only 10 molecules of the protein Clearly P and D bind very tightly have a high affinity as re- flected by the low value of the dissociation constant for their binding reaction. The large size of biological macromolecules such as pro- teins can result in the availability of multiple surfaces for complementary intermolecular interactions. As a conse- quence many macromolecules have the capacity to bind multiple other molecules simultaneously. In some cases these binding reactions are independent with their own dis- tinct K d values that are constant. In other cases binding of a molecule at one site on a macromolecule can change the three-dimensional shape of a distant site thus altering the binding interactions at that distant site. This is an important mechanism by which one molecule can alter regulate the activity of a second molecule e.g. a protein by changing its capacity to interact with a third molecule. We examine this regulatory mechanism in more detail in Chapter 3. Biological Fluids Have Characteristic pH Values The solvent inside cells and in all extracellular fluids is water. An important characteristic of any aqueous solution is the concentration of positively charged hydrogen ions H and negatively charged hydroxyl ions OH . Because these ions are the dissociation products of H 2 O they are constituents of all living systems and they are liberated by many reactions that take place between organic molecules within cells. When a water molecule dissociates one of its polar HOO bonds breaks. The resulting hydrogen ion often re- ferred to as a proton has a short lifetime as a free particle and quickly combines with a water molecule to form a hy- dronium ion H 3 O . For convenience however we refer to the concentration of hydrogen ions in a solution H even though this really represents the concentration of hydronium ions H 3 O . Dissociation of H 2 O generates one OH ion along with each H . The dissociation of water is a reversible reaction H 2 O H OH At 25 ºC H OH 10 14 M 2 so that in pure water H OH 10 7 M. The concentration of hydrogen ions in a solution is ex- pressed conventionally as its pH defined as the negative log of the hydrogen ion concentration. The pH of pure water at 25 ºC is 7: It is important to keep in mind that a 1 unit difference in pH represents a tenfold difference in the concentration of pH logH log 1 H log 1 10 7 7 2.3 • Chemical Equilibrium 47 a Test tube equilibrium concentrations b Intracellular steady-state concentrations A A A B B B B B B B B B A A B B B B B B C C C C ▲ FIGURE 2-21 Comparison of reactions at equilibrium and steady state. a In the test tube a biochemical reaction A n B eventually will reach equilibrium in which the rates of the forward and reverse reactions are equal as indicated by the reaction arrows of equal length. b In metabolic pathways within cells the product B commonly would be consumed in this example by conversion to C. A pathway of linked reactions is at steady state when the rate of formation of the intermediates e.g. B equals their rate of consumption. As indicated by the unequal length of the arrows the individual reversible reactions constituting a metabolic pathway do not reach equilibrium. Moreover the concentrations of the intermediates at steady state differ from what they would be at equilibrium.

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protons. On the pH scale 7.0 is considered neutral: pH val- ues below 7.0 indicate acidic solutions higher H and values above 7.0 indicate basic alkaline solutions. For in- stance gastric juice which is rich in hydrochloric acid HCl has a pH of about 1. Its H is roughly a millionfold greater than that of cytoplasm with a pH of about 7. Although the cytosol of cells normally has a pH of about 7.2 the pH is much lower about 4.5 in the interior of lyso- somes one type of organelle in eukaryotic cells. The many degradative enzymes within lysosomes function optimally in an acidic environment whereas their action is inhibited in the near neutral environment of the cytoplasm. This illus- trates that maintenance of a specific pH is imperative for proper functioning of some cellular structures. On the other hand dramatic shifts in cellular pH may play an important role in controlling cellular activity. For example the pH of the cytoplasm of an unfertilized sea urchin egg is 6.6. Within 1 minute of fertilization however the pH rises to 7.2 that is the H concentration decreases to about one-fourth its orig- inal value a change that is necessary for subsequent growth and division of the egg. Hydrogen Ions Are Released by Acids and Taken Up by Bases In general an acid is any molecule ion or chemical group that tends to release a hydrogen ion H such as hy- drochloric acid HCl and the carboxyl group OCOOH which tends to dissociate to form the negatively charged car- boxylate ion OCOO . Likewise a base is any molecule ion or chemical group that readily combines with a H such as the hydroxyl ion OH ammonia NH 3 which forms an ammonium ion NH 4 and the amino group ONH 2 . When acid is added to an aqueous solution the H in- creases the pH goes down. Conversely when a base is added to a solution the H decreases the pH goes up. Be- cause H OH 10 14 M 2 any increase in H is cou- pled with a decrease in OH and vice versa. Many biological molecules contain both acidic and basic groups. For example in neutral solutions pH 7.0 amino acids exist predominantly in the doubly ionized form in which the carboxyl group has lost a proton and the amino group has accepted one: where R represents the side chain. Such a molecule contain- ing an equal number of positive and negative ions is called a zwitterion. Zwitterions having no net charge are neutral. At extreme pH values only one of these two ionizable groups of an amino acid will be charged. The dissociation reaction for an acid or acid group in a larger molecule HA can be written as HA H A . NH 3 COO C H R The equilibrium constant for this reaction denoted K a sub- script a for “acid” is defined as K a H A /HA. Tak- ing the logarithm of both sides and rearranging the result yields a very useful relation between the equilibrium constant and pH: 2-5 where pK a equals –log K a . From this expression commonly known as the Henderson- Hasselbalch equation it can be seen that the pK a of any acid is equal to the pH at which half the molecules are dissociated and half are neutral undissociated. This is because when pK a pH then log A /HA 0 and therefore A HA. The Henderson-Hasselbalch equation allows us to calculate the degree of dissociation of an acid if both the pH of the solution and the pK a of the acid are known. Experimentally by meas- uring the A and HA as a function of the solution’s pH one can calculate the pK a of the acid and thus the equilibrium constant K a for the dissociation reaction. Buffers Maintain the pH of Intracellular and Extracellular Fluids A growing cell must maintain a constant pH in the cyto- plasm of about 7.2–7.4 despite the metabolic production of many acids such as lactic acid and carbon dioxide the lat- ter reacts with water to form carbonic acid H 2 CO 3 . Cells have a reservoir of weak bases and weak acids called buffers which ensure that the cell’s pH remains relatively constant despite small fluctuations in the amounts of H or OH being generated by metabolism or by the uptake or se- cretion of molecules and ions by the cell. Buffers do this by “soaking up” excess H or OH when these ions are added to the cell or are produced by metabolism. If additional acid or base is added to a solution that contains a buffer at its pK a value a 1:1 mixture of HA and A the pH of the solution changes but it changes less than it would if the buffer had not been present. This is because protons released by the added acid are taken up by the ion- ized form of the buffer A likewise hydroxyl ions gener- ated by the addition of base are neutralized by protons released by the undissociated buffer HA. The capacity of a substance to release hydrogen ions or take them up depends partly on the extent to which the substance has already taken up or released protons which in turn depends on the pH of the solution. The ability of a buffer to minimize changes in pH its buffering capacity depends on the relationship be- tween its pK a value and the pH which is expressed by the Henderson-Hasselbalch equation. The titration curve for acetic acid shown in Figure 2-22 illustrates the effect of pH on the fraction of molecules in the un-ionized HA and ionized forms A . At one pH unit below the pK a of an acid 91 percent of the molecules are in the HA form at one pH unit above the pK a 91 percent are pH pK a log A HA 48 CHAPTER 2 • Chemical Foundations

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in the A form. At pH values more than one unit above or below the pK a the buffering capacity of weak acids and bases declines rapidly. In other words the addition of the same number of moles of acid to a solution containing a mix- ture of HA and A that is at a pH near the pK a will cause less of a pH change than it would if the HA and A were not present or if the pH were far from the pK a value. All biological systems contain one or more buffers. Phos- phate ions the ionized forms of phosphoric acid are pres- ent in considerable quantities in cells and are an important factor in maintaining or buffering the pH of the cytoplasm. Phosphoric acid H 3 PO 4 has three protons that are capable of dissociating but they do not dissociate simultaneously. Loss of each proton can be described by a discrete dissocia- tion reaction and pK a as shown in Figure 2-23. The titration curve for phosphoric acid shows that the pK a for the disso- ciation of the second proton is 7.2. Thus at pH 7.2 about 50 percent of cellular phosphate is H 2 PO 4 and about 50 per- cent is HPO 4 2 according to the Henderson-Hasselbalch equation. For this reason phosphate is an excellent buffer at pH values around 7.2 the approximate pH of the cyto- plasm of cells and at pH 7.4 the pH of human blood. KEY CONCEPTS OF SECTION 2.3 Chemical Equilibrium ■ A chemical reaction is at equilibrium when the rate of the forward reaction is equal to the rate of the reverse re- action no net change in the concentration of the reactants or products. ■ The equilibrium constant K eq of a reaction reflects the ratio of products to reactants at equilibrium and thus is a measure of the extent of the reaction and the relative sta- bilities of the reactants and products. ■ The K eq depends on the temperature pressure and chemical properties of the reactants and products but is independent of the reaction rate and of the initial concen- trations of reactants and products. ■ For any reaction the equilibrium constant K eq equals the ratio of the forward rate constant to the reverse rate constant k f /k r . ■ Within cells the linked reactions in metabolic pathways generally are at steady state not equilibrium at which rate of formation of the intermediates equals their rate of con- sumption see Figure 2-21. ■ The dissociation constant K d for a reaction involving the noncovalent binding of two molecules is a measure of the stability of the complex formed between the molecules e.g. ligand-receptor or protein-DNA complexes. ■ The pH is the negative logarithm of the concentration of hydrogen ions –log H . The pH of the cytoplasm is normally about 7.2–7.4 whereas the interior of lysosomes has a pH of about 4.5. 2.3 • Chemical Equilibrium 49 14 12 10 8 6 4 2 0 pH Added OH − HPO 4 2 − PO 4 3− + H + H 2 PO 4 − HPO 4 2− + H + H 3 PO 4 H 2 PO 4 − + H + pK a 12.7 pK a 7.2 pK a 2.1 ▲ FIGURE 2-23 The titration curve of phosphoric acid H 3 PO 4 . This biologically ubiquitous molecule has three hydrogen atoms that dissociate at different pH values thus phosphoric acid has three pK a values as noted on the graph. The shaded areas denote the pH ranges—within one pH unit of the three pK a values—where the buffering capacity of phosphoric acid is high. In these regions the addition of acid or base will cause relatively small changes in the pH. CH 3 COO − + H + CH 3 COOH pH 2 4 6 8 1.0 0.8 0.6 0.4 0.2 0 pK a 4.75 Added OH − Fraction of dissociated CH 3 COOH ▲ FIGURE 2-22 The titration curve of acetic acid CH 3 COOH. The pK a for the dissociation of acetic acid to hydrogen and acetate ions is 4.75. At this pH half the acid molecules are dissociated. Because pH is measured on a logarithmic scale the solution changes from 91 percent CH 3 COOH at pH 3.75 to 9 percent CH 3 COOH at pH 5.75. The acid has maximum buffering capacity in this pH range.

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■ Acids release protons H and bases bind them. In bio- logical molecules the carboxyl and phosphate groups are the most common acidic groups the amino group is the most common basic group. ■ Buffers are mixtures of a weak acid HA and its corre- sponding base form A which minimize the change in pH of a solution when acid or alkali is added. Biological systems use various buffers to maintain their pH within a very narrow range. Biochemical Energetics The production of energy its storage and its use are central to the economy of the cell. Energy may be defined as the abil- ity to do work a concept applicable to automobile engines and electric power plants in our physical world and to cel- lular engines in the biological world. The energy associated with chemical bonds can be harnessed to support chemical work and the physical movements of cells. Several Forms of Energy Are Important in Biological Systems There are two principal forms of energy: kinetic and poten- tial. Kinetic energy is the energy of movement—the motion of molecules for example. The second form of energy po- tential energy or stored energy is particularly important in the study of biological or chemical systems. Kinetic Energy Heat or thermal energy is a form of kinetic energy—the energy of the motion of molecules. For heat to do work it must flow from a region of higher temperature— where the average speed of molecular motion is greater—to one of lower temperature. Although differences in tempera- ture can exist between the internal and external environments of cells these thermal gradients do not usually serve as the source of energy for cellular activities. The thermal energy in warm-blooded animals which have evolved a mechanism for thermoregulation is used chiefly to maintain constant organ- ismic temperatures. This is an important function since the rates of many cellular activities are temperature-dependent. For example cooling mammalian cells from their normal body temperature of 37 ºC to 4 ºC can virtually “freeze” or stop many cellular processes e.g. intracellular membrane movements. Radiant energy is the kinetic energy of photons or waves of light and is critical to biology. Radiant energy can be con- verted to thermal energy for instance when light is absorbed by molecules and the energy is converted to molecular motion. During photosynthesis light energy absorbed by specialized molecules e.g. chlorophyll is subsequently con- verted into the energy of chemical bonds Chapter 8. Mechanical energy a major form of kinetic energy in bi- ology usually results from the conversion of stored chemical 2.4 energy. For example changes in the lengths of cytoskeletal filaments generates forces that push or pull on membranes and organelles Chapter 19. Electric energy—the energy of moving electrons or other charged particles—is yet another major form of kinetic energy. Potential Energy Several forms of potential energy are bio- logically significant. Central to biology is chemical potential energy the energy stored in the bonds connecting atoms in molecules. Indeed most of the biochemical reactions de- scribed in this book involve the making or breaking of at least one covalent chemical bond. We recognize this energy when chemicals undergo energy-releasing reactions. For ex- ample the high potential energy in the covalent bonds of glu- cose can be released by controlled enzymatic combustion in cells see later discussion. This energy is harnessed by the cell to do many kinds of work. A second biologically important form of potential energy is the energy in a concentration gradient. When the concen- tration of a substance on one side of a barrier such as a membrane is different from that on the other side a con- centration gradient exists. All cells form concentration gra- dients between their interior and the external fluids by selectively exchanging nutrients waste products and ions with their surroundings. Also organelles within cells e.g. mitochondria lysosomes frequently contain different con- centrations of ions and other molecules the concentration of protons within a lysosome as we saw in the last section is about 500 times that of the cytoplasm. A third form of potential energy in cells is an electric potential—the energy of charge separation. For instance there is a gradient of electric charge of ≈200000 volts per cm across the plasma membrane of virtually all cells. We discuss how concentration gradients and the potential difference across cell membranes are generated and maintained in Chapter 7. Cells Can Transform One Type of Energy into Another According to the first law of thermodynamics energy is nei- ther created nor destroyed but can be converted from one form to another. In nuclear reactions mass is converted to energy but this is irrelevant to biological systems. In pho- tosynthesis for example the radiant energy of light is trans- formed into the chemical potential energy of the covalent bonds between the atoms in a sucrose or starch molecule. In muscles and nerves chemical potential energy stored in co- valent bonds is transformed respectively into the kinetic energy of muscle contraction and the electric energy of nerve transmission. In all cells potential energy released by break- ing certain chemical bonds is used to generate potential en- ergy in the form of concentration and electric potential gradients. Similarly energy stored in chemical concentration gradients or electric potential gradients is used to synthesize 50 CHAPTER 2 • Chemical Foundations

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chemical bonds or to transport molecules from one side of a membrane to another to generate a concentration gradient. This latter process occurs during the transport of nutrients such as glucose into certain cells and transport of many waste products out of cells. Because all forms of energy are interconvertible they can be expressed in the same units of measurement. Although the standard unit of energy is the joule biochemists have tradi- tionally used an alternative unit the calorie 1 joule 0.239 calories. Throughout this book we use the kilocalorie to measure energy changes 1 kcal 1000 cal. The Change in Free Energy Determines the Direction of a Chemical Reaction Because biological systems are generally held at constant temperature and pressure it is possible to predict the direc- tion of a chemical reaction from the change in the free energy G named after J. W. Gibbs who showed that “all systems change in such a way that free energy G is minimized.” In the case of a chemical reaction reactants products the change in free energy G is given by G G products G reactants The relation of G to the direction of any chemical reaction can be summarized in three statements: ■ If G is negative the forward reaction from left to right as written will tend to occur spontaneously. ■ If G is positive the reverse reaction from right to left as written will tend to occur. ■ If G is zero both forward and reverse reactions occur at equal rates the reaction is at equilibrium. The standard free-energy change of a reaction Gº is the value of the change in free energy under the conditions of 298 K 25 ºC 1 atm pressure pH 7.0 as in pure water and initial concentrations of 1 M for all reactants and products except protons which are kept at 10 7 M pH 7.0. Most bi- ological reactions differ from standard conditions particu- larly in the concentrations of reactants which are normally less than 1 M. The free energy of a chemical system can be defined as G H TS where H is the bond energy or enthalpy of the system T is its temperature in degrees Kelvin K and S is the entropy a measure of its randomness or disorder. If temperature remains constant a reaction proceeds sponta- neously only if the free-energy change G in the following equation is negative: G H T S 2-6 In an exothermic reaction the products contain less bond en- ergy than the reactants the liberated energy is usually con- verted to heat the energy of molecular motion and H is negative. In an endothermic reaction the products contain more bond energy than the reactants heat is absorbed and H is positive. The combined effects of the changes in the en- thalpy and entropy determine if the G for a reaction is pos- itive or negative. An exothermic reaction H 0 in which entropy increases S 0 occurs spontaneously G 0. An endothermic reaction H 0 will occur spontaneously if S increases enough so that the T S term can overcome the positive H. Many biological reactions lead to an increase in order and thus a decrease in entropy S 0. An obvious exam- ple is the reaction that links amino acids together to form a protein. A solution of protein molecules has a lower entropy than does a solution of the same amino acids unlinked be- cause the free movement of any amino acid in a protein is restricted when it is bound into a long chain. Often cells compensate for decreases in entropy by “coupling” such syn- thetic reactions with independent reactions that have a very highly negative G see below. In this fashion cells can con- vert sources of energy in their environment into the building of highly organized structures and metabolic pathways that are essential for life. The actual change in free energy G during a reaction is influenced by temperature pressure and the initial con- centrations of reactants and products and usually differs from Gº . Most biological reactions—like others that take place in aqueous solutions—also are affected by the pH of the solution. We can estimate free-energy changes for dif- ferent temperatures and initial concentrations using the equation G Gº RT ln Q Gº RTln 2-7 where R is the gas constant of 1.987 cal/degree·mol T is the temperature in degrees Kelvin and Q is the initial ratio of products to reactants. For a reaction A B C in which two molecules combine to form a third Q in Equation 2-7 equals C/AB. In this case an increase in the initial concentration of either A or B will result in a large nega- tive value for G and thus drive the reaction toward more formation of C. Regardless of the Gº for a particular biochemical reaction it will proceed spontaneously within cells only if G is negative given the usual intracellular concentrations of reactants and products. For example the conversion of glyceraldehyde 3-phosphate G3P to dihydroxyacetone phosphate DHAP two intermediates in the breakdown of glucose G3P DHAP has a Gº of 1840 cal/mol. If the initial concentrations of G3P and DHAP are equal then G Gº because RT ln 1 0 in this situation the reversible reaction G3P DHAP will proceed in the direction of DHAP formation until equi- librium is reached. However if the initial DHAP is 0.1 M and the initial G3P is 0.001 M with other conditions being standard then Q in Equation 2-7 equals 0.1/0.001 100 products reactants 2.4 • Biochemical Energetics 51

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giving a G of 887 cal/mole. Under these conditions the reaction will proceed in the direction of formation of G3P. The G for a reaction is independent of the reaction rate. Indeed under usual physiological conditions few if any of the biochemical reactions needed to sustain life would occur without some mechanism for increasing reaction rates. As we describe in Chapter 3 the rates of reactions in biological sys- tems are usually determined by the activity of enzymes the protein catalysts that accelerate the formation of products from reactants without altering the value of G. The Gº of a Reaction Can Be Calculated from Its K eq A chemical mixture at equilibrium is already in a state of minimal free energy that is no free energy is being generated or released. Thus for a system at equilibrium G 0 Q K eq we can write Gº 2.3RT log K eq 1362 log K eq 2-8 under standard conditions note the change to base 10 loga- rithms. Thus if the concentrations of reactants and prod- ucts at equilibrium i.e. the K eq are determined the value of Gº can be calculated. For example K eq for the intercon- version of glyceraldehyde 3-phosphate to dihydroxyacetone phosphate G3P DHAP is 22.2 under standard condi- tions. Substituting this value into Equation 2-8 we can eas- ily calculate the Gº for this reaction as 1840 cal/mol. By rearranging Equation 2-8 and taking the antiloga- rithm we obtain K eq 10 Gº /2.3RT 2-9 From this expression it is clear that if Gº is negative the exponent will be positive and hence K eq will be greater than 1. Therefore at equilibrium there will be more products than reactants in other words the formation of products from re- actants is favored. Conversely if Gº is positive the expo- nent will be negative and K eq will be less than 1. An Unfavorable Chemical Reaction Can Proceed If It Is Coupled with an Energetically Favorable Reaction Many processes in cells are energetically unfavorable G 0 and will not proceed spontaneously. Examples include the synthesis of DNA from nucleotides and transport of a sub- stance across the plasma membrane from a lower to a higher concentration. Cells can carry out an energy-requiring reac- tion G 1 0 by coupling it to an energy-releasing reac- tion G 2 0 if the sum of the two reactions has a net negative G. Suppose for example that the reaction A B X has a G of 5 kcal/mol and that the reaction X Y Z has a G of 10 kcal/mol. 1A B X G 5 kcal/mol 2X Y Z G 10 kcal/mol Sum:A B Y Z Gº 5 kcal/mol In the absence of the second reaction there would be much more A than B at equilibrium. However because the conver- sion of X to Y Z is such a favorable reaction it will pull the first process toward the formation of B and the con- sumption of A. Energetically unfavorable reactions in cells often are coupled to the hydrolysis of ATP as we discuss next. Hydrolysis of ATP Releases Substantial Free Energy and Drives Many Cellular Processes In almost all organisms adenosine triphosphate or ATP is the most important molecule for capturing transiently stor- ing and subsequently transferring energy to perform work e.g. biosynthesis mechanical motion. The useful energy in an ATP molecule is contained in phosphoanhydride bonds which are covalent bonds formed from the condensation of two molecules of phosphate by the loss of water: An ATP molecule has two key phosphoanhydride bonds Figure 2-24. Hydrolysis of a phosphoanhydride bond in each of the following reactions has a highly negative Gº of about 7.3 kcal/mol: O O P O O O O O P O O P O O O P O H 2 O OH HO 52 CHAPTER 2 • Chemical Foundations CH 2 NH 2 O H HO H H OH H N N N N C C C HC CH Adenosine triphosphate ATP O O O P O O P O O Phosphoanhydride bonds O O P O ▲ FIGURE 2-24 Adenosine triphosphate ATP. The two phosphoanhydride bonds red in ATP which link the three phosphate groups each has a G˚ of 7 .3 kcal/mol for hydroly- sis. Hydrolysis of these bonds especially the terminal one drives many energy-requiring reactions in biological systems.

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Appp H 2 O On App P i H ATP ADP Appp H 2 O On Ap PP i H ATP AMP App H 2 O On Ap P i H ADP AMP In these reactions P i stands for inorganic phosphate PO 4 3 and PP i for inorganic pyrophosphate two phosphate groups linked by a phosphodiester bond. As the top two reactions show the removal of a phosphate or a pyrophosphate group from ATP leaves adenosine diphosphate ADP or adenosine monophosphate AMP respectively. A phosphoanhydride bond or other high-energy bond commonly denoted by is not intrinsically different from other covalent bonds. High-energy bonds simply release es- pecially large amounts of energy when broken by addition of water hydrolyzed. For instance the Gº for hydrolysis of a phosphoanhydride bond in ATP 7.3 kcal/mol is more than three times the Gº for hydrolysis of the phosphoester bond red in glycerol 3-phosphate 2.2 kcal/mol: A principal reason for this difference is that ATP and its hy- drolysis products ADP and P i are highly charged at neutral pH. During synthesis of ATP a large input of energy is re- quired to force the negative charges in ADP and P i together. Conversely much energy is released when ATP is hydrolyzed to ADP and P i . In comparison formation of the phospho- ester bond between an uncharged hydroxyl in glycerol and P i requires less energy and less energy is released when this bond is hydrolyzed. Cells have evolved protein-mediated mechanisms for transferring the free energy released by hydrolysis of phos- phoanhydride bonds to other molecules thereby driving re- actions that would otherwise be energetically unfavorable. For example if the G for the reaction B C On D is pos- itive but less than the G for hydrolysis of ATP the reaction can be driven to the right by coupling it to hydrolysis of the terminal phosphoanhydride bond in ATP. In one common mechanism of such energy coupling some of the energy stored in this phosphoanhydride bond is transferred to the one of the reactants by breaking the bond in ATP and form- ing a covalent bond between the released phosphate group and one of the reactants. The phosphorylated intermediate generated in this fashion can then react with C to form D P i in a reaction that has a negative G : B Appp On Bp App Bp C On D P i HO P CH 2 OH CH 2 O O O OH CH Glycerol 3-phosphate The overall reaction B C ATP D ADP P i is energetically favorable G 0. An alternative mechanism of energy coupling is to use the energy released by ATP hydrolysis to change the conforma- tion of the molecule to an “energy-rich” stressed state. In turn the energy stored as conformational stress can be re- leased as the molecule “relaxes” back into its unstressed con- formation. If this relaxation process can be mechanistically coupled to another reaction the released energy can be har- nessed to drive important cellular processes. As with many biosynthetic reactions transport of mole- cules into or out of the cell often has a positive G and thus requires an input of energy to proceed. Such simple transport reactions do not directly involve the making or breaking of covalent bonds thus the Gº is 0. In the case of a substance moving into a cell Equation 2-7 becomes G 2-10 where C in is the initial concentration of the substance in- side the cell and C out is its concentration outside the cell. We can see from Equation 2-10 that G is positive for transport of a substance into a cell against its concentration gradient when C in C out the energy to drive such “uphill” trans- port often is supplied by the hydrolysis of ATP. Conversely when a substance moves down its concentration gradient C out C in G is negative. Such “downhill” transport releases energy that can be coupled to an energy-requiring re- action say the movement of another substance uphill across a membrane or the synthesis of ATP itself see Chapter 7. ATP Is Generated During Photosynthesis and Respiration Clearly to continue functioning cells must constantly re- plenish their ATP supply. The initial energy source whose en- ergy is ultimately transformed into the phosphoanhydride bonds of ATP and bonds in other compounds in nearly all cells is sunlight. In photosynthesis plants and certain mi- croorganisms can trap the energy in light and use it to syn- thesize ATP from ADP and P i . Much of the ATP produced in photosynthesis is hydrolyzed to provide energy for the conversion of carbon dioxide to six-carbon sugars a process called carbon fixation: In animals the free energy in sugars and other molecules de- rived from food is released in the process of respiration. All synthesis of ATP in animal cells and in nonphotosynthetic microorganisms results from the chemical transformation of energy-rich compounds in the diet e.g. glucose starch. We discuss the mechanisms of photosynthesis and cellular respi- ration in Chapter 8. 6 CO 2 6 H 2 O ATP P i ADP C 6 H 12 O 6 6 O 2 RT ln C in C out 2.4 • Biochemical Energetics 53

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The complete oxidation of glucose to yield carbon dioxide C 6 H 12 O 6 6 O 2 On 6 CO 2 6 H 2 O has a Gº of 686 kcal/mol and is the reverse of photo- synthetic carbon fixation. Cells employ an elaborate set of enzyme-catalyzed reactions to couple the metabolism of 1 molecule of glucose to the synthesis of as many as 30 molecules of ATP from 30 molecules of ADP. This oxygen-dependent aerobic degradation catabolism of glucose is the major pathway for generating ATP in all an- imal cells nonphotosynthetic plant cells and many bac- terial cells. Light energy captured in photosynthesis is not the only source of chemical energy for all cells. Certain microorgan- isms that live in deep ocean vents where sunlight is com- pletely absent derive the energy for converting ADP and P i into ATP from the oxidation of reduced inorganic com- pounds. These reduced compounds originate in the center of the earth and are released at the vents. NAD and FAD Couple Many Biological Oxidation and Reduction Reactions In many chemical reactions electrons are transferred from one atom or molecule to another this transfer may or may not accompany the formation of new chemical bonds. The loss of electrons from an atom or a molecule is called oxida- tion and the gain of electrons by an atom or a molecule is called reduction. Because electrons are neither created nor destroyed in a chemical reaction if one atom or molecule is oxidized another must be reduced. For example oxygen draws electrons from Fe 2 ferrous ions to form Fe 3 fer- ric ions a reaction that occurs as part of the process by which carbohydrates are degraded in mitochondria. Each oxygen atom receives two electrons one from each of two Fe 2 ions: 2 Fe 2 1 ⁄2 O 2 On 2 Fe 3 O 2 Thus Fe 2 is oxidized and O 2 is reduced. Such reactions in which one molecule is reduced and another oxidized often are referred to as redox reactions. Oxygen is an electron ac- ceptor in many redox reactions in aerobic cells. Many biologically important oxidation and reduction re- actions involve the removal or the addition of hydrogen atoms protons plus electrons rather than the transfer of iso- lated electrons on their own. The oxidation of succinate to fumarate which also occurs in mitochondria is an example Figure 2-25. Protons are soluble in aqueous solutions as H 3 O but electrons are not and must be transferred di- 54 CHAPTER 2 • Chemical Foundations Succinate O O C O 2 e 2 H C O C H H C H H Fumarate O O C O C O C H C H ▲ FIGURE 2-25 Conversion of succinate to fumarate. In this oxidation reaction which occurs in mitochondria as part of the citric acid cycle succinate loses two electrons and two protons. These are transferred to FAD reducing it to FADH 2 . Oxidized: NAD Nicotinamide NAD H 2 e NADH Flavin H 2 e 2 H 2 e Ribitol Adenosine 2P Ribitol Adenosine 2P FAD 2 H 2 e FADH 2 a Reduced: FADH 2 Oxidized: FAD b H 3 C H 3 C H H H H O O O O O N N N N H 3 C H 3 C H H H H H N N N N C NH 2 N + Reduced: NADH H H O C NH 2 N • • Ribose Adenosine 2P Ribose Adenosine 2P ▲ FIGURE 2-26 The electron-carrying coenzymes NAD and FAD. a NAD nicotinamide adenine dinucleotide is reduced to NADH by addition of two electrons and one proton simultaneously. In many biological redox reactions e.g. succinate n fumarate a pair of hydrogen atoms two protons and two electrons are removed from a molecule. One of the protons and both electrons are transferred to NAD the other proton is released into solution. b FAD flavin adenine dinucleotide is reduced to FADH 2 by addition of two electrons and two protons. In this two-step reaction addition of one electron together with one proton first generates a short-lived semiquinone intermediate not shown which then accepts a second electron and proton.

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KEY CONCEPTS OF SECTION 2.4 Biochemical Energetics ■ The change in free energy G is the most useful meas- ure for predicting the direction of chemical reactions in bi- ological systems. Chemical reactions tend to proceed in the direction for which G is negative. ■ Directly or indirectly light energy captured by photo- synthesis in plants and photosynthetic bacteria is the ulti- mate source of chemical energy for almost all cells. ■ The chemical free-energy change Gº equals 2.3 RT log K eq . Thus the value of Gº can be calculated from the experimentally determined concentrations of reactants and products at equilibrium. ■ A chemical reaction having a positive G can proceed if it is coupled with a reaction having a negative G of larger magnitude. ■ Many otherwise energetically unfavorable cellular pro- cesses are driven by hydrolysis of phosphoanhydride bonds in ATP see Figure 2-24. ■ An oxidation reaction loss of electrons is always cou- pled with a reduction reaction gain of electrons. ■ Biological oxidation and reduction reactions often are coupled by electron-carrying coenzymes such as NAD and FAD see Figure 2-26. ■ Oxidation-reduction reactions with a positive E have a negative G and thus tend to proceed spontaneously. KEY TERMS acid 48 carbon atom C 38 amino acids 38 amphipathic 29 base 48 buffers 48 chemical potential energy 50 covalent bond 30 dehydration reaction 37 G free-energy change 51 disulfide bond 38 energy coupling 53 enthalpy H 51 entropy S 51 equilibrium constant 46 fatty acids 43 hydrogen bond 33 hydrophilic 29 hydrophobic 29 Key Terms 55 rectly from one atom or molecule to another without a water-dissolved intermediate. In this type of oxidation reac- tion electrons often are transferred to small electron-carry- ing molecules sometimes referred to as coenzymes. The most common of these electron carriers are NAD nicotinamide adenine dinucleotide which is reduced to NADH and FAD flavin adenine dinucleotide which is reduced to FADH 2 Figure 2-26. The reduced forms of these coenzymes can transfer protons and electrons to other molecules thereby re- ducing them. To describe redox reactions such as the reaction of fer- rous ion Fe 2 and oxygen O 2 it is easiest to divide them into two half-reactions: Oxidation of Fe 2 :2 Fe 2 On 2 Fe 3 2 e Reduction of O 2 :2 e 1 ⁄2 O 2 On O 2 In this case the reduced oxygen O 2 readily reacts with two protons to form one water molecule H 2 O. The readi- ness with which an atom or a molecule gains an electron is its reduction potential E. The tendency to lose electrons the ox- idation potential has the same magnitude but opposite sign as the reduction potential for the reverse reaction. Reduction potentials are measured in volts V from an arbitrary zero point set at the reduction potential of the fol- lowing half-reaction under standard conditions 25 ºC 1 atm and reactants at 1 M: reduction H e 399999994 1 ⁄2 H 2 oxidation The value of E for a molecule or an atom under standard conditions is its standard reduction potential E 0. A mole- cule or ion with a positive E 0 has a higher affinity for elec- trons than the H ion does under standard conditions. Conversely a molecule or ion with a negative E 0 has a lower affinity for electrons than the H ion does under standard conditions. Like the values of Gº standard reduction po- tentials may differ somewhat from those found under the conditions in a cell because the concentrations of reactants in a cell are not 1 M. In a redox reaction electrons move spontaneously to- ward atoms or molecules having more positive reduction potentials. In other words a compound having a more neg- ative reduction potential can transfer electrons to i.e. re- duce a compound with a more positive reduction potential. In this type of reaction the change in electric po- tential E is the sum of the reduction and oxidation poten- tials for the two half-reactions. The E for a redox reaction is related to the change in free energy G by the following expression: G cal/mol n 23064 E volts 2-11 where n is the number of electrons transferred. Note that a redox reaction with a positive E value will have a negative G and thus will tend to proceed from left to right. hydrophobic effect 35 ionic interactions 33 molecular complementarity 36 monosaccharides 41 nucleosides 40 nucleotides 40 oxidation 55 pH 47 phosphoanhydride bonds 52 phospholipid bilayers 45 polar 32 polymer 37 redox reaction 54 reduction 55 saturated 43 steady state 46 stereoisomers 31 unsaturated 43 van der Waals interactions 34

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REVIEW THE CONCEPTS 1. The gecko is a reptile with an amazing ability to climb smooth surfaces including glass. Recent discoveries indicate that geckos stick to smooth surfaces via van der Waals in- teractions between septae on their feet and the smooth sur- face. How is this method of stickiness advantageous over covalent interactions Given that van der Waals forces are among the weakest molecular interactions how can the gecko’s feet stick so effectively 2. The K channel is an example of a transmembrane pro- tein a protein that spans the phospholipid bilayer of the plasma membrane. What types of amino acids are likely to be found a lining the channel through which K passes b in contact with the phospholipid bilayer containing fatty acid c in the cytosolic domain of the protein and d in the extracellular domain of the protein 3. V-M-Y-Y-E-N: This is the single-letter amino acid ab- breviation for a peptide. Draw the structure of this peptide. What is the net charge of this peptide at pH 7.0 An enzyme called a protein tyrosine kinase can attach phosphates to the hydroxyl groups of tyrosine. What is the net charge of the peptide at pH 7.0 after it has been phosphorylated by a ty- rosine kinase What is the likely source of phosphate utilized by the kinase for this reaction 4. Disulfide bonds help to stabilize the three-dimensional structure of proteins. What amino acids are involved in the formation of disulfide bonds Does the formation of a disul- fide bond increase or decrease entropy S 5. In the 1960s the drug thalidomide was prescribed to pregnant women to treat morning sickness. However thalidomide caused severe limb defects in the children of some women who took the drug and its use for morning sickness was discontinued. It is now known that thalidomide was administered as a mixture of two stereoisomeric com- pounds one of which relieved morning sickness and the other of which was responsible for the birth defects. What are stereoisomers Why might two such closely related com- pounds have such different physiologic effects 6. Name the compound shown below. Is this nucleotide a component of DNA RNA or both Name one other func- tion of this compound. H 2 N OH OH H HH H O N N N CH 2 HN C C C O C CH 1 1 2 5 4 3 6 7 9 8 4 2 3 5 OP O O O O P O O O O P O O O 7. The chemical basis of blood-group specificity resides in the carbohydrates displayed on the surface of red blood cells. Carbohydrates have the potential for great structural diver- sity. Indeed the structural complexity of the oligosaccharides that can be formed from four sugars is greater than that for oligopeptides from four amino acids. What properties of car- bohydrates make this great structural diversity possible 8. Ammonia NH 3 is a weak base that under acidic con- ditions becomes protonated to the ammonium ion in the fol- lowing reaction: NH 3 H n NH 4 NH 3 freely permeates biological membranes including those of lysosomes. The lysosome is a subcellular organelle with a pH of about 5.0 the pH of cytoplasm is 7.0. What is the ef- fect on the pH of the fluid content of lysosomes when cells are exposed to ammonia Note: Protonated ammonia does not diffuse freely across membranes. 9. Consider the binding reaction L R n LR where L is a ligand and R is its receptor. When 1 10 3 M L is added to a solution containing 5 10 2 M R 90 of the L binds to form LR. What is the K eq of this reaction How will the K eq be affected by the addition of a protein that catalyzes this binding reaction What is the K d 10. What is the ionization state of phosphoric acid in the cy- toplasm Why is phosphoric acid such a physiologically im- portant compound 11. The G for the reaction X Y n XY is 1000 cal/mol. What is the G at 25 C 298 Kelvin starting with 0.01 M each X Y and XY Suggest two ways one could make this reaction energetically favorable. REFERENCES Alberty R. A. and R. J. Silbey. 2000. Physical Chemistry 3d ed. Wiley. Atkins P. W. 2000. The Elements of Physical Chemistry 3d ed. W. H. Freeman and Company. Berg J. M. J. L. Tymoczko and L. Stryer. 2002. Biochemistry 5th ed. W. H. Freeman and Company. Cantor P. R. and C. R. Schimmel. 1980. Biophysical Chemistry. W. H. Freeman and Company. Davenport H. W. 1974. ABC of Acid-Base Chemistry 6th ed. University of Chicago Press. Edsall J. T. and J. Wyman. 1958. Biophysical Chemistry vol. 1. Academic Press. Eisenberg D. and D. Crothers. 1979. Physical Chemistry with Applications to the Life Sciences. Benjamin-Cummings. Gennis R. B. 1989. Biomembranes: Molecular Structure and Function. Springer-Verlag New York. Guyton A. C. and J. E. Hall. 2000. Textbook of Medical Phys- iology 10th ed. Saunders. Hill T. J. 1977. Free Energy Transduction in Biology. Academic Press. Klotz I. M. 1978. Energy Changes in Biochemical Reactions. Academic Press. 56 CHAPTER 2 • Chemical Foundations

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Lehninger A. L. D. L. Nelson and M. M. Cox. 2000. Princi- ples of Biochemistry 3d ed. Worth. Murray R. K. et al. 1999. Harper’s Biochemistry 25th ed. Lange. Nicholls D. G. and S. J. Ferguson. 1992. Bioenergetics 2. Aca- demic Press. Oxtoby D. H. Gillis and N. Nachtrieb. 2003. Principles of Modern Chemistry 5th ed. Saunders. Sharon N. 1980. Carbohydrates. Sci. Am. 2435:90–116. Tanford C. 1980. The Hydrophobic Effect: Formation of Mi- celles and Biological Membranes 2d ed. Wiley. Tinoco I. K. Sauer and J. Wang. 2001. Physical Chemistry— Principles and Applications in Biological Sciences 4th ed. Prentice Hall. Van Holde K. W. Johnson and P. Ho. 1998. Principles of Phys- ical Biochemistry. Prentice Hall. Voet D. and J. Voet. 1995. Biochemistry 2d ed. Wiley. Watson J. D. et al. 2003. Molecular Biology of the Gene 5th ed. Benjamin-Cummings. Wood W. B. et al. 1981. Biochemistry: A Problems Approach 2d ed. Benjamin-Cummings. References 57

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3 Electron density map of the F 1 -ATPase associated with a ring of 10 c-subunits from the F 0 domain of ATP synthase a molecular machine that carries out the synthesis of ATP in eubacteria chloroplasts and mitochondria. Courtesy of Andrew Leslie MRC Laboratory of Molecular Biology Cambridge UK. PROTEIN STRUCTURE AND FUNCTION P roteins the working molecules of a cell carry out the program of activities encoded by genes. This program requires the coordinated effort of many different types of proteins which first evolved as rudimentary molecules that facilitated a limited number of chemical reactions. Grad- ually many of these primitive proteins evolved into a wide array of enzymes capable of catalyzing an incredible range of intracellular and extracellular chemical reactions with a speed and specificity that is nearly impossible to attain in a test tube. With the passage of time other proteins acquired specialized abilities and can be grouped into several broad functional classes: structural proteins which provide struc- tural rigidity to the cell transport proteins which control the flow of materials across cellular membranes regulatory pro- teins which act as sensors and switches to control protein activity and gene function signaling proteins including cell- surface receptors and other proteins that transmit external signals to the cell interior and motor proteins which cause motion. A key to understanding the functional design of proteins is the realization that many have “moving” parts and are ca- pable of transmitting various forces and energy in an orderly fashion. However several critical and complex cell processes—synthesis of nucleic acids and proteins signal transduction and photosynthesis—are carried out by huge macromolecular assemblies sometimes referred to as molec- ular machines. A fundamental goal of molecular cell biologists is to un- derstand how cells carry out various processes essential for life. A major contribution toward achieving this goal is the identification of all of an organism’s proteins—that is a list of the parts that compose the cellular machinery. The com- pilation of such lists has become feasible in recent years with the sequencing of entire genomes—complete sets of genes— of more and more organisms. From a computer analysis of 59 OUTLINE 3.1 Hierarchical Structure of Proteins 3.2 Folding Modification and Degradation of Proteins 3.3 Enzymes and the Chemical Work of Cells 3.4 Molecular Motors and the Mechanical Work of Cells 3.5 Common Mechanisms for Regulating Protein Function 3.6 Purifying Detecting and Characterizing Proteins

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60 CHAPTER 3 • Protein Structure and Function genome sequences researchers can deduce the number and primary structure of the encoded proteins Chapter 9. The term proteome was coined to refer to the entire protein com- plement of an organism. For example the proteome of the yeast Saccharomyces cerevisiae consists of about 6000 dif- ferent proteins the human proteome is only about five times as large comprising about 32000 different proteins. By comparing protein sequences and structures scientists can classify many proteins in an organism’s proteome and deduce their functions by homology with proteins of known func- tion. Although the three-dimensional structures of relatively few proteins are known the function of a protein whose structure has not been determined can often be inferred from its interactions with other proteins from the effects result- ing from genetically mutating it from the biochemistry of the complex to which it belongs or from all three. In this chapter we begin our study of how the structure of a protein gives rise to its function a theme that recurs throughout this book Figure 3-1. The first section examines how chains of amino acid building blocks are arranged and the various higher-order folded forms that the chains assume. The next section deals with special proteins that aid in the folding of proteins modifications that take place after the protein chain has been synthesized and mechanisms that de- grade proteins. The third section focuses on proteins as cat- alysts and reviews the basic properties exhibited by all enzymes. We then introduce molecular motors which con- vert chemical energy into motion. The structure and function of these functional classes of proteins and others are detailed in numerous later chapters. Various mechanisms that cells use to control the activity of proteins are covered next. The chapter concludes with a section on commonly used tech- niques in the biologist’s tool kit for isolating proteins and characterizing their properties. Hierarchical Structure of Proteins Although constructed by the polymerization of only 20 dif- ferent amino acids into linear chains proteins carry out an incredible array of diverse tasks. A protein chain folds into a unique shape that is stabilized by noncovalent interactions between regions in the linear sequence of amino acids. This spatial organization of a protein—its shape in three dimen- sions—is a key to understanding its function. Only when a protein is in its correct three-dimensional structure or con- formation is it able to function efficiently. A key concept in understanding how proteins work is that function is derived from three-dimensional structure and three-dimensional structure is specified by amino acid sequence. Here we con- sider the structure of proteins at four levels of organization starting with their monomeric building blocks the amino acids. The Primary Structure of a Protein Is Its Linear Arrangement of Amino Acids We reviewed the properties of the amino acids used in syn- thesizing proteins and their linkage by peptide bonds into lin- ear chains in Chapter 2. The repeated amide N carbon C and carbonyl C atoms of each amino acid residue form the backbone of a protein molecule from which the various side-chain groups project Figure 3-2. As a consequence of the peptide linkage the backbone exhibits directionality be- cause all the amino groups are located on the same side of the C atoms. Thus one end of a protein has a free unlinked amino group the N-terminus and the other end has a free carboxyl group the C-terminus. The sequence of a protein chain is conventionally written with its N-terminal amino acid on the left and its C-terminal amino acid on the right. 3.1 60 CHAPTER 3 • Protein Structure and Function MOLECULAR STRUCTURE Primary sequence Secondary local folding Tertiary long-range folding Quaternary multimeric organization FUNCTION Signaling Catalysis Structure Movement Regulation Transport Supramolecular large-scale assemblies "on" "off" A B a b ▲ FIGURE 3-1 Overview of protein structure and function. a The linear sequence of amino acids primary structure folds into helices or sheets secondary structure which pack into a globular or fibrous domain tertiary structure. Some individual proteins self-associate into complexes quaternary structure that can consist of tens to hundreds of subunits supramolecular assemblies. b Proteins display functions that include catalysis of chemical reactions enzymes flow of small molecules and ions transport sensing and reaction to the environment signaling control of protein activity regulation organization of the genome lipid bilayer membrane and cytoplasm structure and generation of force for movement motor proteins. These functions and others arise from specific binding interactions and conformational changes in the structure of a properly folded protein.

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The primary structure of a protein is simply the linear arrangement or sequence of the amino acid residues that compose it. Many terms are used to denote the chains formed by the polymerization of amino acids. A short chain of amino acids linked by peptide bonds and having a defined sequence is called a peptide longer chains are referred to as polypeptides. Peptides generally contain fewer than 20–30 amino acid residues whereas polypeptides contain as many as 4000 residues. We generally reserve the term protein for a polypeptide or for a complex of polypeptides that has a well-defined three-dimensional structure. It is implied that proteins and peptides are the natural products of a cell. The size of a protein or a polypeptide is reported as its mass in daltons a dalton is 1 atomic mass unit or as its mo- lecular weight MW which is a dimensionless number. For example a 10000-MW protein has a mass of 10000 daltons Da or 10 kilodaltons kDa. In the last section of this chap- ter we will consider different methods for measuring the sizes and other physical characteristics of proteins. The known and predicted proteins encoded by the yeast genome have an av- erage molecular weight of 52728 and contain on average 466 amino acid residues. The average molecular weight of amino acids in proteins is 113 taking into account their aver- age relative abundance. This value can be used to estimate the number of residues in a protein from its molecular weight or conversely its molecular weight from the number of residues. Secondary Structures Are the Core Elements of Protein Architecture The second level in the hierarchy of protein structure consists of the various spatial arrangements resulting from the fold- ing of localized parts of a polypeptide chain these arrange- ments are referred to as secondary structures. A single polypeptide may exhibit multiple types of secondary struc- ture depending on its sequence. In the absence of stabilizing noncovalent interactions a polypeptide assumes a random- coil structure. However when stabilizing hydrogen bonds form between certain residues parts of the backbone fold into one or more well-defined periodic structures: the alpha helix the beta sheet or a short U-shaped turn. In an average protein 60 percent of the polypeptide chain exist as helices and sheets the remainder of the molecule is in random coils and turns. Thus helices and sheets are the major internal supportive elements in proteins. In this sec- tion we explore forces that favor the formation of secondary structures. In later sections we examine how these structures can pack into larger arrays. The Helix In a polypeptide segment folded into an helix the carbonyl oxygen atom of each peptide bond is hydrogen- bonded to the amide hydrogen atom of the amino acid four residues toward the C-terminus. This periodic arrangement of bonds confers a directionality on the helix because all the hydrogen-bond donors have the same orientation Figure 3-3. 3.1 • Hierarchical Structure of Proteins 61 aa 1 aa 2 aa 3 Peptide bond Peptide bond R R R ▲ FIGURE 3-2 Structure of a tripeptide. Peptide bonds yellow link the amide nitrogen atom blue of one amino acid aa with the carbonyl carbon atom gray of an adjacent one in the linear polymers known as peptides or polypeptides depending on their length. Proteins are polypeptides that have folded into a defined three-dimensional structure conformation. The side chains or R groups green extending from the carbon atoms black of the amino acids composing a protein largely determine its properties. At physiological pH values the terminal amino and carboxyl groups are ionized. 3.6 residues per helical turn R R R R R R R R R R R R R ▲ FIGURE 3-3 The helix a common secondary structure in proteins. The polypeptide backbone red is folded into a spiral that is held in place by hydrogen bonds between backbone oxygen and hydrogen atoms. The outer surface of the helix is covered by the side-chain R groups green.

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62 CHAPTER 3 • Protein Structure and Function The stable arrangement of amino acids in the helix holds the backbone in a rodlike cylinder from which the side chains point outward. The hydrophobic or hydrophilic quality of the helix is determined entirely by the side chains because the polar groups of the peptide backbone are already engaged in hydrogen bonding in the helix. The Sheet Another type of secondary structure the sheet consists of laterally packed strands. Each strand is a short 5- to 8-residue nearly fully extended polypeptide segment. Hydrogen bonding between backbone atoms in adjacent strands within either the same polypeptide chain or between different polypeptide chains forms a sheet Figure 3-4a. The planarity of the peptide bond forces a sheet to be pleated hence this structure is also called a pleated sheet or simply a pleated sheet. Like helices strands have a directionality de- fined by the orientation of the peptide bond. Therefore in a pleated sheet adjacent strands can be oriented in the same parallel or opposite antiparallel directions with respect to each other. In both arrangements the side chains project from both faces of the sheet Figure 3-4b. In some proteins sheets form the floor of a binding pocket the hydrophobic core of other proteins contains multiple sheets. Turns Composed of three or four residues turns are located on the surface of a protein forming sharp bends that redirect the polypeptide backbone back toward the interior. These short U-shaped secondary structures are stabilized by a hy- drogen bond between their end residues see Figure 3-4a. Glycine and proline are commonly present in turns. The lack of a large side chain in glycine and the presence of a built-in bend in proline allow the polypeptide backbone to fold into a tight U shape. Turns allow large proteins to fold into highly compact structures. A polypeptide backbone also may con- tain longer bends or loops. In contrast with turns which ex- hibit just a few well-defined structures loops can be formed in many different ways. Overall Folding of a Polypeptide Chain Yields Its Tertiary Structure Tertiary structure refers to the overall conformation of a polypeptide chain—that is the three-dimensional arrange- ment of all its amino acid residues. In contrast with second- ary structures which are stabilized by hydrogen bonds tertiary structure is primarily stabilized by hydrophobic in- teractions between the nonpolar side chains hydrogen bonds between polar side chains and peptide bonds. These stabi- lizing forces hold elements of secondary structure— helices strands turns and random coils—compactly together. Because the stabilizing interactions are weak however the tertiary structure of a protein is not rigidly fixed but under- goes continual and minute fluctuation. This variation in structure has important consequences in the function and regulation of proteins. Different ways of depicting the conformation of proteins convey different types of information. The simplest way to represent three-dimensional structure is to trace the course of the backbone atoms with a solid line Figure 3-5a the most complex model shows every atom Figure 3-5b. The former a C trace shows the overall organization of the polypeptide chain without consideration of the amino acid side chains the latter a ball-and-stick model details the interactions be- tween side-chain atoms which stabilize the protein’s confor- mation as well as the atoms of the backbone. Even though both views are useful the elements of secondary structure are not easily discerned in them. Another type of representation uses common shorthand symbols for depicting secondary structure—for example coiled ribbons or solid cylinders for helices flat ribbons or arrows for strands and flexible a R R R R R R R R R R R R R R R R R R R R R R R R R R R b FIGURE 3-4 The sheet another common secondary structure in proteins. a T op view of a simple two-stranded sheet with antiparallel strands. The stabilizing hydrogen bonds between the strands are indicated by green dashed lines. The short turn between the strands also is stabilized by a hydrogen bond. b Side view of a sheet. The projection of the R groups green above and below the plane of the sheet is obvious in this view. The fixed angle of the peptide bond produces a pleated contour.

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thin strands for turns and loops Figure 3-5c. This type of representation makes the secondary structures of a protein easy to see. However none of these three ways of representing pro- tein structure convey much information about the protein surface which is of interest because it is where other mole- cules bind to a protein. Computer analysis can identify the surface atoms that are in contact with the watery environ- ment. On this water-accessible surface regions having a common chemical character hydrophobicity or hydrophilic- ity and electrical character basic or acidic can be mapped. Such models reveal the topography of the protein surface and the distribution of charge both important features of bind- ing sites as well as clefts in the surface where small mole- cules often bind Figure 3-5d. This view represents a protein as it is “seen” by another molecule. Motifs Are Regular Combinations of Secondary Structures Particular combinations of secondary structures called mo- tifs or folds build up the tertiary structure of a protein. In some cases motifs are signatures for a specific function. For example the helix-loop-helix is a Ca 2 -binding motif marked by the presence of certain hydrophilic residues at in- variant positions in the loop Figure 3-6a. Oxygen atoms in the invariant residues bind a Ca 2 ion through ionic bonds. This motif also called the EF hand has been found in more than 100 calcium-binding proteins. In another common motif the zinc finger three secondary structures—an helix and two strands with an antiparallel orientation—form a fingerlike bundle held together by a zinc ion Figure 3-6b. This motif is most commonly found in proteins that bind RNA or DNA. Many proteins especially fibrous proteins self-associate into oligomers by using a third motif the coiled coil. In these proteins each polypeptide chain contains -helical segments in which the hydrophobic residues although apparently randomly arranged are in a regular pattern—a repeated heptad sequence. In the heptad a hydrophobic residue— sometimes valine alanine or methionine—is at position 1 and a leucine residue is at position 4. Because hydrophilic side chains extend from one side of the helix and hydropho- bic side chains extend from the opposite side the overall hel- ical structure is amphipathic. The amphipathic character of these helices permits two three or four helices to wind around each other forming a coiled coil hence the name of this motif Figure 3-6c. We will encounter numerous additional motifs in later discussions of other proteins in this chapter and other chap- ters. The presence of the same motif in different proteins with similar functions clearly indicates that these useful 3.1 • Hierarchical Structure of Proteins 63 a C α backbone trace b Ball and stick c Ribbons d Solvent-accessible surface FIGURE 3-5 Various graphic representations of the structure of Ras a monomeric guanine nucleotide-binding protein. The inactive guanosine diphosphate GDP–bound form is shown in all four panels with GDP always depicted in blue spacefill. a The C backbone trace demonstrates how the polypeptide is packed into the smallest possible volume. b A ball-and-stick representation reveals the location of all atoms. c A ribbon representation emphasizes how strands blue and helices red are organized in the protein. Note the turns and loops connecting pairs of helices and strands. d A model of the water-accessible surface reveals the numerous lumps bumps and crevices on the protein surface. Regions of positive charge are shaded blue regions of negative charge are shaded red.

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64 CHAPTER 3 • Protein Structure and Function combinations of secondary structures have been conserved in evolution. To date hundreds of motifs have been cataloged and proteins are now classified according to their motifs. Structural and Functional Domains Are Modules of Tertiary Structure The tertiary structure of proteins larger than 15000 MW is typically subdivided into distinct regions called domains. Structurally a domain is a compactly folded region of polypeptide. For large proteins domains can be recognized in structures determined by x-ray crystallography or in im- ages captured by electron microscopy. Although these dis- crete regions are well distinguished or physically separated from one another they are connected by intervening seg- ments of the polypeptide chain. Each of the subunits in hemagglutinin for example contains a globular domain and a fibrous domain Figure 3-7a. A structural domain consists of 100–150 residues in var- ious combinations of motifs. Often a domain is characterized by some interesting structural feature: an unusual abundance of a particular amino acid e.g. a proline-rich domain an acidic domain sequences common to conserved in many proteins e.g. SH3 or Src homology region 3 or a particu- lar secondary-structure motif e.g. zinc-finger motif in the kringle domain. Domains are sometimes defined in functional terms on the basis of observations that an activity of a protein is lo- calized to a small region along its length. For instance a par- ticular region or regions of a protein may be responsible for its catalytic activity e.g. a kinase domain or binding ability e.g. a DNA-binding domain a membrane-binding domain. Functional domains are often identified experimentally by whittling down a protein to its smallest active fragment with the aid of proteases enzymes that cleave the polypeptide backbone. Alternatively the DNA encoding a protein can be Leu 4 Leu 4 Leu 4 Leu 4 Val 1 Asn 1 Val 1 His His Cys Cys N C NN C C N C Asp Asp Asn Thr Glu H 2 O Ca 2+ Zn 2+ a Helix-loop-helix motif c Coiled coil motif b Zinc-finger motif Consensus sequence: D/N - D/N - D/N/S - backbone O - - - - E/D Consensus sequence: F/Y - C - - C - - - - F/Y - - - - - - - - H - - - H - Heptad repeat: V/N/M - - L - - - ▲ FIGURE 3-6 Motifs of protein secondary structure. a Two helices connected by a short loop in a specific conformation constitute a helix-loop-helix motif. This motif exists in many calcium-binding and DNA-binding regulatory proteins. In calcium-binding proteins such as calmodulin oxygen atoms from five loop residues and one water molecule form ionic bonds with a Ca 2 ion. b The zinc-finger motif is present in many DNA-binding proteins that help regulate transcription. A Zn 2 ion is held between a pair of strands blue and a single helix red by a pair of cysteine residues and a pair of histidine residues. The two invariant cysteine residues are usually at positions 3 and 6 and the two invariant histidine residues are at positions 20 and 24 in this 25-residue motif. c The parallel two-stranded coiled-coil motif found in the transcription factor Gcn4 is characterized by two helices wound around one another. Helix packing is stabilized by interactions between hydrophobic side chains red and blue present at regular intervals along the surfaces of the intertwined helices. Each helix exhibits a characteristic heptad repeat sequence with a hydrophobic residue at positions 1 and 4. See A. Lewit-Bentley and S. Rety 2000 EF-hand calcium-binding proteins Curr. Opin. Struct. Biol. 10:637–643 S. A. Wolfe L. Nekludova and C. O. Pabo 2000 DNA recognition by Cys2His2 zinc finger proteins Ann. Rev. Biophys. Biomol. Struct. 29:183–212.

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subjected to mutagenesis so that segments of the protein’s backbone are removed or changed. The activity of the trun- cated or altered protein product synthesized from the mu- tated gene is then monitored and serves as a source of insight about which part of a protein is critical to its function. The organization of large proteins into multiple do- mains illustrates the principle that complex molecules are built from simpler components. Like motifs of secondary structure domains of tertiary structure are incorporated as modules into different proteins. In Chapter 10 we consider the mechanism by which the gene segments that correspond to domains became shuffled in the course of evolution re- sulting in their appearance in many proteins. The modular approach to protein architecture is particularly easy to rec- ognize in large proteins which tend to be mosaics of dif- ferent domains and thus can perform different functions simultaneously. The epidermal growth factor EGF domain is one exam- ple of a module that is present in several proteins Figure 3-8. EGF is a small soluble peptide hormone that binds to cells in the embryo and in skin and connective tissue in adults caus- ing them to divide. It is generated by proteolytic cleavage be- tween repeated EGF domains in the EGF precursor protein which is anchored in the cell membrane by a membrane- spanning domain. EGF modules are also present in other proteins and are liberated by proteolysis these proteins in- clude tissue plasminogen activator TPA a protease that is used to dissolve blood clots in heart attack victims Neu protein which takes part in embryonic differentiation and Notch protein a receptor protein in the plasma mem- brane that functions in developmentally important signaling Chapter 14. Besides the EGF domain these proteins con- tain domains found in other proteins. For example TPA pos- sesses a trypsin domain a common feature in enzymes that degrade proteins. 3.1 • Hierarchical Structure of Proteins 65 PROXIMAL C HA 1 N HA 2 N Globular domain Fibrous domain DISTAL a Viral membrane b Sialic acid FIGURE 3-7 Tertiary and quaternary levels of structure in hemagglutinin HA a surface protein on influenza virus. This long multimeric molecule has three identical subunits each composed of two polypeptide chains HA 1 and HA 2 . a Tertiary structure of each HA subunit constitutes the folding of its helices and strands into a compact structure that is 13.5 nm long and divided into two domains. The membrane-distal domain is folded into a globular conformation. The membrane-proximal domain has a fibrous stemlike conformation owing to the alignment of two long helices cylinders of HA 2 with strands in HA 1 . Short turns and longer loops which usually lie at the surface of the molecule connect the helices and strands in a given chain. b Quaternary structure of HA is stabilized by lateral interactions between the long helices cylinders in the fibrous domains of the three subunits yellow blue and green forming a triple-stranded coiled- coil stalk. Each of the distal globular domains in HA binds sialic acid red on the surface of target cells. Like many membrane proteins HA contains several covalently linked carbohydrate chains not shown. EGF Neu TPA EGF precursor ▲ FIGURE 3-8 Schematic diagrams of various proteins illustrating their modular nature. Epidermal growth factor EGF is generated by proteolytic cleavage of a precursor protein containing multiple EGF domains green and a membrane- spanning domain blue. The EGF domain is also present in Neu protein and in tissue plasminogen activator TPA. These proteins also contain other widely distributed domains indicated by shape and color. Adapted from I. D. Campbell and P . Bork 1993 Curr. Opin. Struct. Biol. 3:385.

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66 CHAPTER 3 • Protein Structure and Function Proteins Associate into Multimeric Structures and Macromolecular Assemblies Multimeric proteins consist of two or more polypeptides or subunits. A fourth level of structural organization quaternary structure describes the number stoichiometry and relative positions of the subunits in multimeric proteins. Hemagglu- tinin for example is a trimer of three identical subunits held together by noncovalent bonds Figure 3-7b. Other multi- meric proteins can be composed of any number of identical or different subunits. The multimeric nature of many proteins is critical to mechanisms for regulating their function. In ad- dition enzymes in the same pathway may be associated as subunits of a large multimeric protein within the cell thereby increasing the efficiency of pathway operation. The highest level of protein structure is the association of proteins into macromolecular assemblies. Typically such structures are very large exceeding 1 mDa in mass ap- proaching 30–300 nm in size and containing tens to hun- dreds of polypeptide chains as well as nucleic acids in some cases. Macromolecular assemblies with a structural function include the capsid that encases the viral genome and bundles of cytoskeletal filaments that support and give shape to the plasma membrane. Other macromolecular assemblies act as molecular machines carrying out the most complex cellular processes by integrating individual functions into one coor- dinated process. For example the transcriptional machine that initiates the synthesis of messenger RNA mRNA con- sists of RNA polymerase itself a multimeric protein and at least 50 additional components including general transcrip- tion factors promoter-binding proteins helicase and other protein complexes Figure 3-9. The transcription factors and promoter-binding proteins correctly position a poly- merase molecule at a promoter the DNA site that determines where transcription of a specific gene begins. After helicase unwinds the double-stranded DNA molecule polymerase si- multaneously translocates along the DNA template strand and synthesizes an mRNA strand. The operational details of this complex machine and of others listed in Table 3-1 are discussed elsewhere. TABLE 3-1 Selected Molecular Machines Machine Main Components Cellular Location Function Replisome 4 Helicase primase DNA polymerase Nucleus DNA replication Transcription initiation Promoter-binding protein helicase Nucleus RNA synthesis complex 11 general transcription factors TFs RNA polymerase large multisubunit mediator complex Spliceosome 12 Pre-mRNA small nuclear RNAs Nucleus mRNA splicing snRNAs protein factors Nuclear pore Nucleoporins 50–100 Nuclear membrane Nuclear import complex 12 and export Ribosome 4 Ribosomal proteins 50 and four Cytoplasm/ER membrane Protein synthesis rRNA molecules eukaryotes organized into large and small subunits associated mRNA and protein factors IFs EFs Chaperonin 3 GroEL GroES bacteria Cytoplasm Protein folding mitochondria endoplasmic reticulum Proteasome 3 Core proteins regulatory cap proteins Cytoplasm Protein degradation Photosystem 8 Light-harvesting complex multiple Thylakoid membrane Photosynthesis proteins and pigments reaction center in plant chloroplasts initial stage multisubunit protein with associated plasma membrane of pigments and electron carriers photosynthetic bacteria MAP kinase Scaffold protein multiple different Cytoplasm Signal transduction cascades 14 protein kinases Sarcomere 19 Thick myosin filaments thin actin Cytoplasm of Contraction filaments Z lines titin/nebulin muscle cells Numbers in parentheses indicate chapters in which various machines are discussed.

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Members of Protein Families Have a Common Evolutionary Ancestor Studies on myoglobin and hemoglobin the oxygen-carrying proteins in muscle and blood respectively provided early ev- idence that function derives from three-dimensional struc- ture which in turn is specified by amino acid sequence. X-ray crystallographic analysis showed that the three- dimensional structures of myoglobin and the and sub- units of hemoglobin are remarkably similar. Subsequent se- quencing of myoglobin and the hemoglobin subunits revealed that many identical or chemically similar residues are found in identical positions throughout the primary structures of both proteins. Similar comparisons between other proteins conclusively confirmed the relation between the amino acid sequence three-dimensional structure and function of proteins. This principle is now commonly employed to predict on the basis of sequence comparisons with proteins of known structure and function the structure and function of pro- teins that have not been isolated Chapter 9. This use of sequence comparisons has expanded substantially in recent years as the genomes of more and more organisms have been sequenced. The molecular revolution in biology during the last decades of the twentieth century also created a new scheme 3.1 • Hierarchical Structure of Proteins 67 HEMOGLOBIN MYOGLOBIN Monocot hemoglobin Dicot hemoglobin Vertebrate αβ Annelid Insect Nematode Fungal Protozoan Algal Bacterial Ancestral oxygen-binding protein Leghemoglobin Myoglobin Hemoglobin α α β β subunit of hemoglobin β LEGHEMOGLOBIN ▲ FIGURE 3-10 Evolution of the globin protein family. Left A primitive monomeric oxygen-binding globin is thought to be the ancestor of modern-day blood hemoglobins muscle myoglobins and plant leghemoglobins. Sequence comparisons have revealed that evolution of the globin proteins parallels the evolution of animals and plants. Major junctions occurred with the divergence of plant globins from animal globins and of myoglobin from hemoglobin. Later gene duplication gave rise to the and subunits of hemoglobin. Right Hemoglobin is a tetramer of two and two subunits. The structural similarity of these subunits with leghemoglobin and myoglobin both of which are monomers is evident. A heme molecule red noncovalently associated with each globin polypeptide is the actual oxygen- binding moiety in these proteins. Left Adapted from R. C. Hardison 1996 Proc. Natl. Acad. Sci. USA 93:5675. General transcription factors Transcription preinitiation complex Mediator complex RNA polymerase ++ Promoter DNA ▲ FIGURE 3-9 The mRNA transcription-initiation machinery. The core RNA polymerase general transcription factors a mediator complex containing about 20 subunits and other protein complexes not depicted here assemble at a promoter in DNA. The polymerase carries out transcription of DNA the associated proteins are required for initial binding of polymerase to a specific promoter thereby initiating transcription.

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68 CHAPTER 3 • Protein Structure and Function of biological classification based on similarities and differ- ences in the amino acid sequences of proteins. Proteins that have a common ancestor are referred to as homologs. The main evidence for homology among proteins and hence their common ancestry is similarity in their sequences or structures. We can therefore describe homologous proteins as belonging to a “family” and can trace their lineage from comparisons of their sequences. The folded three-dimen- sional structures of homologous proteins are similar even if parts of their primary structure show little evidence of homology. The kinship among homologous proteins is most easily visualized by a tree diagram based on sequence analyses. For example the amino acid sequences of globins from bacteria plants and animals suggest that they evolved from an an- cestral monomeric oxygen-binding protein Figure 3-10. With the passage of time the gene for this ancestral protein slowly changed initially diverging into lineages leading to animal and plant globins. Subsequent changes gave rise to myoglobin a monomeric oxygen-storing protein in muscle and to the and subunits of the tetrameric hemoglobin molecule 2 2 of the circulatory system. KEY CONCEPTS OF SECTION 3.1 Hierarchical Structure of Proteins ■ A protein is a linear polymer of amino acids linked together by peptide bonds. Various mostly noncovalent interactions between amino acids in the linear sequence stabilize a specific folded three-dimensional structure con- formation for each protein. ■ The helix strand and sheet and turn are the most prevalent elements of protein secondary structure which is stabilized by hydrogen bonds between atoms of the pep- tide backbone. ■ Certain combinations of secondary structures give rise to different motifs which are found in a variety of pro- teins and are often associated with specific functions see Figure 3-6. ■ Protein tertiary structure results from hydrophobic in- teractions between nonpolar side groups and hydrogen bonds between polar side groups that stabilize folding of the secondary structure into a compact overall arrange- ment or conformation. ■ Large proteins often contain distinct domains independ- ently folded regions of tertiary structure with characteristic structural or functional properties or both see Figure 3-7. ■ The incorporation of domains as modules in different proteins in the course of evolution has generated diversity in protein structure and function. ■ Quaternary structure encompasses the number and or- ganization of subunits in multimeric proteins. ■ Cells contain large macromolecular assemblies in which all the necessary participants in complex cellular processes e.g. DNA RNA and protein synthesis photosynthesis signal transduction are integrated to form molecular ma- chines see Table 3-1. ■ The sequence of a protein determines its three-dimensional structure which determines its function. In short function derives from structure structure derives from sequence. ■ Homologous proteins which have similar sequences structures and functions evolved from a common ancestor. Folding Modification and Degradation of Proteins A polypeptide chain is synthesized by a complex process called translation in which the assembly of amino acids in a particu- lar sequence is dictated by messenger RNA mRNA. The in- tricacies of translation are considered in Chapter 4. Here we describe how the cell promotes the proper folding of a na- scent polypeptide chain and in many cases modifies residues or cleaves the polypeptide backbone to generate the final pro- tein. In addition the cell has error-checking processes that eliminate incorrectly synthesized or folded proteins. Incor- rectly folded proteins usually lack biological activity and in some cases may actually be associated with disease. Protein misfolding is suppressed by two distinct mechanisms. First cells have systems that reduce the chances for misfolded pro- teins to form. Second any misfolded proteins that do form as well as cytosolic proteins no longer needed by a cell are de- graded by a specialized cellular garbage-disposal system. The Information for Protein Folding Is Encoded in the Sequence Any polypeptide chain containing n residues could in prin- ciple fold into 8 n conformations. This value is based on the fact that only eight bond angles are stereochemically allowed in the polypeptide backbone. In general however all mole- cules of any protein species adopt a single conformation called the native state for the vast majority of proteins the native state is the most stably folded form of the molecule. What guides proteins to their native folded state The an- swer to this question initially came from in vitro studies on protein refolding. Thermal energy from heat extremes of pH that alter the charges on amino acid side chains and chemi- cals such as urea or guanidine hydrochloride at concentra- tions of 6–8 M can disrupt the weak noncovalent interactions that stabilize the native conformation of a protein. The denaturation resulting from such treatment causes a protein to lose both its native conformation and its biological activity. Many proteins that are completely unfolded in 8 M urea and -mercaptoethanol which reduces disulfide bonds spon- taneously renature refold into their native states when the de- naturing reagents are removed by dialysis. Because no cofactors 3.2

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or other proteins are required in vitro protein folding is a self- directed process. In other words sufficient information must be contained in the protein’s primary sequence to direct cor- rect refolding. The observed similarity in the folded three- dimensional structures of proteins with similar amino acid sequences noted in Section 3.1 provided other evidence that the primary sequence also determines protein folding in vivo. Folding of Proteins in Vivo Is Promoted by Chaperones Although protein folding occurs in vitro only a minority of unfolded molecules undergo complete folding into the native conformation within a few minutes. Clearly cells require a faster more efficient mechanism for folding proteins into their correct shapes otherwise cells would waste much en- ergy in the synthesis of nonfunctional proteins and in the degradation of misfolded or unfolded proteins. Indeed more than 95 percent of the proteins present within cells have been shown to be in their native conformation despite high pro- tein concentrations 200–300 mg/ml which favor the pre- cipitation of proteins in vitro. The explanation for the cell’s remarkable efficiency in promoting protein folding probably lies in chaperones a class of proteins found in all organisms from bacteria to hu- mans. Chaperones are located in every cellular compartment bind a wide range of proteins and function in the general protein-folding mechanism of cells. Two general families of chaperones are reconized: ■ Molecular chaperones which bind and stabilize un- folded or partly folded proteins thereby preventing these proteins from aggregating and being degraded ■ Chaperonins which directly facilitate the folding of proteins Molecular chaperones consist of Hsp70 and its homologs: Hsp70 in the cytosol and mitochondrial matrix BiP in the en- doplasmic reticulum and DnaK in bacteria. First identified by their rapid appearance after a cell has been stressed by heat shock Hsp70 and its homologs are the major chaperones in all organisms. Hsc70 is a constitutively expressed homolog of Hsp70. When bound to ATP Hsp70-like proteins assume an open form in which an exposed hydrophobic pocket tran- siently binds to exposed hydrophobic regions of the unfolded target protein. Hydrolysis of the bound ATP causes molecu- lar chaperones to assume a closed form in which a target pro- tein can undergo folding. The exchange of ATP for ADP releases the target protein Figure 3-11a top. This cycle is 3.2 • Folding Modification and Degradation of Proteins 69 a Ribosome Partially folded protein ATP ADP b GroEL "tight" conformation GroEL "relaxed" conformation GroEL GroES ADP + P i Properly folded protein Properly folded protein Protein Protein P i ATP Hsp 70-ATP ▲ FIGURE 3-11 Chaperone- and chaperonin-mediated protein folding. a Many proteins fold into their proper three- dimensional structures with the assistance of Hsp70-like proteins top. These molecular chaperones transiently bind to a nascent polypeptide as it emerges from a ribosome. Proper folding of other proteins bottom depends on chaperonins such as the prokaryotic GroEL a hollow barrel-shaped complex of 14 identical 60000-MW subunits arranged in two stacked rings. One end of GroEL is transiently blocked by the co- chaperonin GroES an assembly of 10000-MW subunits. b In the absence of ATP or presence of ADP GroEL exists in a “tight” conformational state that binds partly folded or misfolded proteins. Binding of ATP shifts GroEL to a more open “relaxed” state which releases the folded protein. See text for details. Part b from A. Roseman et al. 1996 Cell 87:241 courtesy of H. Saibil. MEDIA CONNECTIONS Focus Animation: Chaperone-Mediated Folding

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70 CHAPTER 3 • Protein Structure and Function speeded by the co-chaperone Hsp40 in eukaryotes. In bacteria an additional protein called GrpE also interacts with DnaK promoting the exchange of ATP for the bacterial co-chaperone DnaJ and possibly its dissociation. Molecular chaperones are thought to bind all nascent polypeptide chains as they are being synthesized on ribosomes. In bacteria 85 percent of the proteins are released from their chaperones and proceed to fold normally an even higher percentage of proteins in eu- karyotes follow this pathway. The proper folding of a large variety of newly synthesized or translocated proteins also requires the assistance of chap- eronins. These huge cylindrical macromolecular assemblies are formed from two rings of oligomers. The eukaryotic chaper- onin TriC consists of eight subunits per ring. In the bacterial mitochondrial and chloroplast chaperonin known as GroEL each ring contains seven identical subunits Figure 3-11b. The GroEL folding mechanism which is better understood than TriC-mediated folding serves as a general model Figure 3-11a bottom. In bacteria a partly folded or misfolded polypeptide is inserted into the cavity of GroEL where it binds to the inner wall and folds into its native conformation. In an ATP-dependent step GroEL undergoes a conformational change and releases the folded protein a process assisted by a co-chaperonin GroES which caps the ends of GroEL. Many Proteins Undergo Chemical Modification of Amino Acid Residues Nearly every protein in a cell is chemically modified after its synthesis on a ribosome. Such modifications which may alter the activity life span or cellular location of proteins entail the linkage of a chemical group to the free –NH 2 or –COOH group at either end of a protein or to a reactive side- chain group in an internal residue. Although cells use the 20 amino acids shown in Figure 2-13 to synthesize proteins analysis of cellular proteins reveals that they contain upward of 100 different amino acids. Chemical modifications after synthesis account for this difference. Acetylation the addition of an acetyl group CH 3 CO to the amino group of the N-terminal residue is the most com- mon form of chemical modification affecting an estimated 80 percent of all proteins: This modification may play an important role in controlling the life span of proteins within cells because nonacetylated proteins are rapidly degraded by intracellular proteases. Residues at or near the termini of some membrane proteins are chemically modified by the addition of long lipidlike groups. The attachment of these hydrophobic “tails” which function to anchor proteins to the lipid bilayer constitutes one way that cells localize certain proteins to membranes Chapter 5. Acetyl groups and a variety of other chemical groups can also be added to specific internal residues in proteins Fig- ure 3-12. An important modification is the phosphorylation of serine threonine tyrosine and histidine residues. We will encounter numerous examples of proteins whose activity is regulated by reversible phosphorylation and dephosphory- lation. The side chains of asparagine serine and threonine are sites for glycosylation the attachment of linear and branched carbohydrate chains. Many secreted proteins and membrane proteins contain glycosylated residues the syn- thesis of such proteins is described in Chapters 16 and 17. Other post-translational modifications found in selected pro- teins include the hydroxylation of proline and lysine residues in collagen the methylation of histidine residues in mem- brane receptors and the -carboxylation of glutamate in prothrombin an essential blood-clotting factor. A special modification discussed shortly marks cytosolic proteins for degradation. Peptide Segments of Some Proteins Are Removed After Synthesis After their synthesis some proteins undergo irreversible changes that do not entail changes in individual amino acid residues. This type of post-translational alteration is some- times called processing. The most common form is enzymatic cleavage of a backbone peptide bond by proteases resulting in the removal of residues from the C- or N-terminus of a NH 2 H 2 C H 2 C OH CH CH COO 3-Hydroxyproline H HC H 3 C CH 2 NH 3 CH C C NN COO 3-Methylhistidine CH 2 NH 3 CH CH COO OOC OOC -Carboxyglutamate NCH 2 NH 3 CH CH 2 CH 2 COO CH 2 Acetyl lysine CH 3 C O P O − O O − CH 2 NH 3 CH COO Phosphoserine O ▲ FIGURE 3-12 Common modifications of internal amino acid residues found in proteins. These modified residues and numerous others are formed by addition of various chemical groups red to the amino acid side chains after synthesis of a polypeptide chain. N C CH 3 O C C H H R O Acetylated N-terminus

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polypeptide chain. Proteolytic cleavage is a common mecha- nism for activating enzymes that function in blood coagula- tion digestion and programmed cell death Chapter 22. Proteolysis also generates active peptide hormones such as EGF and insulin from larger precursor polypeptides. An unusual and rare type of processing termed protein self-splicing takes place in bacteria and some eukaryotes. This process is analogous to editing film: an internal segment of a polypeptide is removed and the ends of the polypeptide are rejoined. Unlike proteolytic processing protein self- splicing is an autocatalytic process which proceeds by itself without the participation of enzymes. The excised peptide appears to eliminate itself from the protein by a mechanism similar to that used in the processing of some RNA mole- cules Chapter 12. In vertebrate cells the processing of some proteins includes self-cleavage but the subsequent ligation step is absent. One such protein is Hedgehog a membrane- bound signaling molecule that is critical to a number of de- velopmental processes Chapter 15. Ubiquitin Marks Cytosolic Proteins for Degradation in Proteasomes In addition to chemical modifications and processing the ac- tivity of a cellular protein depends on the amount present which reflects the balance between its rate of synthesis and rate of degradation in the cell. The numerous ways that cells regulate protein synthesis are discussed in later chapters. In this section we examine protein degradation focusing on the major pathways for degrading cytosolic proteins. The life span of intracellular proteins varies from as short as a few minutes for mitotic cyclins which help regulate pas- sage through mitosis to as long as the age of an organism for proteins in the lens of the eye. Eukaryotic cells have several intracellular proteolytic pathways for degrading misfolded or denatured proteins normal proteins whose concentration must be decreased and extracellular proteins taken up by the cell. One major intracellular pathway is degradation by en- zymes within lysosomes membrane-limited organelles whose acidic interior is filled with hydrolytic enzymes. Lysosomal degradation is directed primarily toward extracellular pro- teins taken up by the cell and aged or defective organelles of the cell see Figure 5-20. Distinct from the lysosomal pathway are cytosolic mecha- nisms for degrading proteins. Chief among these mechanisms is a pathway that includes the chemical modification of a ly- sine side chain by the addition of ubiquitin a 76-residue polypeptide followed by degradation of the ubiquitin-tagged protein by a specialized proteolytic machine. Ubiquitination is a three-step process Figure 3-13a: ■ Activation of ubiquitin-activating enzyme E1 by the addition of a ubitiquin molecule a reaction that requires ATP ■ Transfer of this ubiquitin molecule to a cysteine residue in ubiquitin-conjugating enzyme E2 ■ Formation of a peptide bond between the ubiquitin molecule bound to E2 and a lysine residue in the target protein a reaction catalyzed by ubiquitin ligase E3 This process is repeated many times with each subsequent ubiquitin molecule being added to the preceding one. The re- sulting polyubiquitin chain is recognized by a proteasome another of the cell’s molecular machines Figure 3-13b. The numerous proteasomes dispersed throughout the cell cytosol proteolytically cleave ubiquitin-tagged proteins in an ATP- dependent process that yields short 7- to 8-residue peptides and intact ubiquitin molecules. 3.2 • Folding Modification and Degradation of Proteins 71 a Cytosolic target protein Ub Ub Ub Ub n NH 2 Ub Ub Ub Proteasome Peptides Ub E1 Ub Ub E1 C O E2 C O + ATP E2 E1 AMP + PP i E3 E2 12 3 4 5 Steps 1 2 3 n times b Cap Core Cap E1 Ubiquitin-activating enzyme E2 Ubiquitin-conjugating enzyme E3 Ubiquitin ligase Ub Ubiquitin ATP ADP C O NH ▲ FIGURE 3-13 Ubiquitin-mediated proteolytic pathway. a Enzyme E1 is activated by attachment of a ubiquitin Ub molecule step and then transfers this Ub molecule to E2 step . Ubiquitin ligase E3 transfers the bound Ub molecule on E2 to the side-chain —NH 2 of a lysine residue in a target protein step . Additional Ub molecules are added to the target protein by repeating steps – forming a polyubiquitin chain that directs the tagged protein to a proteasome step . Within this large complex the protein is cleaved into numerous small peptide fragments step . b Computer-generated image reveals that a proteasome has a cylindrical structure with a cap at each end of a core region. Proteolysis of ubiquitin-tagged proteins occurs along the inner wall of the core. Part b from W. Baumeister et al. 1998 Cell 92:357 courtesy of W. Baumeister. 5 4 3 1 3 2 1 MEDIA CONNECTIONS Overview Animation: Life Cycle of a Protein

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72 CHAPTER 3 • Protein Structure and Function Cellular proteins degraded by the ubiquitin-mediated pathway fall into one of two general categories: 1 native cy- tosolic proteins whose life spans are tightly controlled and 2 proteins that become misfolded in the course of their syn- thesis in the endoplasmic reticulum ER. Both contain se- quences recognized by the ubiquitinating enzyme complex. The cyclins for example are cytosolic proteins whose amounts are tightly controlled throughout the cell cycle. These proteins contain the internal sequence Arg-X-X-Leu- Gly-X-Ile-Gly-Asp/Asn X can be any amino acid which is recognized by specific ubiquitinating enzyme complexes. At a specific time in the cell cycle each cyclin is phosphorylated by a cyclin kinase. This phosphorylation is thought to cause a conformational change that exposes the recognition se- quence to the ubiquitinating enzymes leading to degradation of the tagged cyclin Chapter 21. Similarly the misfolding of proteins in the endoplasmic reticulum exposes hydrophobic sequences normally buried within the folded protein. Such proteins are transported to the cytosol where ubiquitinat- ing enzymes recognize the exposed hydrophobic sequences. The immune system also makes use of the ubiquitin- mediated pathway in the response to altered self-cells par- ticularly virus-infected cells. Viral proteins within the cytosol of infected cells are ubiquitinated and then degraded in pro- teasomes specially designed for this role. The resulting anti- genic peptides are transported to the endoplasmic reticulum where they bind to class I major histocompatibility complex MHC molecules within the ER membrane. Subsequently the peptide-MHC complexes move to the cell membrane where the antigenic peptides can be recognized by cytotoxic T lymphocytes which mediate the destruction of the infected cells. Digestive Proteases Degrade Dietary Proteins The major extracellular pathway for protein degradation is the system of digestive proteases that breaks down ingested pro- teins into peptides and amino acids in the intestinal tract. Three classes of proteases function in digestion. Endoproteases attack selected peptide bonds within a polypeptide chain. The principal endoproteases are pepsin which preferentially cleaves the backbone adjacent to phenylalanine and leucine residues and trypsin and chymotrypsin which cleave the backbone adjacent to basic and aromatic residues. Exopepti- dases sequentially remove residues from the N-terminus aminopeptidases or C-terminus carboxypeptidases of a protein. Peptidases split oligopeptides containing as many as about 20 amino acids into di- and tripeptides and individual amino acids. These small molecules are then transported across the intestinal lining into the bloodstream. To protect a cell from degrading itself endoproteases and carboxypeptidases are synthesized and secreted as inactive forms zymogens: pepsin by chief cells in the lining of the stomach the others by pancreatic cells. Proteolytic cleavage of the zymogens within the gastic or intestinal lumen yields the active enzymes. Intestinal epithelial cells produce aminopeptidases and the di- and tripeptidases. Alternatively Folded Proteins Are Implicated in Slowly Developing Diseases As noted earlier each protein species normally folds into a single energetically favorable conformation that is specified by its amino acid sequence. Recent evidence suggests however that a protein may fold into an al- ternative three-dimensional structure as the result of muta- tions inappropriate post-translational modification or other as-yet-unidentified reasons. Such “misfolding” not only leads to a loss of the normal function of the protein but also marks it for proteolytic degradation. The subsequent accumulation of proteolytic fragments contributes to certain degenerative diseases characterized by the presence of insoluble protein plaques in various organs including the liver and brain. ❚ Some neurodegenerative diseases including Alzheimer’s disease and Parkinson’s disease in humans and transmissible spongiform encephalopathy “mad cow” disease in cows 20 m a 100 nm b ▲ EXPERIMENTAL FIGURE 3-14 Alzheimer’s disease is characterized by the formation of insoluble plaques composed of amyloid protein. a At low resolution an amyloid plaque in the brain of an Alzheimer’s patient appears as a tangle of filaments. b The regular structure of filaments from plaques is revealed in the atomic force microscope. Proteolysis of the naturally occurring amyloid precursor protein yields a short fragment called -amyloid protein that for unknown reasons changes from an -helical to a -sheet conformation. This alternative structure aggregates into the highly stable filaments amyloid found in plaques. Similar pathologic changes in other proteins cause other degenerative diseases. Courtesy of K. Kosik.

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and sheep are marked by the formation of tangled filamen- tous plaques in a deteriorating brain Figure 3-14. The amy- loid filaments composing these structures derive from abundant natural proteins such as amyloid precursor pro- tein which is embedded in the plasma membrane Tau a microtubule-binding protein and prion protein an “infec- tious” protein whose inheritance follows Mendelian genetics. Influenced by unknown causes these helix–containing pro- teins or their proteolytic fragments fold into alternative sheet–containing structures that polymerize into very stable filaments. Whether the extracellular deposits of these fila- ments or the soluble alternatively folded proteins are toxic to the cell is unclear. KEY CONCEPTS OF SECTION 3.2 Folding Modification and Degradation of Proteins ■ The amino acid sequence of a protein dictates its fold- ing into a specific three-dimensional conformation the na- tive state. ■ Protein folding in vivo occurs with assistance from mo- lecular chaperones Hsp70 proteins which bind to nas- cent polypeptides emerging from ribosomes and prevent their misfolding see Figure 3-11. Chaperonins large com- plexes of Hsp60-like proteins shelter some partly folded or misfolded proteins in a barrel-like cavity providing ad- ditional time for proper folding. ■ Subsequent to their synthesis most proteins are modi- fied by the addition of various chemical groups to amino acid residues. These modifications which alter protein structure and function include acetylation hydroxylation glycosylation and phosphorylation. ■ The life span of intracellular proteins is largely deter- mined by their susceptibility to proteolytic degradation by various pathways. ■ Viral proteins produced within infected cells normal cy- tosolic proteins and misfolded proteins are marked for de- struction by the covalent addition of a polyubiquitin chain and then degraded within proteasomes large cylindrical complexes with multiple proteases in their interiors see Figure 3-13. ■ Some neurodegenerative diseases are caused by aggre- gates of proteins that are stably folded in an alternative conformation. Enzymes and the Chemical Work of Cells Proteins are designed to bind every conceivable molecule— from simple ions and small metabolites sugars fatty acids to large complex molecules such as other proteins and nucleic acids. Indeed the function of nearly all proteins depends on their ability to bind other molecules or ligands with a high 3.3 degree of specificity. For instance an enzyme must first bind specifically to its target molecule which may be a small mole- cule e.g. glucose or a macromolecule before it can execute its specific task. Likewise the many different types of hor- mone receptors on the surface of cells display a high degree of sensitivity and discrimination for their ligands. And as we will examine in Chapter 11 the binding of certain regulatory proteins to specific sequences in DNA is a major mechanism for controlling genes. Ligand binding often causes a change in the shape of a protein. Ligand-driven conformational changes are integral to the mechanism of action of many proteins and are important in regulating protein activity. After consider- ing the general properties of protein–ligand binding we take a closer look at how enzymes are designed to function as the cell’s chemists. Specificity and Affinity of Protein–Ligand Binding Depend on Molecular Complementarity Two properties of a protein characterize its interaction with ligands. Specificity refers to the ability of a protein to bind one molecule in preference to other molecules. Affinity refers to the strength of binding. The K d for a protein– ligand complex which is the inverse of the equilibrium con- stant K eq for the binding reaction is the most common quantitative measure of affinity Chapter 2. The stronger the interaction between a protein and ligand the lower the value of K d . Both the specificity and the affinity of a protein for a ligand depend on the structure of the ligand-binding site which is designed to fit its partner like a mold. For high-affinity and highly specific interactions to take place the shape and chemical surface of the binding site must be complementary to the ligand molecule a property termed molecular complementarity. The ability of proteins to distinguish different molecules is perhaps most highly developed in the blood proteins called antibodies which animals produce in response to antigens such as infectious agents e.g. a bacterium or a virus and certain foreign substances e.g. proteins or polysaccharides in pollens. The presence of an antigen causes an organism to make a large quantity of different antibody proteins each of which may bind to a slightly different region or epitope of the antigen. Antibodies act as specific sensors for antigens forming antibody–antigen complexes that initiate a cascade of protective reactions in cells of the immune system. All antibodies are Y-shaped molecules formed from two identical heavy chains and two identical light chains Figure 3-15a. Each arm of an antibody molecule contains a single light chain linked to a heavy chain by a disulfide bond. Near the end of each arm are six highly variable loops called complementarity-determining regions CDRs which form the antigen-binding sites. The sequences of the six loops are highly variable among antibodies making them specific for different antigens. The interaction between an antibody and an epitope in an antigen is complementary in all cases that is the surface of the antibody’s antigen-binding site physically matches the corresponding epitope like a glove 3.3 • Enzymes and the Chemical Work of Cells 73

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74 CHAPTER 3 • Protein Structure and Function Figure 3-15b. The intimate contact between these two sur- faces stabilized by numerous noncovalent bonds is respon- sible for the exquisite binding specificity exhibited by an antibody. The specificity of antibodies is so precise that they can distinguish between the cells of individual members of a species and in some cases can distinguish between proteins that differ by only a single amino acid. Because of their speci- ficity and the ease with which they can be produced anti- bodies are highly useful reagents in many of the experiments discussed in subsequent chapters. Enzymes Are Highly Efficient and Specific Catalysts In contrast with antibodies which bind and simply present their ligands to other components of the immune system en- zymes promote the chemical alteration of their ligands called substrates. Almost every chemical reaction in the cell is catalyzed by a specific enzyme. Like all catalysts enzymes do not affect the extent of a reaction which is determined by the change in free energy G between reactants and products Chapter 2. For reactions that are energetically favorable G enzymes increase the reaction rate by lowering the activation energy Figure 3-16. In the test tube catalysts such as charcoal and platinum facilitate reactions but usually only at high temperatures or pressures at extremes of high or low pH or in organic solvents. As the cell’s protein cata- lysts however enzymes must function effectively in aqueous environment at 37 C 1 atmosphere pressure and pH 6.5–7.5. Two striking properties of enzymes enable them to func- tion as catalysts under the mild conditions present in cells: their enormous catalytic power and their high degree of specificity. The immense catalytic power of enzymes causes the rates of enzymatically catalyzed reactions to be 10 6 –10 12 times that of the corresponding uncatalyzed reactions under otherwise similar conditions. The exquisite specificity of enzymes—their ability to act selectively on one substrate or a small number of chemically similar substrates—is exempli- fied by the enzymes that act on amino acids. As noted in Chapter 2 amino acids can exist as two stereoisomers des- ignated L and D although only L isomers are normally found in biological systems. Not surprisingly enzyme-catalyzed re- actions of L-amino acids take place much more rapidly than do those of D-amino acids even though both stereoisomers of a given amino acid are the same size and possess the same R groups see Figure 2-12. Approximately 3700 different types of enzymes each of which catalyzes a single chemical reaction or set of closely re- lated reactions have been classified in the enzyme database. Certain enzymes are found in the majority of cells because they catalyze the synthesis of common cellular products e.g. proteins nucleic acids and phospholipids or take part in the ▲ FIGURE 3-15 Antibody structure and antibody-antigen interaction. a Ribbon model of an antibody. Every antibody molecule consists of two identical heavy chains red and two identical light chains blue covalently linked by disulfide bonds. b The hand-in-glove fit between an antibody and an epitope on its antigen—in this case chicken egg-white lysozyme. Regions where the two molecules make contact are shown as surfaces. The antibody contacts the antigen with residues from all its complementarity-determining regions CDRs. In this view the complementarity of the antigen and antibody is especially apparent where “fingers” extending from the antigen surface are opposed to “clefts” in the antibody surface.

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production of energy by the conversion of glucose and oxy- gen into carbon dioxide and water. Other enzymes are pres- ent only in a particular type of cell because they catalyze chemical reactions unique to that cell type e.g. the enzymes that convert tyrosine into dopamine a neurotransmitter in nerve cells. Although most enzymes are located within cells some are secreted and function in extracellular sites such as the blood the lumen of the digestive tract or even outside the organism. The catalytic activity of some enzymes is critical to cellu- lar processes other than the synthesis or degradation of mole- cules. For instance many regulatory proteins and intracellular signaling proteins catalyze the phosphorylation of proteins and some transport proteins catalyze the hydrolysis of ATP coupled to the movement of molecules across membranes. An Enzyme’s Active Site Binds Substrates and Carries Out Catalysis Certain amino acid side chains of an enzyme are important in determining its specificity and catalytic power. In the na- tive conformation of an enzyme these side chains are brought into proximity forming the active site. Active sites thus consist of two functionally important regions: one that recognizes and binds the substrate or substrates and an- other that catalyzes the reaction after the substrate has been bound. In some enzymes the catalytic region is part of the substrate-binding region in others the two regions are struc- turally as well as functionally distinct. To illustrate how the active site binds a specific substrate and then promotes a chemical change in the bound substrate we examine the action of cyclic AMP–dependent protein ki- nase now generally referred to as protein kinase A PKA. This enzyme and other protein kinases which add a phos- phate group to serine threonine or tyrosine residues in pro- teins are critical for regulating the activity of many cellular proteins often in response to external signals. Because the eukaryotic protein kinases belong to a common superfam- ily the structure of the active site and mechanism of phos- phorylation are very similar in all of them. Thus protein kinase A can serve as a general model for this important class of enzymes. The active site of protein kinase A is located in the 240- residue “kinase core” of the catalytic subunit. The kinase core which is largely conserved in all protein kinases is re- sponsible for the binding of substrates ATP and a target pep- tide sequence and the subsequent transfer of a phosphate group from ATP to a serine threonine or tyrosine residue in the target sequence. The kinase core consists of a large do- main and small one with an intervening deep cleft the active site comprises residues located in both domains. Substrate Binding by Protein Kinases The structure of the ATP-binding site in the catalytic kinase core complements the structure of the nucleotide substrate. The adenine ring of ATP sits snugly at the base of the cleft between the large and the small domains. A highly conserved sequence Gly-X-Gly-X- X-Gly-X-Val X can be any amino acid dubbed the “glycine lid” closes over the adenine ring and holds it in position Fig- ure 3-17a. Other conserved residues in the binding pocket stabilize the highly charged phosphate groups. Although ATP is a common substrate for all protein ki- nases the sequence of the target peptide varies among dif- ferent kinases. The peptide sequence recognized by protein kinase A is Arg-Arg-X-Ser-Y where X is any amino acid and Y is a hydrophobic amino acid. The part of the polypeptide chain containing the target serine or threonine residue is bound to a shallow groove in the large domain of the kinase core. The peptide specificity of protein kinase A is conferred by several glutamic acid residues in the large domain which form salt bridges with the two arginine residues in the tar- get peptide. Different residues determine the specificity of other protein kinases. The catalytic core of protein kinase A exists in an “open” and “closed” conformation Figure 3-17b. In the open con- formation the large and small domains of the core region are separated enough that substrate molecules can enter and bind. When the active site is occupied by substrate the do- mains move together into the closed position. This change in tertiary structure an example of induced fit brings the tar- get peptide sequence sufficiently close to accept a phosphate 3.3 • Enzymes and the Chemical Work of Cells 75 Progress of reaction Reactants Transition state uncatalyzed Transition state catalyzed Products ∆ G uncat ∆ G cat Free energy G ▲ FIGURE 3-16 Effect of a catalyst on the activation energy of a chemical reaction. This hypothetical reaction pathway depicts the changes in free energy G as a reaction proceeds. A reaction will take place spontaneously only if the total G of the products is less than that of the reactants G. However all chemical reactions proceed through one or more high-energy transition states and the rate of a reaction is inversely proportional to the activation energy G ‡ which is the difference in free energy between the reactants and the highest point along the pathway. Enzymes and other catalysts accelerate the rate of a reaction by reducing the free energy of the transition state and thus G ‡ .

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76 CHAPTER 3 • Protein Structure and Function group from the bound ATP. After the phosphorylation reac- tion has been completed the presence of the products causes the domains to rotate to the open position from which the products are released. The rotation from the open to the closed position also causes movement of the glycine lid over the ATP-binding cleft. The glycine lid controls the entry of ATP and release of ADP at the active site. In the open position ATP can enter and bind the active site cleft in the closed position the glycine lid pre- vents ATP from leaving the cleft. Subsequent to phosphoryl transfer from the bound ATP to the bound peptide sequence the glycine lid must rotate back to the open position before ADP can be released. Kinetic measurements show that the rate of ADP release is 20-fold slower than that of phosphoryl trans- fer indicating the influence of the glycine lid on the rate of ki- nase reactions. Mutations in the glycine lid that inhibit its flexibility slow catalysis by protein kinase A even further. Phosphoryl T ransfer by Protein Kinases After substrates have bound and the catalytic core of protein kinase A has assumed the closed conformation the phosphorylation of a serine or threonine residue on the target peptide can take place Figure 3-18. As with all chemical reactions phosphoryl transfer cat- alyzed by protein kinase A proceeds through a transition state in which the phosphate group to be transferred and the ac- ceptor hydroxyl group are brought into close proximity. Bind- ing and stabilization of the intermediates by protein kinase A reduce the activation energy of the phosphoryl transfer reac- tion permitting it to take place at measurable rates under the mild conditions present within cells see Figure 3-16. Forma- tion of the products induces the enzyme to revert to its open conformational state allowing ADP and the phosphorylated target peptide to diffuse from the active site. V max and K m Characterize an Enzymatic Reaction The catalytic action of an enzyme on a given substrate can be described by two parameters: V max the maximal velocity of the reaction at saturating substrate concentrations and K m the Michaelis constant a measure of the affinity of an en- zyme for its substrate Figure 3-19. The K m is defined as the substrate concentration that yields a half-maximal reaction rate i.e. V max . The smaller the value of K m the more avidly an enzyme can bind substrate from a dilute solution and the smaller the substrate concentration needed to reach half-maximal velocity. The concentrations of the various small molecules in a cell vary widely as do the K m values for the different en- zymes that act on them. Generally the intracellular concen- tration of a substrate is approximately the same as or greater than the K m value of the enzyme to which it binds. Enzymes in a Common Pathway Are Often Physically Associated with One Another Enzymes taking part in a common metabolic process e.g. the degradation of glucose to pyruvate are generally located in the same cellular compartment e.g. in the cytosol at a membrane within a particular organelle. Within a com- partment products from one reaction can move by diffusion to the next enzyme in the pathway. However diffusion en- tails random movement and is a slow inefficient process for 1 2 Small domain Large domain Target peptide Glycine lid Nucleotide- binding pocket Small domain Active site Glycine lid Large domain Open Closed a b ▲ FIGURE 3-17 Protein kinase A and conformational change induced by substrate binding. a Model of the catalytic subunit of protein kinase A with bound substrates the conserved kinase core is indicated as a molecular surface. An overhanging glycine-rich sequence blue traps ATP green in a deep cleft between the large and small domains of the core. Residues in the large domain bind the target peptide red. The structure of the kinase core is largely conserved in other eukaryotic protein kinases. b Schematic diagrams of open and closed conformations of the kinase core. In the absence of substrate the kinase core is in the open conformation. Substrate binding causes a rotation of the large and small domains that brings the ATP- and peptide-binding sites closer together and causes the glycine lid to move over the adenine residue of ATP thereby trapping the nucleotide in the binding cleft. The model in part a is in the closed conformation.

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moving molecules between widely dispersed enzymes Figure 3-20a. To overcome this impediment cells have evolved mechanisms for bringing enzymes in a common pathway into close proximity. In the simplest such mechanism polypeptides with differ- ent catalytic activities cluster closely together as subunits of a multimeric enzyme or assemble on a common “scaffold” Figure 3-20b. This arrangement allows the products of one reaction to be channeled directly to the next enzyme in the pathway. The first approach is illustrated by pyruvate 3.3 • Enzymes and the Chemical Work of Cells 77 V max E 1.0 unit V max E 0.25 unit Rate of formation of reaction product P relative units 2.0 1.5 1.0 0.5 0 K m a Concentration of substrate S V max 1.0 0.8 0.6 0.2 0.4 0 K m for S’ b Concentration of substrate S or S’ Rate of reaction High-affinity substrate S Low-affinity substrate S’ K m for S ▲ EXPERIMENTAL FIGURE 3-19 The K m and V max for an enzyme-catalyzed reaction are determined from plots of the initial velocity versus substrate concentration. The shape of these hypothetical kinetic curves is characteristic of a simple enzyme-catalyzed reaction in which one substrate S is converted into product P. The initial velocity is measured immediately after addition of enzyme to substrate before the substrate concentration changes appreciably. a Plots of the initial velocity at two different concentrations of enzyme E as a function of substrate concentration S. The S that yields a half- maximal reaction rate is the Michaelis constant K m a measure of the affinity of E for S. Doubling the enzyme concentration causes a proportional increase in the reaction rate and so the maximal velocity V max is doubled the K m however is unaltered. b Plots of the initial velocity versus substrate concentration with a substrate S for which the enzyme has a high affinity and with a substrate S for which the enzyme has a low affinity. Note that the V max is the same with both substrates but that K m is higher for S the low-affinity substrate. ADP Phosphorylated peptide Phosphate transfer End state Intermediate state ATP Formation of transition state Initial state ATP O O O O O O O P P C CH 2 O O O Mg 2+ Mg 2+ O P O O O C CH 2 O O O O P O O O O P P C CH 2 O O O Mg 2+ Lys-168 Mg 2+ O P O O α γ β O O O O O P P + Asp-166 Ser or Thr of target peptide − Asp-184 Lys-72 − − − − 2− 2− 2− + H + + + + ▲ FIGURE 3-18 Mechanism of phosphorylation by protein kinase A. Top Initially ATP and the target peptide bind to the active site see Figure 3-17a. Electrons of the phosphate group are delocalized by interactions with lysine side chains and Mg 2 . Colored circles represent the residues in the kinase core critical to substrate binding and phosphoryl transfer. Note that these residues are not adjacent to one another in the amino acid sequence. Middle A new bond then forms between the serine or threonine side-chain oxygen atom and phosphate yielding a pentavalent intermediate. Bottom The phosphoester bond between the and phosphates is broken yielding the products ADP and a peptide with a phosphorylated serine or threonine side chain. The catalytic mechanism of other protein kinases is similar.

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78 CHAPTER 3 • Protein Structure and Function dehydrogenase a complex of three distinct enzymes that con- verts pyruvate into acetyl CoA in mitochondria Figure 3-21. The scaffold approach is employed by MAP kinase signal- transduction pathways discussed in Chapter 14. In yeast three protein kinases assembled on the Ste5 scaffold protein form a kinase cascade that transduces the signal triggered by the binding of mating factor to the cell surface. In some cases separate proteins have been fused together at the genetic level to create a single multidomain multi- functional enzyme Figure 3-20c. For instance the isomer- ization of citrate to isocitrate in the citric acid cycle is catalyzed by aconitase a single polypeptide that carries out two separate reactions: 1 the dehydration of citrate to form cis-aconitate and then 2 the hydration of cis-aconitate to yield isocitrate see Figure 8-9. KEY CONCEPTS OF SECTION 3.3 Enzymes and the Chemical Work of Cells ■ The function of nearly all proteins depends on their abil- ity to bind other molecules ligands. Ligand-binding sites on proteins and the corresponding ligands are chemically and topologically complementary. ■ The affinity of a protein for a particular ligand refers to the strength of binding its specificity refers to the prefer- ential binding of one or a few closely related ligands. ■ Enzymes are catalytic proteins that accelerate the rate of cellular reactions by lowering the activation energy and stabilizing transition-state intermediates see Figure 3-16. ■ An enzyme active site comprises two functional parts: a substrate-binding region and a catalytic region. The amino acids composing the active site are not necessarily adjacent in the amino acid sequence but are brought into proxim- ity in the native conformation. ■ From plots of reaction rate versus substrate concen- tration two characteristic parameters of an enzyme can be determined: the Michaelis constant K m a measure of the enzyme’s affinity for substrate and the maximal ve- locity V max a measure of its catalytic power see Figure 3-19. Reactants Products A B C C C A B Reactants Products A Scaffold C C B B A Reactants Products Reactants Products OR a b c ▲ FIGURE 3-20 Evolution of multifunctional enzyme. In the hypothetical reaction pathways illustrated here the initial reactants are converted into final products by the sequential action of three enzymes: A B and C. a When the enzymes are free in solution or even constrained within the same cellular compartment the intermediates in the reaction sequence must diffuse from one enzyme to the next an inherently slow process. b Diffusion is greatly reduced or eliminated when the enzymes associate into multisubunit complexes. c The closest integration of different catalytic activities occurs when the enzymes are fused at the genetic level becoming domains in a single protein. O HSCoA CH 3 C SCoA Acetyl CoA NADH + H + NAD + O CH 3 C COO − Pyruvate + NAD + + CoA CO 2 + NADH + acetyl CoA CO 2 E 1 E 2 E 2 E 3 Pyruvate b a E 1 E 3 Net reaction: ▲ FIGURE 3-21 Structure and function of pyruvate dehydrogenase a large multimeric enzyme complex that converts pyruvate into acetyl CoA. a The complex consists of 24 copies of pyruvate decarboxylase E 1 24 copies of lipoamide transacetylase E 2 and 12 copies of dihydrolipoyl dehydrogenase E 3 . The E 1 and E 3 subunits are bound to the outside of the core formed by the E 2 subunits. b The reactions catalyzed by the complex include several enzyme-bound intermediates not shown. The tight structural integration of the three enzymes increases the rate of the overall reaction and minimizes possible side reactions.

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■ Enzymes in a common pathway are located within spe- cific cell compartments and may be further associated as domains of a monomeric protein subunits of a multimeric protein or components of a protein complex assembled on a common scaffold see Figure 3-20. Molecular Motors and the Mechanical Work of Cells A common property of all cells is motility the ability to move in a specified direction. Many cell processes exhibit some type of movement at either the molecular or the cellular level all move- ments result from the application of a force. In Brownian mo- tion for instance thermal energy constantly buffets molecules and organelles in random directions and for very short dis- tances. On the other hand materials within a cell are trans- ported in specific directions and for longer distances. This type of movement results from the mechanical work carried out by proteins that function as motors. We first briefly describe the types and general properties of molecular motors and then look at how one type of motor protein generates force for movement. Molecular Motors Convert Energy into Motion At the nanoscale of cells and molecules movement is effected by much different forces from those in the macroscopic world. For example the high protein concentration 200–300 mg/ml of the cytoplasm prevents organelles and vesicles from diffus- ing faster than 100 m/3 hours. Even a micrometer-sized bac- terium experiences a drag force from water that stops its forward movement within a fraction of a nanometer when it stops actively swimming. To generate the forces necessary for many cellular movements cells depend on specialized enzymes commonly called motor proteins. These mechanochemical en- zymes convert energy released by the hydrolysis of ATP or from ion gradients into a mechanical force. Motor proteins generate either linear or rotary motion Table 3-2. Some motor proteins are components of macro- 3.4 molecular assemblies but those that move along cytoskeletal fibers are not. This latter group comprises the myosins ki- nesins and dyneins—linear motor proteins that carry at- tached “cargo” with them as they proceed along either microfilaments or microtubules Figure 3-22a. DNA and RNA polymerases also are linear motor proteins because they translocate along DNA during replication and tran- scription. In contrast rotary motors revolve to cause the beat of bacterial flagella to pack DNA into the capsid of a virus and to synthesize ATP. The propulsive force for bacterial swimming for instance is generated by a rotary motor pro- tein complex in the bacterial membrane. Ions flow down an electrochemical gradient through an immobile ring of pro- teins the stator which is located in the membrane. Torque generated by the stator rotates an inner ring of proteins and the attached flagellum Figure 3-22b. Similarly in the mito- chondrial ATP synthase or F 0 F 1 complex a flux of ions across the inner mitochondrial membrane is transduced by the F 0 part into rotation of the subunit which projects into a surrounding ring of and subunits in the F 1 part. Inter- actions between the subunit and the subunits directs the synthesis of ATP Chapter 8. From the observed activities of motor proteins we can infer three general properties that they possess: ■ The ability to transduce a source of energy either ATP or an ion gradient into linear or rotary movement ■ The ability to bind and translocate along a cytoskeletal filament nucleic acid strand or protein complex ■ Net movement in a given direction The motor proteins that attach to cytoskeletal fibers also bind to and carry along cargo as they translocate. The cargo in muscle cells and eukaryotic flagella consists of thick fila- ments and B tubules respectively see Figure 3-22a. These motor proteins can also transport cargo chromosomes and membrane-limited vesicles as they move along microtubules or microfilaments Figure 3-23. 3.4 • Molecular Motors and the Mechanical Work of Cells 79 a b Stator Flagellum Ions Rotor Thick filament or B tubule Actin filament or A tubule ATP ADP Myosin or dynein ▲ FIGURE 3-22 Comparison of linear and rotary molecular motors. a In muscle and eukaryotic flagella the head domains of motor proteins blue bind to an actin thin filament muscle or the A tubule of a doublet microtubule flagella. ATP hydrolysis in the head causes linear movement of the cytoskeletal fiber orange relative to the attached thick filament or B tubule of an adjacent doublet microtubule. b In the rotary motor in the bacterial membrane the stator blue is immobile in the membrane. Ion flow through the stator generates a torque that powers rotation of the rotor orange and the flagellum attached to it. MEDIA CONNECTIONS Video: Rotary Motor Action: Flagellum

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80 CHAPTER 3 • Protein Structure and Function Cargo Cargo binding Fiber binding Cytoskeletal fiber ATP hydrolysis Motor protein Tail Head Neck FIGURE 3-23 Motor protein-dependent movement of cargo. The head domains of myosin dynein and kinesin motor proteins bind to a cytoskeletal fiber microfilaments or microtubules and the tail domain attaches to one of various types of cargo—in this case a membrane-limited vesicle. Hydrolysis of ATP in the head domain causes the head domain to “walk” along the track in one direction by a repeating cycle of conformational changes. TABLE 3-2 Selected Molecular Motors Energy Motor Source Structure/Components Cellular Location Movement Generated LINEAR MOTORS DNA polymerase 4 ATP Multisubunit polymerase Nucleus Translocation along DNA within replisome during replication RNA polymerase 4 ATP Multisubunit polymerase Nucleus Translocation along DNA within transcription during transcription elongation complex Ribosome 4 GTP Elongation factor 2 EF2 Cytoplasm/ER Translocation along mRNA bound to ribosome membrane during translation Myosins 3 19 ATP Heavy and light chains Cytoplasm Transport of cargo head domains with ATPase vesicles contraction activity and microfilament- binding site Kinesins 20 ATP Heavy and light chains head Cytoplasm Transport of cargo domains with ATPase activity vesicles and chromosomes and microtubule-binding site during mitosis Dyneins 20 ATP Multiple heavy intermediate Cytoplasm Transport of cargo and light chains head domains vesicles beating of cilia with ATPase activity and and eukaryotic flagella microtubule-binding site ROTARY MOTORS Bacterial flagellar H /Na Stator and rotor proteins Plasma membrane Rotation of flagellum motor gradient flagellum attached to rotor ATP synthase H Multiple subunits forming Inner mitochondrial Rotation of subunit F 0 F 1 8 gradientF 0 and F 1 particles membrane thylakoid leading to ATP synthesis membrane bacterial plasma membrane Viral capsid motor ATP Connector prohead Capsid Rotation of connector RNA ATPase leading to DNA packaging Numbers in parentheses indicate chapters in which various motors are discussed.

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All Myosins Have Head Neck and Tail Domains with Distinct Functions To further illustrate the properties of motor proteins we con- sider myosin II which moves along actin filaments in muscle cells during contraction. Other types of myosin can transport vesicles along actin filaments in the cytoskeleton. Myosin II and other members of the myosin superfamily are composed of one or two heavy chains and several light chains. The heavy chains are organized into three structurally and func- tionally different types of domains Figure 3-24a. The two globular head domains are specialized ATPases that couple the hydrolysis of ATP with motion. A critical fea- ture of the myosin ATPase activity is that it is actin activated. In the absence of actin solutions of myosin slowly convert ATP into ADP and phosphate. However when myosin is complexed with actin the rate of myosin ATPase activity is four to five times as fast as it is in the absence of actin. The actin-activation step ensures that the myosin ATPase oper- ates at its maximal rate only when the myosin head do- main is bound to actin. Adjacent to the head domain lies the -helical neck region which is associated with the light chains. These light chains are crucial for converting small conformational changes in the head into large movements of the molecule and for regulating the activity of the head do- main. The rodlike tail domain contains the binding sites that determine the specific activities of a particular myosin. The results of studies of myosin fragments produced by proteolysis helped elucidate the functions of the domains. X-ray crystallographic analysis of the S1 fragment of myosin II which consists of the head and neck domains revealed its shape the positions of the light chains and the locations of the ATP-binding and actin-binding sites. The elongated myosin head is attached at one end to the -helical neck Fig- ure 3-24b. Two light-chain molecules lie at the base of the head wrapped around the neck like C-clamps. In this posi- tion the light chains stiffen the neck region and are therefore able to regulate the activity of the head domain. Conformational Changes in the Myosin Head Couple ATP Hydrolysis to Movement The results of studies of muscle contraction provided the first evidence that myosin heads slide or walk along actin fila- ments. Unraveling the mechanism of muscle contraction was greatly aided by the development of in vitro motility as- says and single-molecule force measurements. On the basis of information obtained with these techniques and the three- dimensional structure of the myosin head researchers devel- oped a general model for how myosin harnesses the energy released by ATP hydrolysis to move along an actin filament. Because all myosins are thought to use the same mechanism to generate movement we will ignore whether the myosin tail is bound to a vesicle or is part of a thick filament as it is in muscle. One assumption in this model is that the hydrol- ysis of a single ATP molecule is coupled to each step taken by a myosin molecule along an actin filament. Evidence sup- porting this assumption is discussed in Chapter 19. As shown in Figure 3-25 myosin undergoes a series of events during each step of movement. In the course of one cycle myosin must exist in at least three conformational states: an ATP state unbound to actin an ADP-P i state bound to actin and a state after the power-generating stroke has been completed. The major question is how the nucleotide-binding pocket and the distant actin-binding site are mutually influenced and how changes at these sites are converted into force. The results of structural studies of myosin in the presence of nucleotides and nucleotide analogs that mimic the various steps in the cycle indicate that the binding and hydrolysis of a nucleotide cause a 3.4 • Molecular Motors and the Mechanical Work of Cells 81 Regulatory light chain Actin- binding site Head Neck Tail a Myosin II b Head domain Essential light chain Heavy chains Heavy chain Essential light chain Nucleotide- binding site Regulatory light chain ▲ FIGURE 3-24 Structure of myosin II. a Myosin II is a dimeric protein composed of two identical heavy chains white and four light chains blue and green. Each of the head domains transduces the energy from ATP hydrolysis into movement. Two light chains are associated with the neck domain of each heavy chain. The coiled-coil sequence of the tail domain organizes myosin II into a dimer. b Three-dimensional model of a single head domain shows that it has a curved elongated shape and is bisected by a large cleft. The nucleotide-binding pocket lies on one side of this cleft and the actin-binding site lies on the other side near the tip of the head. Wrapped around the shaft of the - helical neck are the two light chains. These chains stiffen the neck so that it can act as a lever arm for the head. Shown here is the ADP-bound conformation.

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82 CHAPTER 3 • Protein Structure and Function KEY CONCEPTS OF SECTION 3.4 Molecular Motors and the Mechanical Work of Cells ■ Motor proteins are mechanochemical enzymes that con- vert energy released by ATP hydrolysis into either linear or rotary movement see Figure 3-22. ■ Linear motor proteins myosins kinesins and dyneins move along cytoskeletal fibers carrying bound cargo which includes vesicles chromosomes thick filaments in muscle and microtubules in eukaryotic flagella. ■ Myosin II consists of two heavy chains and several light chains. Each heavy chain has a head motor domain which is an actin-activated ATPase a neck domain which is associated with light chains and a long rodlike tail do- main that organizes the dimeric molecule and binds to thick filaments in muscle cells see Figure 3-24. ■ Movement of myosin relative to an actin filament results from the attachment of the myosin head to an actin fila- ment rotation of the neck region and detachment in a cyclical ATP-dependent process see Figure 3-25. The same general mechanism is thought to account for all myosin- and kinesin-mediated movement. Common Mechanisms for Regulating Protein Function Most processes in cells do not take place independently of one another or at a constant rate. Instead the catalytic ac- tivity of enzymes or the assembly of a macromolecular com- plex is so regulated that the amount of reaction product or the appearance of the complex is just sufficient to meet the needs of the cell. As a result the steady-state concentrations 3.5 small conformational change in the head domain that is amplified into a large movement of the neck region. The small conformational change in the head domain is local- ized to a “switch” region consisting of the nucleotide- and actin-binding sites. A “converter” region at the base of the head acts like a fulcrum that causes the leverlike neck to bend and rotate. Homologous switch converter and lever arm structures in kinesin are responsible for the movement of kinesin motor proteins along microtubules. The structural basis for dynein movement is unknown because the three-dimensional struc- ture of dynein has not been determined. FIGURE 3-25 Operational model for the coupling of ATP hydrolysis to movement of myosin along an actin filament. Shown here is the cycle for a myosin II head that is part of a thick filament in muscle but other myosins that attach to other cargo e.g. the membrane of a vesicle are thought to operate according to the same cyclical mechanism. In the absence of bound nucleotide a myosin head binds actin tightly in a “rigor” state. Step : Binding of ATP opens the cleft in the myosin head disrupting the actin-binding site and weakening the interaction with actin. Step : Freed of actin the myosin head hydrolyzes ATP causing a conformational change in the head that moves it to a new position closer to the end of the actin filament where it rebinds to the filament. Step : As phosphate P i dissociates from the ATP-binding pocket the myosin head undergoes a second conformational change—the power stroke— which restores myosin to its rigor conformation. Because myosin is bound to actin this conformational change exerts a force that causes myosin to move the actin filament. Step : Release of ADP completes the cycle. Adapted from R. D. Vale and R. A. Milligan 2002 Science 288:88. 4 3 2 1 ATP-binding site Myosin head Actin thin filament Head pivots and binds a new actin subunit Hydrolysis Head pivots and moves filament power stroke P i release ADP release ATP ADP•P i P i Head dissociates from filament Nucleotide binding ADP ADP 1 2 3 4 Thick filament MEDIA CONNECTIONS Focus Animation: Myosin Crossbridge Cycle

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of substrates and products will vary depending on cellular conditions. The flow of material in an enzymatic pathway is controlled by several mechanisms some of which also regu- late the functions of nonenzymatic proteins. One of the most important mechanisms for regulating protein function entails allostery. Broadly speaking allostery refers to any change in a protein’s tertiary or quaternary structure or both induced by the binding of a ligand which may be an activator inhibitor substrate or all three. Al- losteric regulation is particularly prevalent in multimeric en- zymes and other proteins. We first explore several ways in which allostery influences protein function and then consider other mechanisms for regulating proteins. Cooperative Binding Increases a Protein’s Response to Small Changes in Ligand Concentration In many cases especially when a protein binds several mol- ecules of one ligand the binding is graded that is the bind- ing of one ligand molecule affects the binding of subsequent ligand molecules. This type of allostery often called cooper- ativity permits many multisubunit proteins to respond more efficiently to small changes in ligand concentration than would otherwise be possible. In positive cooperativity se- quential binding is enhanced in negative cooperativity sequential binding is inhibited. Hemoglobin presents a classic example of positive coop- erative binding. Each of the four subunits in hemoglobin contains one heme molecule which consists of an iron atom held within a porphyrin ring see Figure 8-16a. The heme groups are the oxygen-binding components of hemoglobin see Figure 3-10. The binding of oxygen to the heme mole- cule in one of the four hemoglobin subunits induces a local conformational change whose effect spreads to the other subunits lowering the K m for the binding of additional oxy- gen molecules and yielding a sigmoidal oxygen-binding curve Figure 3-26. Consequently the sequential binding of oxy- gen is facilitated permitting hemoglobin to load more oxy- gen in peripheral tissues than it otherwise could at normal oxygen concentrations. Ligand Binding Can Induce Allosteric Release of Catalytic Subunits or Transition to a State with Different Activity Previously we looked at protein kinase A to illustrate bind- ing and catalysis by the active site of an enzyme. This enzyme can exist as an inactive tetrameric protein composed of two catalytic subunits and two regulatory subunits. Each regula- tory subunit contains a pseudosubstrate sequence that binds to the active site in a catalytic subunit. By blocking substrate binding the regulatory subunit inhibits the activity of the catalytic subunit. Inactive protein kinase A is turned on by cyclic AMP cAMP a small second-messenger molecule. The binding of cAMP to the regulatory subunits induces a conformational change in the pseudosubstrate sequence so that it can no longer bind the catalytic subunit. Thus in the presence of cAMP the inactive tetramer dissociates into two monomeric active catalytic subunits and a dimeric regulatory subunit Figure 3-27. As discussed in Chapter 13 the binding of var- ious hormones to cell-surface receptors induces a rise in the intracellular concentration of cAMP leading to the activa- tion of protein kinase A. When the signaling ceases and the cAMP level decreases the activity of protein kinase A is turned off by reassembly of the inactive tetramer. The bind- ing of cAMP to the regulatory subunits exhibits positive co- operativity thus small changes in the concentration of this allosteric molecule produce a large change in the activity of protein kinase A. Many multimeric enzymes undergo allosteric transitions that alter the relation of the subunits to one another but do not cause dissociation as in protein kinase A. In this type of allostery the activity of a protein in the ligand-bound state differs from that in the unbound state. An example is the GroEL chaperonin discussed earlier. This barrel-shaped 3.5 • Common Mechanisms for Regulating Protein Function 83 pO 2 torr pO 2 in capillaries of active muscles pO 2 in alveoli of lungs P 50 26 Saturation 50 020 40 6080 100 100 ▲ EXPERIMENTAL FIGURE 3-26 Sequential binding of oxygen to hemoglobin exhibits positive cooperativity. Each hemoglobin molecule has four oxygen-binding sites at saturation all the sites are loaded with oxygen. The oxygen concentration is commonly measured as the partial pressure pO 2 . P 50 is the pO 2 at which half the oxygen-binding sites at a given hemoglobin concentration are occupied it is equivalent to the K m for an enzymatic reaction. The large change in the amount of oxygen bound over a small range of pO 2 values permits efficient unloading of oxygen in peripheral tissues such as muscle. The sigmoidal shape of a plot of percent saturation versus ligand concentration is indicative of cooperative binding. In the absence of cooperative binding a binding curve is a hyperbola similar to the simple kinetic curves in Figure 3-19. Adapted from L. Stryer Biochemistry 4th ed. 1995 W. H. Freeman and Company.

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84 CHAPTER 3 • Protein Structure and Function protein-folding machine comprises two back-to-back multi- subunit rings which can exist in a “tight” peptide-binding state and a “relaxed” peptide-releasing state see Figure 3-11. The binding of ATP and the co-chaperonin GroES to one of the rings in the tight state causes a twofold expansion of the GroEL cavity shifting the equilibrium toward the re- laxed peptide-folding state. Calcium and GTP Are Widely Used to Modulate Protein Activity In the preceding examples oxygen cAMP and ATP cause al- losteric changes in the activity of their target proteins he- moglobin protein kinase A and GroEL respectively. Two additional allosteric ligands Ca 2 and GTP act through two types of ubiquitous proteins to regulate many cellular processes. Calmodulin-Mediated Switching The concentration of Ca 2 free in the cytosol is kept very low ≈10 7 M by mem- brane transport proteins that continually pump Ca 2 out of the cell or into the endoplasmic reticulum. As we learn in Chapter 7 the cytosolic Ca 2 level can increase from 10- to 100-fold by the release of Ca 2 from ER stores or by its im- port from the extracellular environment. This rise in cytoso- lic Ca 2 is sensed by Ca 2 -binding proteins particularly those of the EF hand family all of which contain the helix- loop-helix motif discussed earlier see Figure 3-6a. The prototype EF hand protein calmodulin is found in all eukaryotic cells and may exist as an individual monomeric protein or as a subunit of a multimeric protein. A dumbbell-shaped molecule calmodulin contains four Ca 2 - binding sites with a K D of ≈10 6 M. The binding of Ca 2 to calmodulin causes a conformational change that permits Ca 2 /calmodulin to bind various target proteins thereby switching their activity on or off Figure 3-28. Calmodulin and similar EF hand proteins thus function as switch pro- teins acting in concert with Ca 2 to modulate the activity of other proteins. Switching Mediated by Guanine Nucleotide–Binding Proteins Another group of intracellular switch proteins con- stitutes the GTPase superfamily. These proteins include monomeric Ras protein see Figure 3-5 and the G subunit of the trimeric G proteins. Both Ras and G are bound to the plasma membrane function in cell signaling and play a key role in cell proliferation and differentiation. Other members + Inactive PKA Active PKA Catalytic site cAMP Pseudo- substrate a HH H H O N N N N NH 2 O O O CH 2 CH HC C C C OH P O cyclic AMP cAMP b R R R R C C C C Nucleotide- binding site + ▲ FIGURE 3-27 Ligand-induced activation of protein kinase A PKA. At low concentrations of cyclic AMP cAMP the PKA is an inactive tetramer. Binding of cAMP to the regulatory R subunits causes a conformational change in these subunits that permits release of the active monomeric catalytic C subunits. b Cyclic AMP is a derivative of adenosine monophosphate. This intracellular signaling molecule whose concentration rises in response to various extracellular signals can modulate the activity of many proteins. EF1 EF2 EF3 EF4 Target peptide Ca 2+ ▲ FIGURE 3-28 Switching mediated by Ca 2 /calmodulin. Calmodulin is a widely distributed cytosolic protein that contains four Ca 2 -binding sites one in each of its EF hands. Each EF hand has a helix-loop-helix motif. At cytosolic Ca 2+ concentrations above about 5 10 7 M binding of Ca 2 to calmodulin changes the protein’s conformation. The resulting Ca 2 /calmodulin wraps around exposed helices of various target proteins thereby altering their activity.

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of the GTPase superfamily function in protein synthesis the transport of proteins between the nucleus and the cytoplasm the formation of coated vesicles and their fusion with target membranes and rearrangements of the actin cytoskeleton. All the GTPase switch proteins exist in two forms Figure 3-29: 1 an active “on” form with bound GTP guanosine triphosphate that modulates the activity of specific target proteins and 2 an inactive “off” form with bound GDP guanosine diphosphate. The GTPase activity of these switch proteins hydrolyzes bound GTP to GDP slowly yield- ing the inactive form. The subsequent exchange of GDP with GTP to regenerate the active form occurs even more slowly. Activation is temporary and is enhanced or depressed by other proteins acting as allosteric regulators of the switch protein. We examine the role of various GTPase switch pro- teins in regulating intracellular signaling and other processes in several later chapters. Cyclic Protein Phosphorylation and Dephosphorylation Regulate Many Cellular Functions As noted earlier one of the most common mechanisms for regulating protein activity is phosphorylation the addition and removal of phosphate groups from serine threonine or tyrosine residues. Protein kinases catalyze phosphorylation and phosphatases catalyze dephosphorylation. Although both reactions are essentially irreversible the counteracting activities of kinases and phosphatases provide cells with a “switch” that can turn on or turn off the function of vari- ous proteins Figure 3-30. Phosphorylation changes a pro- tein’s charge and generally leads to a conformational change these effects can significantly alter ligand binding by a pro- tein leading to an increase or decrease in its activity. Nearly 3 percent of all yeast proteins are protein kinases or phosphatases indicating the importance of phosphorylation and dephosphorylation reactions even in simple cells. All classes of proteins—including structural proteins enzymes membrane channels and signaling molecules—are regulated by kinase/phosphatase switches. Different protein kinases and phosphatases are specific for different target proteins and can thus regulate a variety of cellular pathways as discussed in later chapters. Some of these enzymes act on one or a few tar- get proteins whereas others have multiple targets. The latter are useful in integrating the activities of proteins that are co- ordinately controlled by a single kinase/phosphatase switch. Frequently another kinase or phosphatase is a target thus cre- ating a web of interdependent controls. Proteolytic Cleavage Irreversibly Activates or Inactivates Some Proteins The regulatory mechanisms discussed so far act as switches reversibly turning proteins on and off. The regulation of some proteins is by a distinctly different mechanism: the ir- reversible activation or inactivation of protein function by proteolytic cleavage. This mechanism is most common in re- gard to some hormones e.g. insulin and digestive pro- teases. Good examples of such enzymes are trypsin and chymotrypsin which are synthesized in the pancreas and se- creted into the small intestine as the inactive zymogens trypsinogen and chymotrypsinogen respectively. Enteroki- nase an aminopeptidase secreted from cells lining the small intestine converts trypsinogen into trypsin which in turn cleaves chymotrypsinogen to form chymotrypsin. The delay in the activation of these proteases until they reach the in- testine prevents them from digesting the pancreatic tissue in which they are made. 3.5 • Common Mechanisms for Regulating Protein Function 85 GTPase GTPase GAPs RGSs GDIs G T P G D P GTP GDP GEFs + + + − Active "on" Inactive "off" ▲ FIGURE 3-29 Cycling of GTPase switch proteins between the active and inactive forms. Conversion of the active into the inactive form by hydrolysis of the bound GTP is accelerated by GAPs GTPase-accelerating proteins and RGSs regulators of G protein–signaling and inhibited by GDIs guanine nucleotide dissociation inhibitors. Reactivation is promoted by GEFs guanine nucleotide–exchange factors. Protein phosphatase Protein kinase ATP ADP P i Active Inactive H 2 O O O − O R R OH O − P ▲ FIGURE 3-30 Regulation of protein activity by kinase/phosphatase switch. The cyclic phosphorylation and dephosphorylation of a protein is a common cellular mechanism for regulating protein activity. In this example the target protein R is inactive light orange when phosphorylated and active dark orange when dephosphorylated some proteins have the opposite pattern.

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86 CHAPTER 3 • Protein Structure and Function Higher-Order Regulation Includes Control of Protein Location and Concentration The activities of proteins are extensively regulated in order that the numerous proteins in a cell can work together har- moniously. For example all metabolic pathways are closely controlled at all times. Synthetic reactions take place when the products of these reactions are needed degradative re- actions take place when molecules must be broken down. All the regulatory mechanisms heretofore described affect a protein locally at its site of action turning its activity on or off. Normal functioning of a cell however also requires the segregation of proteins to particular compartments such as the mitochondria nucleus and lysosomes. In regard to en- zymes compartmentation not only provides an opportunity for controlling the delivery of substrate or the exit of product but also permits competing reactions to take place simulta- neously in different parts of a cell. We describe the mecha- nisms that cells use to direct various proteins to different compartments in Chapters 16 and 17. In addition to compartmentation cellular processes are regulated by protein synthesis and degradation. For example proteins are often synthesized at low rates when a cell has lit- tle or no need for their activities. When the cell faces in- creased demand e.g. appearance of substrate in the case of enzymes stimulation of B lymphocytes by antigen the cell responds by synthesizing new protein molecules. Later the protein pool is lowered when levels of substrate decrease or the cell becomes inactive. Extracellular signals are often in- strumental in inducing changes in the rates of protein syn- thesis and degradation Chapters 13–15. Such regulated changes play a key role in the cell cycle Chapter 21 and in cell differentiation Chapter 22. KEY CONCEPTS OF SECTION 3.5 Common Mechanisms for Regulating Protein Function ■ In allostery the binding of one ligand molecule a sub- strate activator or inhibitor induces a conformational change or allosteric transition that alters a protein’s ac- tivity or affinity for other ligands. ■ In multimeric proteins such as hemoglobin that bind multiple ligand molecules the binding of one ligand mol- ecule may modulate the binding affinity for subsequent lig- and molecules. Enzymes that cooperatively bind substrates exhibit sigmoidal kinetics similar to the oxygen-binding curve of hemoglobin see Figure 3-26. ■ Several allosteric mechanisms act as switches turning protein activity on and off in a reversible fashion. ■ The binding of allosteric ligand molecules may lead to the conversion of a protein from one conformational/ activity state into another or to the release of active sub- units see Figure 3-27. ■ Two classes of intracellular switch proteins regulate a variety of cellular processes: 1 calmodulin and related Ca 2 -binding proteins in the EF hand family and 2 mem- bers of the GTPase superfamily e.g. Ras and G which cycle between active GTP-bound and inactive GDP-bound forms see Figure 3-29. ■ The phosphorylation and dephosphorylation of amino acid side chains by protein kinases and phosphatases pro- vide reversible on/off regulation of numerous proteins. ■ Nonallosteric mechanisms for regulating protein activ- ity include proteolytic cleavage which irreversibly converts inactive zymogens into active enzymes compartmentation of proteins and signal-induced modulation of protein syn- thesis and degradation. Purifying Detecting and Characterizing Proteins A protein must be purified before its structure and the mechanism of its action can be studied. However because proteins vary in size charge and water solubility no single method can be used to isolate all proteins. To isolate one particular protein from the estimated 10000 different pro- teins in a cell is a daunting task that requires methods both for separating proteins and for detecting the presence of spe- cific proteins. Any molecule whether protein carbohydrate or nucleic acid can be separated or resolved from other molecules on the basis of their differences in one or more physical or chemical characteristics. The larger and more numerous the differences between two proteins the easier and more effi- cient their separation. The two most widely used character- istics for separating proteins are size defined as either length or mass and binding affinity for specific ligands. In this sec- tion we briefly outline several important techniques for sep- arating proteins these techniques are also useful for the separation of nucleic acids and other biomolecules. Special- ized methods for removing membrane proteins from mem- branes are described in the next chapter after the unique properties of these proteins are discussed. We then consider general methods for detecting or assaying specific proteins including the use of radioactive compounds for tracking biological activity. Finally we consider several techniques for characterizing a protein’s mass sequence and three- dimensional structure. Centrifugation Can Separate Particles and Molecules That Differ in Mass or Density The first step in a typical protein purification scheme is centrifugation. The principle behind centrifugation is that 3.6

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two particles in suspension cells organelles or mole- cules with different masses or densities will settle to the bottom of a tube at different rates. Remember mass is the weight of a sample measured in grams whereas density is the ratio of its weight to volume grams/liter. Proteins vary greatly in mass but not in density. Unless a protein has an attached lipid or carbohydrate its density will not vary by more than 15 percent from 1.37 g/cm 3 the aver- age protein density. Heavier or more dense molecules set- tle or sediment more quickly than lighter or less dense molecules. A centrifuge speeds sedimentation by subjecting particles in suspension to centrifugal forces as great as 1000000 times the force of gravity g which can sediment particles as small as 10 kDa. Modern ultracentrifuges achieve these forces by reaching speeds of 150000 revolutions per minute rpm or greater. However small particles with masses of 5 kDa or less will not sediment uniformly even at such high rotor speeds. Centrifugation is used for two basic purposes: 1 as a preparative technique to separate one type of material from others and 2 as an analytical technique to measure physi- cal properties e.g. molecular weight density shape and equilibrium binding constants of macromolecules. The sed- imentation constant s of a protein is a measure of its sedi- mentation rate. The sedimentation constant is commonly expressed in svedbergs S: 1 S 10 13 seconds. Differential Centrifugation The most common initial step in protein purification is the separation of soluble proteins from insoluble cellular material by differential centrifugation. A starting mixture commonly a cell homogenate is poured into a tube and spun at a rotor speed and for a period of time that forces cell organelles such as nuclei to collect as a pellet at the bottom the soluble proteins remain in the supernatant Figure 3-31a. The supernatant fraction then is poured off and can be subjected to other purification methods to sepa- rate the many different proteins that it contains. Rate-Zonal Centrifugation On the basis of differences in their masses proteins can be separated by centrifugation through a solution of increasing density called a density gra- dient. A concentrated sucrose solution is commonly used to form density gradients. When a protein mixture is layered on top of a sucrose gradient in a tube and subjected to centrifu- gation each protein in the mixture migrates down the tube at a rate controlled by the factors that affect the sedimenta- tion constant. All the proteins start from a thin zone at the top of the tube and separate into bands or zones actually disks of proteins of different masses. In this separation tech- nique called rate-zonal centrifugation samples are cen- trifuged just long enough to separate the molecules of interest into discrete zones Figure 3-31b. If a sample is cen- trifuged for too short a time the different protein molecules will not separate sufficiently. If a sample is centrifuged much longer than necessary all the proteins will end up in a pellet at the bottom of the tube. Although the sedimentation rate is strongly influenced by particle mass rate-zonal centrifugation is seldom effective in determining precise molecular weights because variations in shape also affect sedimentation rate. The exact effects of shape are hard to assess especially for proteins and single- stranded nucleic acid molecules that can assume many com- plex shapes. Nevertheless rate-zonal centrifugation has proved to be the most practical method for separating many different types of polymers and particles. A second density- gradient technique called equilibrium density-gradient cen- trifugation is used mainly to separate DNA or organelles see Figure 5-37. Electrophoresis Separates Molecules on the Basis of Their Charge :Mass Ratio Electrophoresis is a technique for separating molecules in a mixture under the influence of an applied electric field. Dis- solved molecules in an electric field move or migrate at a speed determined by their charge:mass ratio. For example if two molecules have the same mass and shape the one with the greater net charge will move faster toward an electrode. SDS-Polyacrylamide Gel Electrophoresis Because many proteins or nucleic acids that differ in size and shape have nearly identical charge:mass ratios electrophoresis of these macromolecules in solution results in little or no separation of molecules of different lengths. However successful separation of proteins and nucleic acids can be accomplished by electrophoresis in various gels semisolid suspensions in water rather than in a liquid solution. Electrophoretic separation of proteins is most commonly performed in polyacrylamide gels. When a mixture of proteins is applied to a gel and an electric current is ap- plied smaller proteins migrate faster through the gel than do larger proteins. Gels are cast between a pair of glass plates by polymer- izing a solution of acrylamide monomers into polyacry- lamide chains and simultaneously cross-linking the chains into a semisolid matrix. The pore size of a gel can be varied by adjusting the concentrations of polyacrylamide and the cross-linking reagent. The rate at which a protein moves through a gel is influenced by the gel’s pore size and the strength of the electric field. By suitable adjustment of these parameters proteins of widely varying sizes can be separated. In the most powerful technique for resolving protein mixtures proteins are exposed to the ionic detergent SDS sodium dodecylsulfate before and during gel electrophore- sis Figure 3-32. SDS denatures proteins causing mul- timeric proteins to dissociate into their subunits and all polypeptide chains are forced into extended conforma- tions with similar charge:mass ratios. SDS treatment thus 3.6 • Purifying Detecting and Characterizing Proteins 87

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88 CHAPTER 3 • Protein Structure and Function Sample is poured into tube a Differential centrifugation More dense particle Less dense particle Decant liquid into container Particles settle according to mass Sample is layered on top of gradient b Rate-zonal centrifugation Decreasing mass of particles Larger particle Smaller particle Centrifuge Centrifugal force Centrifugal force Sucrose gradient Particles settle according to mass Centrifuge Stop centrifuge 2 3 2 3 11 Collect fractions and do assay Stop centrifuge ▲ EXPERIMENTAL FIGURE 3-31 Centrifugation techniques separate particles that differ in mass or density. a In differential centrifugation a cell homogenate or other mixture is spun long enough to sediment the denser particles e.g. cell organelles cells which collect as a pellet at the bottom of the tube step . The less dense particles e.g. soluble proteins nucleic acids remain in the liquid supernatant which can be 2 transferred to another tube step . b In rate-zonal centrifugation a mixture is spun just long enough to separate molecules that differ in mass but may be similar in shape and density e.g. globular proteins RNA molecules into discrete zones within a density gradient commonly formed by a concentrated sucrose solution step . Fractions are removed from the bottom of the tube and assayed step . 5 2 3 eliminates the effect of differences in shape and so chain length which corresponds to mass is the sole determinant of the migration rate of proteins in SDS-polyacrylamide elec- trophoresis. Even chains that differ in molecular weight by less than 10 percent can be separated by this technique. Moreover the molecular weight of a protein can be esti- mated by comparing the distance that it migrates through a gel with the distances that proteins of known molecular weight migrate. Two-Dimensional Gel Electrophoresis Electrophoresis of all cellular proteins through an SDS gel can separate proteins having relatively large differences in mass but cannot resolve proteins having similar masses e.g. a 41-kDa protein from a 42-kDa protein. To separate proteins of similar masses another physical characteristic must be exploited. Most com- monly this characteristic is electric charge which is deter- mined by the number of acidic and basic residues in a protein. Two unrelated proteins having similar masses are

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unlikely to have identical net charges because their se- quences and thus the number of acidic and basic residues are different. In two-dimensional electrophoresis proteins are sepa- rated sequentially first by their charges and then by their masses Figure 3-33a. In the first step a cell extract is fully denatured by high concentrations 8 M of urea and then layered on a gel strip that contains an continuous pH gradient. The gradient is formed by ampholytes a mixture of polyanionic and polycationic molecules that are cast into the gel with the most acidic ampholyte at one end and the most basic ampholyte at the opposite end. A charged protein will migrate through the gradient until it reaches its isoelectric point pI the pH at which the net charge of the protein is zero. This technique called iso- electric focusing IEF can resolve proteins that differ by only one charge unit. Proteins that have been separated on an IEF gel can then be separated in a second dimension on the basis of their molecular weights. To accomplish this separation the IEF gel strip is placed lengthwise on a poly- acrylamide slab gel this time saturated with SDS. When an electric field is imposed the proteins will migrate from the IEF gel into the SDS slab gel and then separate according to their masses. The sequential resolution of proteins by charge and mass can achieve excellent separation of cellular proteins Figure 3-33b. For example two-dimensional gels have been very useful in comparing the proteomes in undifferentiated and differentiated cells or in normal and cancer cells because as many as 1000 proteins can be resolved simultaneously. 3.6 • Purifying Detecting and Characterizing Proteins 89 SDS-coated proteins Place mixture of proteins on gel apply electric field Direction of migration Partially separated proteins Cross-linked polyacrylamide gel _ + Stain to visualize separated bands 1 2 3 Denature sample with sodium dodecylsulfate Decreasing size EXPERIMENTAL FIGURE 3-32 SDS- polyacrylamide gel electrophoresis separates proteins solely on the basis of their masses. Initial treatment with SDS a negatively charged detergent dissociates multimeric proteins and denatures all the polypeptide chains step . During electrophoresis the SDS-protein complexes migrate through the polyacrylamide gel step . Small proteins are able to move through the pores more easily and faster than larger proteins. Thus the proteins separate into bands according to their sizes as they migrate through the gel. The separated protein bands are visualized by staining with a dye step . 3 2 1 MEDIA CONNECTIONS Technique Animation: SDS Gel Electrophoresis

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90 CHAPTER 3 • Protein Structure and Function Liquid Chromatography Resolves Proteins by Mass Charge or Binding Affinity A third common technique for separating mixtures of pro- teins as well as other molecules is based on the principle that molecules dissolved in a solution will interact bind and dissociate with a solid surface. If the solution is allowed to flow across the surface then molecules that interact fre- quently with the surface will spend more time bound to the surface and thus move more slowly than molecules that in- teract infrequently with the surface. In this technique called liquid chromatography the sample is placed on top of a tightly packed column of spherical beads held within a glass cylinder. The nature of these beads determines whether the separation of proteins depends on differences in mass charge or binding affinity. Gel Filtration Chromatography Proteins that differ in mass can be separated on a column composed of porous beads made from polyacrylamide dextran a bacterial polysaccha- ride or agarose a seaweed derivative a technique called gel filtration chromatography. Although proteins flow around the spherical beads in gel filtration chromatography they spend some time within the large depressions that cover a bead’s sur- face. Because smaller proteins can penetrate into these depres- sions more easily than can larger proteins they travel through a gel filtration column more slowly than do larger proteins Figure 3-34a. In contrast proteins migrate through the pores in an electrophoretic gel thus smaller proteins move faster than larger ones. The total volume of liquid required to elute a protein from a gel filtration column depends on its mass: the smaller the mass the greater the elution volume. By use of proteins of known mass the elution volume can be used to estimate the mass of a protein in a mixture. Ion-Exchange Chromatography In a second type of liquid chromatography called ion-exchange chromatography pro- teins are separated on the basis of differences in their charges. This technique makes use of specially modified beads whose surfaces are covered by amino groups or car- boxyl groups and thus carry either a positive charge NH 3 or a negative charge COO at neutral pH. The proteins in a mixture carry various net charges at any given pH. When a solution of a protein mixture flows through a column of positively charged beads only proteins with a net negative charge acidic proteins adhere to the beads neutral and positively charged basic proteins flow unimpeded through the column Figure 3-34b. The acidic proteins are then eluted selectively by passing a gradient of increasing concentrations of salt through the column. At low a Apply first gel to top of second pH 4.0 pH 10.0 pH 4.0 Isoelectric focusing IEF SDS electrophoresis pH 10.0 Protein mixture Separate in first dimension by charge Separate in second dimension by size 1 2 3 3 1 b 66 43 30 16 4.2 5.9 7.4 pI Molecular weight 10 3 SDS electrophoresis Isoelectric focusing ▲ EXPERIMENTAL FIGURE 3-33 Two-dimensional gel electrophoresis can separate proteins of similar mass. a In this technique proteins are first separated on the basis of their charges by isoelectric focusing step . The resulting gel strip is applied to an SDS-polyacrylamide gel and the proteins are separated into bands by mass step . b In this two- 3 1 dimensional gel of a protein extract from cultured cells each spot represents a single polypeptide. Polypeptides can be detected by dyes as here or by other techniques such as autoradiography. Each polypeptide is characterized by its isoelectric point pI and molecular weight. Part b courtesy of J. Celis.

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3.6 • Purifying Detecting and Characterizing Proteins 91 salt concentrations protein molecules and beads are at- tracted by their opposite charges. At higher salt concentra- tions negative salt ions bind to the positively charged beads displacing the negatively charged proteins. In a gradient of increasing salt concentration weakly charged proteins are eluted first and highly charged proteins are eluted last. Simi- larly a negatively charged column can be used to retain and fractionate basic proteins. b Ion-exchange chromatography c Antibody-affinity chromatography Add buffer to wash proteins through column Layer sample on column Collect fractions a Gel filtration chromatography Layer sample on column Collect positively charged proteins Elute negatively charged protein with salt solution NaCl Large protein Small protein Positively charged protein Negatively charged protein Load in pH 7 buffer Protein recognized by antibody Cl − Na + Protein not recognized by antibody Polymer gel bead 321 4321 Antibody Wash Elute with pH 3 buffer Positively charged gel bead 321 ▲ EXPERIMENTAL FIGURE 3-34 Three commonly used liquid chromatographic techniques separate proteins on the basis of mass charge or affinity for a specific ligand. a Gel filtration chromatography separates proteins that differ in size. A mixture of proteins is carefully layered on the top of a glass cylinder packed with porous beads. Smaller proteins travel through the column more slowly than larger proteins. Thus different proteins have different elution volumes and can be collected in separate liquid fractions from the bottom. b Ion- exchange chromatography separates proteins that differ in net charge in columns packed with special beads that carry either a positive charge shown here or a negative charge. Proteins having the same net charge as the beads are repelled and flow through the column whereas proteins having the opposite charge bind to the beads. Bound proteins—in this case negatively charged—are eluted by passing a salt gradient usually of NaCl or KCl through the column. As the ions bind to the beads they desorb the protein. c In antibody-affinity chromatography a specific antibody is covalently attached to beads packed in a column. Only protein with high affinity for the antibody is retained by the column all the nonbinding proteins flow through. The bound protein is eluted with an acidic solution which disrupts the antigen–antibody complexes.

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92 CHAPTER 3 • Protein Structure and Function Affinity Chromatography The ability of proteins to bind specifically to other molecules is the basis of affinity chro- matography. In this technique ligand molecules that bind to the protein of interest are covalently attached to the beads used to form the column. Ligands can be enzyme substrates or other small molecules that bind to specific proteins. In a widely used form of this technique antibody-affinity chro- matography the attached ligand is an antibody specific for the desired protein Figure 3-34c. An affinity column will retain only those proteins that bind the ligand attached to the beads the remaining pro- teins regardless of their charges or masses will pass through the column without binding to it. However if a re- tained protein interacts with other molecules forming a complex then the entire complex is retained on the column. The proteins bound to the affinity column are then eluted by adding an excess of ligand or by changing the salt concen- tration or pH. The ability of this technique to separate par- ticular proteins depends on the selection of appropriate ligands. Highly Specific Enzyme and Antibody Assays Can Detect Individual Proteins The purification of a protein or any other molecule requires a specific assay that can detect the molecule of interest in col- umn fractions or gel bands. An assay capitalizes on some highly distinctive characteristic of a protein: the ability to bind a particular ligand to catalyze a particular reaction or to be recognized by a specific antibody. An assay must also be simple and fast to minimize errors and the possibility that the protein of interest becomes denatured or degraded while the assay is performed. The goal of any purification scheme is to isolate sufficient amounts of a given protein for study thus a useful assay must also be sensitive enough that only a small proportion of the available material is consumed. Many common protein assays require just from 10 9 to 10 12 g of material. Chromogenic and Light-Emitting Enzyme Reactions Many assays are tailored to detect some functional aspect of a pro- tein. For example enzyme assays are based on the ability to detect the loss of substrate or the formation of product. Some enzyme assays utilize chromogenic substrates which change color in the course of the reaction. Some substrates are naturally chromogenic if they are not they can be linked to a chromogenic molecule. Because of the specificity of an enzyme for its substrate only samples that contain the en- zyme will change color in the presence of a chromogenic sub- strate and other required reaction components the rate of the reaction provides a measure of the quantity of enzyme present. Such chromogenic enzymes can also be fused or chemi- cally linked to an antibody and used to “report” the presence or location of the antigen. Alternatively luciferase an en- zyme present in fireflies and some bacteria can be linked to an antibody. In the presence of ATP and luciferin luciferase catalyzes a light-emitting reaction. In either case after the antibody binds to the protein of interest substrates of the linked enzyme are added and the appearance of color or Electrophoresis/transfer SDS-polyacrylamide gel Electric current Membrane Antibody detection React with substrate for Ab 2 -linked enzyme Incubate with Ab 1 wash excess Incubate with enzyme- linked Ab 2 wash excess 1 2 3 4 Chromogenic detection ▲ EXPERIMENTAL FIGURE 3-35 Western blotting immunoblotting combines several techniques to resolve and detect a specific protein. Step : After a protein mixture has been electrophoresed through an SDS gel the separated bands are transferred blotted from the gel onto a porous membrane. Step : The membrane is flooded with a solution of antibody Ab 1 specific for the desired protein. Only the band containing this protein binds the antibody forming a layer of antibody molecules although their position 2 1 cannot be seen at this point. After sufficient time for binding the membrane is washed to remove unbound Ab 1 . Step : The membrane is incubated with a second antibody Ab 2 that binds to the bound Ab 1 . This second antibody is covalently linked to alkaline phosphatase which catalyzes a chromogenic reaction. Step : Finally the substrate is added and a deep purple precipitate forms marking the band containing the desired protein. 4 3 MEDIA CONNECTIONS Technique Animation: Immunoblotting

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emitted light is monitored. A variation of this technique par- ticularly useful in detecting specific proteins within living cells makes use of green fluorescent protein GFP a natu- rally fluorescent protein found in jellyfish see Figure 5-46. Western Blotting A powerful method for detecting a par- ticular protein in a complex mixture combines the superior resolving power of gel electrophoresis the specificity of an- tibodies and the sensitivity of enzyme assays. Called Western blotting or immunoblotting this multistep procedure is commonly used to separate proteins and then identify a spe- cific protein of interest. As shown in Figure 3-35 two dif- ferent antibodies are used in this method one specific for the desired protein and the other linked to a reporter enzyme. Radioisotopes Are Indispensable Tools for Detecting Biological Molecules A sensitive method for tracking a protein or other biologi- cal molecule is by detecting the radioactivity emitted from ra- dioisotopes introduced into the molecule. At least one atom in a radiolabeled molecule is present in a radioactive form called a radioisotope. Radioisotopes Useful in Biological Research Hundreds of biological compounds e.g. amino acids nucleosides and numerous metabolic intermediates labeled with various ra- dioisotopes are commercially available. These preparations vary considerably in their specific activity which is the amount of radioactivity per unit of material measured in dis- integrations per minute dpm per millimole. The specific ac- tivity of a labeled compound depends on the probability of decay of the radioisotope indicated by its half-life which is the time required for half the atoms to undergo radioactive decay. In general the shorter the half-life of a radioisotope the higher its specific activity Table 3-3. The specific activity of a labeled compound must be high enough that sufficient radioactivity is incorporated into cel- lular molecules to be accurately detected. For example me- thionine and cysteine labeled with sulfur-35 35 S are widely used to label cellular proteins because preparations of these amino acids with high specific activities 10 15 dpm/mmol are available. Likewise commercial preparations of 3 H- labeled nucleic acid precursors have much higher specific activities than those of the corresponding 14 C-labeled prep- arations. In most experiments the former are preferable be- cause they allow RNA or DNA to be adequately labeled after a shorter time of incorporation or require a smaller cell sam- ple. Various phosphate-containing compounds in which every phosphorus atom is the radioisotope phosphorus-32 are readily available. Because of their high specific activity 32 P-labeled nucleotides are routinely used to label nucleic acids in cell-free systems. Labeled compounds in which a radioisotope replaces atoms normally present in the molecule have the same chem- ical properties as the corresponding nonlabeled compounds. Enzymes for instance cannot distinguish between substrates labeled in this way and their nonlabeled substrates. In con- trast labeling with the radioisotope iodine-125 125 I re- quires the covalent addition of 125 I to a protein or nucleic acid. Because this labeling procedure modifies the chemical structure of a protein or nucleic acid the biological activity of the labeled molecule may differ somewhat from that of the nonlabeled form. Labeling Experiments and Detection of Radiolabeled Molecules Whether labeled compounds are detected by au- toradiography a semiquantitative visual assay or their radio- activity is measured in an appropriate “counter” a highly quantitative assay that can determine the concentration of a radiolabeled compound in a sample depends on the nature of the experiment. In some experiments both types of de- tection are used. In one use of autoradiography a cell or cell constituent is labeled with a radioactive compound and then overlaid with a photographic emulsion sensitive to radiation. Devel- opment of the emulsion yields small silver grains whose dis- tribution corresponds to that of the radioactive material. Autoradiographic studies of whole cells were crucial in de- termining the intracellular sites where various macromole- cules are synthesized and the subsequent movements of these macromolecules within cells. Various techniques employing fluorescent microscopy which we describe in the next chap- ter have largely supplanted autoradiography for studies of this type. However autoradiography is commonly used in various assays for detecting specific isolated DNA or RNA sequences Chapter 9. Quantitative measurements of the amount of radioactiv- ity in a labeled material are performed with several different instruments. A Geiger counter measures ions produced in a gas by the particles or rays emitted from a radioisotope. In a scintillation counter a radiolabeled sample is mixed with a liquid containing a fluorescent compound that emits a flash of light when it absorbs the energy of the particles or rays released in the decay of the radioisotope a phototube in the instrument detects and counts these light flashes. Phosphor- imagers are used to detect radiolabeled compounds on a sur- face storing digital data on the number of decays in 3.6 • Purifying Detecting and Characterizing Proteins 93 ` TABLE 3-3 Radioisotopes Commonly Used in Biological Research Isotope Half-Life Phosphorus-32 14.3 days Iodine-125 60.4 days Sulfur-35 87.5 days Tritium hydrogen-3 12.4 years Carbon-14 5730.4 years

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94 CHAPTER 3 • Protein Structure and Function disintegrations per minute per small pixel of surface area. These instruments which can be thought of as a kind of reusable electronic film are commonly used to quantitate ra- dioactive molecules separated by gel electrophoresis and are replacing photographic emulsions for this purpose. A combination of labeling and biochemical techniques and of visual and quantitative detection methods is often em- ployed in labeling experiments. For instance to identify the major proteins synthesized by a particular cell type a sample of the cells is incubated with a radioactive amino acid e.g. 35 Smethionine for a few minutes. The mixture of cellular proteins is then resolved by gel electrophoresis and the gel is subjected to autoradiography or phosphorimager analysis. The radioactive bands correspond to newly synthesized pro- teins which have incorporated the radiolabeled amino acid. Alternatively the proteins can be resolved by liquid chro- matography and the radioactivity in the eluted fractions can be determined quantitatively with a counter. Pulse-chase experiments are particularly useful for trac- ing changes in the intracellular location of proteins or the transformation of a metabolite into others over time. In this experimental protocol a cell sample is exposed to a radiola- beled compound—the “pulse”—for a brief period of time then washed with buffer to remove the labeled pulse and fi- nally incubated with a nonlabeled form of the compound— the “chase” Figure 3-36. Samples taken periodically are assayed to determine the location or chemical form of the radiolabel. A classic use of the pulse-chase technique was in studies to elucidate the pathway traversed by secreted pro- teins from their site of synthesis in the endoplasmic reticulum to the cell surface Chapter 17. Mass Spectrometry Measures the Mass of Proteins and Peptides A powerful technique for measuring the mass of molecules such as proteins and peptides is mass spectrometry. This ER Golgi Secretory granule Pulse Chase T 0 add 3 H-leucine T 5 min wash out 3 H-leucine T 10 min T 45 min ▲ EXPERIMENTAL FIGURE 3-36 Pulse-chase experiments can track the pathway of protein movement within cells. To determine the pathway traversed by secreted proteins subsequent to their synthesis on the rough endoplasmic reticulum ER cells are briefly incubated in a medium containing a radiolabeled amino acid e.g. 3 Hleucine the pulse which will label any protein synthesized during this period. The cells are then washed with buffer to remove the pulse and transferred to medium lacking a radioactive precursor the chase. Samples taken periodically are analyzed by autoradiography to determine the cellular location of labeled protein. At the beginning of the experiment t 0 no protein is labeled as indicated by the green dotted lines. At the end of the pulse t 5 minutes all the labeled protein red lines appears in the ER. At subsequent times this newly synthesized labeled protein is visualized first in the Golgi complex and then in secretory vesicles. Because any protein synthesized during the chase period is not labeled the movement of the labeled protein can be defined quite precisely. Metal target Sample Laser Detection Time Intensity Ionization 1 2 3 Acceleration Lightest ions arrive at detector first ++ + ▲ EXPERIMENTAL FIGURE 3-37 The molecular weight of proteins and peptides can be determined by time-of-flight mass spectrometry. In a laser-desorption mass spectrometer pulses of light from a laser ionize a protein or peptide mixture that is absorbed on a metal target . An electric field accelerates the molecules in the sample toward the detector and . The time to the detector is inversely proportional to the mass of a molecule. For molecules having the same charge the time to the detector is inversely proportional to the mass. The molecular weight is calculated using the time of flight of a standard. 3 2 1

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technique requires a method for ionizing the sample usually a mixture of peptides or proteins accelerating the molecu- lar ions and then detecting the ions. In a laser desorption mass spectrometer the protein sample is mixed with an or- ganic acid and then dried on a metal target. Energy from a laser ionizes the proteins and an electric field accelerates the ions down a tube to a detector Figure 3-37. Alternatively in an electrospray mass spectrometer a fine mist containing the sample is ionized and then introduced into a separation chamber where the positively charged molecules are acceler- ated by an electric field. In both instruments the time of flight is inversely proportional to a protein’s mass and di- rectly proportional to its charge. As little as 1 10 15 mol 1 femtomole of a protein as large as 200000 MW can be measured with an error of 0.1 percent. Protein Primary Structure Can Be Determined by Chemical Methods and from Gene Sequences The classic method for determining the amino acid sequence of a protein is Edman degradation. In this procedure the free amino group of the N-terminal amino acid of a polypeptide is labeled and the labeled amino acid is then cleaved from the polypeptide and identified by high-pressure liquid chro- matography. The polypeptide is left one residue shorter with a new amino acid at the N-terminus. The cycle is repeated on the ever shortening polypeptide until all the residues have been identified. Before about 1985 biologists commonly used the Edman chemical procedure for determining protein sequences. Now however protein sequences are determined primarily by analysis of genome sequences. The complete genomes of sev- eral organisms have already been sequenced and the data- base of genome sequences from humans and numerous model organisms is expanding rapidly. As discussed in Chap- ter 9 the sequences of proteins can be deduced from DNA sequences that are predicted to encode proteins. A powerful approach for determining the primary struc- ture of an isolated protein combines mass spectroscopy and the use of sequence databases. First mass spectrometry is used to determine the peptide mass fingerprint of the protein. A peptide mass fingerprint is a compilation of the molecular weights of peptides that are generated by a specific protease. The molecular weights of the parent protein and its prote- olytic fragments are then used to search genome databases for any similarly sized protein with identical or similar pep- tide mass maps. Peptides with a Defined Sequence Can Be Synthesized Chemically Synthetic peptides that are identical with peptides synthe- sized in vivo are useful experimental tools in studies of pro- teins and cells. For example short synthetic peptides of 10–15 residues can function as antigens to trigger the pro- duction of antibodies in animals. A synthetic peptide when coupled to a large protein carrier can trick an animal into producing antibodies that bind the full-sized natural protein antigen. As we’ll see throughout this book antibodies are ex- tremely versatile reagents for isolating proteins from mix- tures by affinity chromatography see Figure 3-34c for separating and detecting proteins by Western blotting see Figure 3-35 and for localizing proteins in cells by micro- scopic techniques described in Chapter 5. Peptides are routinely synthesized in a test tube from monomeric amino acids by condensation reactions that form peptide bonds. Peptides are constructed sequentially by cou- pling the C-terminus of a monomeric amino acid with the N- terminus of the growing peptide. To prevent unwanted reactions entailing the amino groups and carboxyl groups of the side chains during the coupling steps a protecting blocking group is attached to the side chains. Without these protecting groups branched peptides would be generated. In the last steps of synthesis the side chain–protecting groups are removed and the peptide is cleaved from the resin on which synthesis takes place. Protein Conformation Is Determined by Sophisticated Physical Methods In this chapter we have emphasized that protein function is dependent on protein structure. Thus to figure out how a protein works its three-dimensional structure must be known. Determining a protein’s conformation requires so- phisticated physical methods and complex analyses of the ex- perimental data. We briefly describe three methods used to generate three-dimensional models of proteins. X-Ray Crystallography The use of x-ray crystallography to determine the three-dimensional structures of proteins was pioneered by Max Perutz and John Kendrew in the 1950s. In this technique beams of x-rays are passed through a protein crystal in which millions of protein molecules are precisely aligned with one another in a rigid array characteristic of the protein. The wavelengths of x-rays are about 0.1–0.2 nm short enough to resolve the atoms in the protein crystal. Atoms in the crystal scatter the x-rays which produce a dif- fraction pattern of discrete spots when they are intercepted by photographic film Figure 3-38. Such patterns are ex- tremely complex—composed of as many as 25000 diffrac- tion spots for a small protein. Elaborate calculations and modifications of the protein such as the binding of heavy metals must be made to interpret the diffraction pattern and to solve the structure of the protein. The process is analogous to reconstructing the precise shape of a rock from the rip- ples that it creates in a pond. To date the detailed three- dimensional structures of more than 10000 proteins have been established by x-ray crystallography. Cryoelectron Microscopy Although some proteins readily crystallize obtaining crystals of others—particularly large multisubunit proteins—requires a time-consuming trial-and- 3.6 • Purifying Detecting and Characterizing Proteins 95

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96 CHAPTER 3 • Protein Structure and Function error effort to find just the right conditions. The structures of such difficult-to-crystallize proteins can be obtained by cryo- electron microscopy. In this technique a protein sample is rapidly frozen in liquid helium to preserve its structure and then examined in the frozen hydrated state in a cryoelectron microscope. Pictures are recorded on film by using a low dose of electrons to prevent radiation-induced damage to the struc- ture. Sophisticated computer programs analyze the images and reconstruct the protein’s structure in three dimensions. Recent advances in cryoelectron microscopy permit re- searchers to generate molecular models that compare with those derived from x-ray crystallography. The use of cryo- electron microscopy and other types of electron microscopy for visualizing cell structures are discussed in Chapter 5. NMR Spectroscopy The three-dimensional structures of small proteins containing about as many as 200 amino acids can be studied with nuclear magnetic resonance NMR spectroscopy. In this technique a concentrated protein solu- tion is placed in a magnetic field and the effects of different radio frequencies on the spin of different atoms are mea- sured. The behavior of any atom is influenced by neighbor- ing atoms in adjacent residues with closely spaced residues being more perturbed than distant residues. From the mag- nitude of the effect the distances between residues can be calculated these distances are then used to generate a model of the three-dimensional structure of the protein. Although NMR does not require the crystallization of a protein a definite advantage this technique is limited to pro- teins smaller than about 20 kDa. However NMR analysis can also be applied to protein domains which tend to be small enough for this technique and can often be obtained as stable structures. KEY CONCEPTS OF SECTION 3.6 Purifying Detecting and Characterizing Proteins ■ Proteins can be separated from other cell components and from one another on the basis of differences in their physical and chemical properties. ■ Centrifugation separates proteins on the basis of their rates of sedimentation which are influenced by their masses and shapes. ■ Gel electrophoresis separates proteins on the basis of their rates of movement in an applied electric field. SDS- polyacrylamide gel electrophoresis can resolve polypeptide chains differing in molecular weight by 10 percent or less see Figure 3-32. ■ Liquid chromatography separates proteins on the basis of their rates of movement through a column packed with spherical beads. Proteins differing in mass are resolved on gel filtration columns those differing in charge on ion- exchange columns and those differing in ligand-binding properties on affinity columns see Figure 3-34. ■ Various assays are used to detect and quantify proteins. Some assays use a light-producing reaction or radioactiv- ity to generate a signal. Other assays produce an amplified colored signal with enzymes and chromogenic substrates. ■ Antibodies are powerful reagents used to detect quan- tify and isolate proteins. They are used in affinity chro- matography and combined with gel electrophoresis in X-ray source X-ray beam Diffracted beams Crystal a Detector e.g. film ▲ EXPERIMENTAL FIGURE 3-38 X-ray crystallography provides diffraction data from which the three-dimensional structure of a protein can be determined. a Basic components of an x-ray crystallographic determination. When a narrow beam of x-rays strikes a crystal part of it passes straight through and the rest is scattered diffracted in various directions. The intensity of the diffracted waves is recorded on an x-ray film or with a solid-state electronic detector. b X-ray diffraction pattern for a topoisomerase crystal collected on a solid-state detector. From complex analyses of patterns like this one the location of every atom in a protein can be determined. Part a adapted from L. Stryer 1995 Biochemistry 4th ed. W. H. Freeman and Company p. 64 part b courtesy of J. Berger.

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Western blotting a powerful method for separating and detecting a protein in a mixture see Figure 3-35. ■ Autoradiography is a semiquantitative technique for de- tecting radioactively labeled molecules in cells tissues or electrophoretic gels. ■ Pulse-chase labeling can determine the intracellular fate of proteins and other metabolites see Figure 3-36. ■ Three-dimensional structures of proteins are obtained by x-ray crystallography cryoelectron microscopy and NMR spectroscopy. X-ray crystallography provides the most de- tailed structures but requires protein crystallization. Cryo- electron microscopy is most useful for large protein com- plexes which are difficult to crystallize. Only relatively small proteins are amenable to NMR analysis. PERSPECTIVES FOR THE FUTURE Impressive expansion of the computational power of com- puters is at the core of advances in determining the three- dimensional structures of proteins. For example vacuum tube computers running on programs punched on cards were used to solve the first protein structures on the basis of x-ray crystallography. In the future researchers aim to predict the structures of proteins only on the basis of amino acid sequences deduced from gene sequences. This computationally challenging problem requires supercom- puters or large clusters of computers working in syn- chrony. Currently only the structures of very small domains containing 100 residues or fewer can be predicted at a low resolution. However continued developments in computing and models of protein folding combined with large-scale efforts to solve the structures of all protein mo- tifs by x-ray crystallography will allow the prediction of the structures of larger proteins. With an exponentially ex- panding database of motifs domains and proteins scien- tists will be able to identify the motifs in an unknown protein match the motif to the sequence and use this head start in predicting the three-dimensional structure of the entire protein. New combined approaches will also help in in determin- ing high-resolution structures of molecular machines such as those listed in Table 3-1. Although these very large macro- molecular assemblies usually are difficult to crystallize and thus to solve by x-ray crystallography they can be imaged in a cryoelectron microscope at liquid helium temperatures and high electron energies. From millions of individual “parti- cles” each representing a random view of the protein com- plex the three-dimensional structure can be built. Because subunits of the complex may already be solved by crystallog- raphy a composite structure consisting of the x-ray-derived subunit structures fitted to the EM-derived model will be gen- erated. An interesting application of this type of study would be the solution of the structures of amyloid and prion pro- teins especially in the early stages in the formation of insolu- ble filaments. Understanding the operation of protein machines will re- quire the measurement of many new characteristics of pro- teins. For example because many machines do nonchemical work of some type biologists will have to identify the en- ergy sources mechanical electrical or thermal and meas- ure the amounts of energy to determine the limits of a particular machine. Because most activities of machines in- clude movement of one type or another the force powering the movement and its relation to biological activity can be a source of insight into how force generation is coupled to chemistry. Improved tools such as optical traps and atomic force microscopes will enable detailed studies of the forces and chemistry pertinent to the operation of individual pro- tein machines. KEY TERMS helix 61 activation energy 74 active site 75 allostery 83 amyloid filament 73 autoradiography 93 sheet 61 chaperone 69 conformation 60 cooperativity 83 domain 63 electrophoresis 87 homology 68 K m 76 ligand 73 liquid chromatography 90 REVIEW THE CONCEPTS 1. The three-dimensional structure of a protein is deter- mined by its primary secondary and tertiary structures. Define the primary secondary and tertiary structures. What are some of the common secondary structures What are the forces that hold together the secondary and tertiary struc- tures What is the quaternary structure 2. Proper folding of proteins is essential for biological activity. Describe the roles of molecular chaperones and chaperonins in the folding of proteins. 3. Proteins are degraded in cells. What is ubiquitin and what role does it play in tagging proteins for degradation What is the role of proteasomes in protein degradation Review the Concepts 97 molecular machine 59 motif 63 motor protein 79 peptide bond 60 polypeptide 61 primary structure 61 proteasome 71 protein 61 proteome 60 quaternary structure 66 rate-zonal centrifugation 87 secondary structure 61 tertiary structure 62 ubiquitin 71 V max 76 x-ray crystallography 95

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98 CHAPTER 3 • Protein Structure and Function + − + − 4pH Nuclear 10 4 pH Cytoplasmic + Drug 10 4. Enzymes can catalyze chemical reactions. How do en- zymes increase the rate of a reaction What constitutes the active site of an enzyme For an enzyme-catalyzed reaction what are K m and V max For enzyme X the K m for substrate A is 0.4 mM and for substrate B is 0.01 mM. Which sub- strate has a higher affinity for enzyme X 5. Motor proteins such as myosin convert energy into a mechanical force. Describe the three general properties char- acteristic of motor proteins. Describe the biochemical events that occur during one cycle of movement of myosin relative to an actin filament. 6. The function of proteins can be regulated in a number of ways. What is cooperativity and how does it influence pro- tein function Describe how protein phosphorylation and proteolytic cleavage can modulate protein function. 7. A number of techniques can separate proteins on the basis of their differences in mass. Describe the use of two of these techniques centrifugation and gel electrophoresis. The blood proteins transferrin MW 76 kDa and lysozyme MW 15 kDa can be separated by rate zonal centrifugation or SDS polyacrylamide gel electrophoresis. Which of the two pro- teins will sediment faster during centrifugation Which will migrate faster during electrophoresis 8. Chromatography is an analytical method used to sepa- rate proteins. Describe the principles for separating proteins by gel filtration ion-exchange and affinity chromatography. 9. Various methods have been developed for detecting proteins. Describe how radioisotopes and autoradiography can be used for labeling and detecting proteins. How does Western blotting detect proteins 10. Physical methods are often used to determine protein conformation. Describe how x-ray crystallography cryoelec- tron microscopy and NMR spectroscopy can be used to determine the shape of proteins. ANALYZE THE DATA Proteomics involves the global analysis of protein expres- sion. In one approach all the proteins in control cells and treated cells are extracted and subsequently separated using two-dimensional gel electrophoresis. Typically hundreds or thousands of protein spots are resolved and the steady-state levels of each protein are compared between control and treated cells. In the following example only a few protein spots are shown for simplicity. Proteins are separated in the first dimension on the basis of charge by isoelectric focusing pH 4–10 and then separated by size by SDS polyacrylamide gel electrophoresis. Proteins are detected with a stain such as Coomassie blue and assigned numbers for identification. a. Cells are treated with a drug “ Drug” or left untreated “Control” and then proteins are extracted and separated by two-dimensional gel electrophoresis. The stained gels are shown below. What do you conclude about the effect of the drug on the steady-state levels of proteins 1–7 b. You suspect that the drug may be inducing a protein kinase and so repeat the experiment in part a in the presence of 32 P-labeled inorganic phosphate. In this experiment the two-dimensional gels are exposed to x-ray film to detect the presence of 32 P-labeled proteins. The x-ray films are shown below. What do you conclude from this experiment about the effect of the drug on proteins 1–7 c. To determine the cellular localization of proteins 1–7 the cells from part a were separated into nuclear and cytoplasmic fractions by differential centrifugation. Two-dimensional gels were run and the stained gels are shown below. What do you conclude about the cellular localization of proteins 1–7 + − + − 4 1 2 4 3 67 5 pH 10 4 pH 10 Control + Drug + − + − 4pH 10 4 pH Control 10 + Drug + − + − 4pH Nuclear 10 4 pH Cytoplasmic Control 10 +

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Weissman A. M. 2001. Themes and variations on ubiquityla- tion. Nature Cell Biol. 2:169–177. Zhang X. F. Beuron and P. S. Freemont. 2002. Machinery of protein folding and unfolding. Curr. Opin. Struct. Biol. 12:231–238. Zwickil P. W. Baumeister and A. Steven. 2000. Dis-assembly lines: The proteasome and related ATPase-assisted proteases. Curr. Opin. Struct. Biol. 10:242–250. Enzymes and the Chemical Work of Cells Dressler D. H. and H. Potter. 1991. Discovering Enzymes. Sci- entific American Library. Fersht A. 1999. Enzyme Structure and Mechanism 3d ed. W. H. Freeman and Company. Smith C. M. et al. 1997. The protein kinase resource. Trends Biochem. Sci. 22:444–446. Taylor S. S. and E. Radzio-Andzelm. 1994. Three protein ki- nase structures define a common motif. Structure 2:345–355. Molecular Motors and the Mechanical Work of Cells Cooke R. 2001. Motor proteins. Encyclopedia Life Sciences. Nature Publishing Group. Spudich J. A. 2001. The myosin swinging cross-bridge model. Nature Rev. Mol. Cell Biol. 2:387–392. Vale R. D. and R. A. Milligan. 2000. The way things move: Looking under the hood of molecular motor proteins. Science 288:88–95. Common Mechanisms for Regulating Protein Function Ackers G. K. 1998. Deciphering the molecular code of hemo- globin allostery. Adv. Protein Chem. 51:185–253. Austin D. J. G. R. Crabtree and S. L. Schreiber. 1994. Prox- imity versus allostery: The role of regulated protein dimerization in biology. Chem. Biol. 1:131–136. Burack W . R. and A. S. Shaw. 2000. Signal transduction: Hang- ing on a scaffold. Curr. Opin. Cell Biol. 12:211–216. Cox S. E. Radzio-Andzelm and S. S. Taylor. 1994. Domain movements in protein kinases. Curr. Opin. Struct. Biol. 4:893–901. Horovitz A. Y. Fridmann G. Kafri and O. Yifrach. 2001. Re- view: Allostery in chaperonins. J. Struct. Biol. 135:104–114. Kawasaki H. S. Nakayama and R. H. Kretsinger. 1998. Clas- sification and evolution of EF-hand proteins. Biometals 11:277–295. Lim W. A. 2002. The modular logic of signaling proteins: Build- ing allosteric switches from simple binding domains. Curr. Opin. Struct. Biol. 12:61–68. Ptashne M. and A. Gann. 1998. Imposing specificity by local- ization: Mechanism and evolvability. Curr. Biol. 8:R812–R822. Saibil H. R. A. L. Horwich and W. A. Fenton. 2001. Allostery and protein substrate conformational change during GroEL/GroES- mediated protein folding. Adv. Protein Chem. 59:45–72. Yap K. L. J. A. B. Ames M. B. Sindells and M. Ikura. 1999. Diversity of conformational states and changes within the EF-hand protein superfamily. Proteins 37:499–507. Purifying Detecting and Characterizing Proteins Hames B. D. A Practical Approach. Oxford University Press. A methods series that describes protein purification methods and assays. d. Summarize the overall properties of proteins 1–7 com- bining the data from parts a b and c. Describe how you could determine the identity of any one of the proteins. REFERENCES General References Berg J. M. J. L. Tymoczko and L. Stryer. 2002. Biochemistry 5th ed. W. H. Freeman and Company chaps. 2–4 7–10. Nelson D. L. and M. M. Cox. 2000. Lehninger Principles of Biochemistry 3d ed. Worth Publishers chaps. 5–8. Web Sites Entry site into the proteins structures genomes and taxonomy: http://www.ncbi.nlm.nih.gov/Entrez/ The protein 3D structure database: http://www.rcsb.org/ Structural classifications of proteins: http://scop.mrclmb.cam.ac. uk/scop/ Sites containing general information about proteins: http://www. expasy.ch/ http://www.proweb.org/ Sites for specific protein families: http://www.pkr.sdsc. edu/html/ index.shtml The protein kinase resource http://www.mrc-lmb.cam. ac.uk/myosin/myosin.html The myosin home page http://www. proweb.org/kinesin// The kinesin home page Hierarchical Structure of Proteins Branden C. and J. Tooze. 1999. Introduction to Protein Struc- ture. Garland. Creighton T. E. 1993. Proteins: Structures and Molecular Prop- erties 2d ed. W. H. Freeman and Company. Hardison R. 1998. Hemoglobins from bacteria to man: Evolu- tion of different patterns of gene expression. J. Exp. Biol. 201: 1099. Lesk A. M. 2001. Introduction to Protein Architecture. Oxford. Macromolecular Machines. 1998. Cell 92:291–423. A special re- view issue on protein machines. Patthy L. 1999. Protein Evolution. Blackwell Science. Folding Modification and Degradation of Proteins Cohen F . E. 1999. Protein misfolding and prion diseases. J. Mol. Biol. 293:313–320. Dobson C. M. 1999. Protein misfolding evolution and disease. Trends Biochem. Sci. 24:329–332. Hartl F. U. and M. Hayer-Hartl. 2002. Molecular chaperones in the cytosol: From nascent chain to folded protein. Science 295:1852–1858. Kirschner M. 1999. Intracellular proteolysis. Trends Cell Biol. 9:M42–M45. Kornitzer D. and A. Ciechanover. 2000. Modes of regulation of ubiqutin-mediated protein degradation. J. Cell Physiol. 182:1–11. Laney J. D. and M. Hochstrasser. 1999. Substrate targeting in the ubiquitin system. Cell 97:427–430. Rochet J.-C. and P. T. Landsbury. 2000. Amyloid fibrillogene- sis: Themes and variations. Curr. Opin. Struct. Biol. 10:60–68. References 99

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4 Electron micrograph of DNA green arrow being tran- scribed into RNA red arrow. O. L. Miller Jr. and Barbara R. Beatty Oak Ridge National Laboratory. BASIC MOLECULAR GENETIC MECHANISMS T he extraordinary versatility of proteins as molecular machines and switches cellular catalysts and compo- nents of cellular structures was described in Chapter 3. In this chapter we consider the nucleic acids. These macro- molecules 1 contain the information for determining the amino acid sequence and hence the structure and function of all the proteins of a cell 2 are part of the cellular struc- tures that select and align amino acids in the correct order as a polypeptide chain is being synthesized and 3 catalyze a number of fundamental chemical reactions in cells includ- ing formation of peptide bonds between amino acids during protein synthesis. Deoxyribonucleic acid DNA contains all the infor- mation required to build the cells and tissues of an organ- ism. The exact replication of this information in any species assures its genetic continuity from generation to generation and is critical to the normal development of an individual. The information stored in DNA is arranged in hereditary units now known as genes that control iden- tifiable traits of an organism. In the process of transcrip- tion the information stored in DNA is copied into ribonu- cleic acid RNA which has three distinct roles in protein synthesis. Messenger RNA mRNA carries the instructions from DNA that specify the correct order of amino acids during protein synthesis. The remarkably accurate stepwise assem- bly of amino acids into proteins occurs by translation of mRNA. In this process the information in mRNA is inter- preted by a second type of RNA called transfer RNA tRNA with the aid of a third type of RNA ribosomal RNA rRNA and its associated proteins. As the correct amino acids are brought into sequence by tRNAs they are linked by peptide bonds to make proteins. Discovery of the structure of DNA in 1953 and subse- quent elucidation of how DNA directs synthesis of RNA which then directs assembly of proteins—the so-called central dogma—were monumental achievements marking the early days of molecular biology. However the simplified represen- tation of the central dogma as DNAnRNAnprotein does not reflect the role of proteins in the synthesis of nucleic acids. Moreover as discussed in later chapters proteins are largely responsible for regulating gene expression the entire process whereby the information encoded in DNA is decoded into the proteins that characterize various cell types. 101 OUTLINE 4.1 Structure of Nucleic Acids 4.2 Transcription of Protein-Coding Genes and Formation of Functional mRNA 4.3 Control of Gene Expression in Prokaryotes 4.4 The Three Roles of RNA in Translation 4.5 Stepwise Synthesis of Proteins on Ribosomes 4.6 DNA Replication 4.7 Viruses: Parasites of the Cellular Genetic System

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In this chapter we first review the basic structures and properties of DNA and RNA. In the next several sections we discuss the basic processes summarized in Figure 4-1: transcription of DNA into RNA precursors processing of these precursors to make functional RNA molecules trans- lation of mRNAs into proteins and the replication of DNA. Along the way we compare gene structure in prokaryotes and eukaryotes and describe how bacteria control transcrip- tion setting the stage for the more complex eukaryotic transcription-control mechanisms discussed in Chapter 11. After outlining the individual roles of mRNA tRNA and rRNA in protein synthesis we present a detailed description of the components and biochemical steps in translation. We also consider the molecular problems involved in DNA repli- 102 CHAPTER 4 • Basic Molecular Genetic Mechanisms cation and the complex cellular machinery for ensuring ac- curate copying of the genetic material. The final section of the chapter presents basic information about viruses which are important model organisms for studying macromolecular synthesis and other cellular processes. Structure of Nucleic Acids DNA and RNA are chemically very similar. The primary structures of both are linear polymers composed of monomers called nucleotides. Cellular RNAs range in length from less than one hundred to many thousands of nu- cleotides. Cellular DNA molecules can be as long as several 4.1 mRNA translation mRNA Ribosomal subunits Translation factors DNA virus tRNA Amino acids Protein AAAAA AAAAA rRNA RNA processing rNTPs Transcription DNA Replication dNTPs pre-mRNA RNA virus 4 1 2 3 Cytosol Nucleolus Nucleus ▲ FIGURE 4-1 Overview of four basic molecular genetic processes. In this chapter we cover the three processes that lead to production of proteins 1 – 3 and the process for replicating DNA 4 . Because viruses utilize host-cell machinery they have been important models for studying these processes. During transcription of a protein-coding gene by RNA polymerase 1 the four-base DNA code specifying the amino acid sequence of a protein is copied into a precursor messenger RNA pre- mRNA by the polymerization of ribonucleoside triphosphate monomers rNTPs. Removal of extraneous sequences and other modifications to the pre-mRNA 2 collectively known as RNA processing produce a functional mRNA which is transported to the cytoplasm. During translation 3 the four-base code of the mRNA is decoded into the 20–amino acid “language” of proteins. Ribosomes the macromolecular machines that translate the mRNA code are composed of two subunits assembled in the nucleolus from riboso- mal RNAs rRNAs and multiple proteins left. After transport to the cytoplasm ribosomal subunits associate with an mRNA and carry out protein synthesis with the help of transfer RNAs tRNAs and various translation factors. During DNA replication 4 which occurs only in cells preparing to divide deoxyribonucleoside triphosphate monomers dNTPs are polymerized to yield two identical copies of each chromosomal DNA molecule. Each daughter cell receives one of the identical copies.

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hundred million nucleotides. These large DNA units in as- sociation with proteins can be stained with dyes and visual- ized in the light microscope as chromosomes so named because of their stainability. A Nucleic Acid Strand Is a Linear Polymer with End-to-End Directionality DNA and RNA each consist of only four different nucleotides. Recall from Chapter 2 that all nucleotides consist of an organic base linked to a five-carbon sugar that has a phos- phate group attached to carbon 5. In RNA the sugar is ribose in DNA deoxyribose see Figure 2-14. The nucleotides used in synthesis of DNA and RNA contain five different bases. The bases adenine A and guanine G are purines which con- 4.1 • Structure of Nucleic Acids 103 tain a pair of fused rings the bases cytosine C thymine T and uracil U are pyrimidines which contain a single ring see Figure 2-15. Both DNA and RNA contain three of these bases—A G and C however T is found only in DNA and U only in RNA. Note that the single-letter abbreviations for these bases are also commonly used to denote the entire nu- cleotides in nucleic acid polymers. A single nucleic acid strand has a backbone composed of repeating pentose-phosphate units from which the purine and pyrimidine bases extend as side groups. Like a polypeptide a nucleic acid strand has an end-to-end chemical orientation: the 5 end has a hydroxyl or phosphate group on the 5 carbon of its terminal sugar the 3 end usually has a hydroxyl group on the 3 carbon of its terminal sugar Figure 4-2. This direc- tionality plus the fact that synthesis proceeds 5 to 3 has given rise to the convention that polynucleotide sequences are written and read in the 5 n3 direction from left to right for example the sequence AUG is assumed to be 5 AUG3 . As we will see the 5 n3 directionality of a nucleic acid strand is an important property of the molecule. The chemical linkage between adjacent nucleotides commonly called a phosphodi- ester bond actually consists of two phosphoester bonds one on the 5 side of the phosphate and another on the 3 side. The linear sequence of nucleotides linked by phosphodi- ester bonds constitutes the primary structure of nucleic acids. Like polypeptides polynucleotides can twist and fold into three-dimensional conformations stabilized by noncovalent bonds. Although the primary structures of DNA and RNA are generally similar their three-dimensional conformations are quite different. These structural differences are critical to the different functions of the two types of nucleic acids. Native DNA Is a Double Helix of Complementary Antiparallel Strands The modern era of molecular biology began in 1953 when James D. Watson and Francis H. C. Crick proposed that DNA has a double-helical structure. Their proposal based on analysis of x-ray diffraction patterns coupled with careful model building proved correct and paved the way for our modern understanding of how DNA functions as the genetic material. DNA consists of two associated polynucleotide strands that wind together to form a double helix. The two sugar- phosphate backbones are on the outside of the double helix and the bases project into the interior. The adjoining bases in each strand stack on top of one another in parallel planes Figure 4-3a. The orientation of the two strands is antipar- allel that is their 5 n3 directions are opposite. The strands are held in precise register by formation of base pairs be- tween the two strands: A is paired with T through two hy- drogen bonds G is paired with C through three hydrogen bonds Figure 4-3b. This base-pair complementarity is a consequence of the size shape and chemical composition of the bases. The presence of thousands of such hydrogen bonds in a DNA molecule contributes greatly to the stability O OO P O H 2 C 5 O H HH H H C 3 3 O O O H HH H H A O 3 O H HH H H OH P O H 2 C 5 H 2 C 5 O O P O O G 5 end a 3 end Phospho- diester bond Phospho- diester bond b C A G P OH 3 5 5 5 3 3 5 C-A-G 3 ▲ FIGURE 4-2 Alternative representations of a nucleic acid strand illustrating its chemical directionality. Shown here is a single strand of DNA containing only three bases: cytosine C adenine A and guanine G. a The chemical structure shows a hydroxyl group at the 3 end and a phosphate group at the 5 end. Note also that two phosphoester bonds link adjacent nucleotides this two-bond linkage commonly is referred to as a phosphodiester bond. b In the “stick” diagram top the sugars are indicated as vertical lines and the phosphodiester bonds as slanting lines the bases are denoted by their single-letter abbre- viations. In the simplest representation bottom only the bases are indicated. By convention a polynucleotide sequence is al- ways written in the 5 n3 direction left to right unless other- wise indicated.

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of the double helix. Hydrophobic and van der Waals inter- actions between the stacked adjacent base pairs further sta- bilize the double-helical structure. In natural DNA A always hydrogen bonds with T and G with C forming A·T and G·C base pairs as shown in Fig- ure 4-3b. These associations between a larger purine and smaller pyrimidine are often called Watson-Crick base pairs. Two polynucleotide strands or regions thereof in which all the nucleotides form such base pairs are said to be comple- mentary. However in theory and in synthetic DNAs other base pairs can form. For example a guanine a purine could theoretically form hydrogen bonds with a thymine a pyrim- idine causing only a minor distortion in the helix. The space available in the helix also would allow pairing between the two pyrimidines cytosine and thymine. Although the non- standard G·T and C·T base pairs are normally not found in DNA G·U base pairs are quite common in double-helical regions that form within otherwise single-stranded RNA. Most DNA in cells is a right-handed helix. The x-ray dif- fraction pattern of DNA indicates that the stacked bases are regularly spaced 0.36 nm apart along the helix axis. The helix makes a complete turn every 3.6 nm thus there are about 10.5 pairs per turn. This is referred to as the B form of DNA the normal form present in most DNA stretches in cells. On the outside of B-form DNA the spaces between the intertwined strands form two helical grooves of different widths described as the major groove and the minor groove see Figure 4-3a. As a consequence the atoms on the edges of each base within these grooves are accessible from out- side the helix forming two types of binding surfaces. DNA- binding proteins can “read” the sequence of bases in duplex DNA by contacting atoms in either the major or the minor grooves. In addition to the major B form three additional DNA structures have been described. Two of these are compared to B DNA in Figure 4-4. In very low humidity the crystallo- graphic structure of B DNA changes to the A form RNA- DNA and RNA-RNA helices exist in this form in cells and in vitro. Short DNA molecules composed of alternating purine- pyrimidine nucleotides especially Gs and Cs adopt an al- ternative left-handed configuration instead of the normal right-handed helix. This structure is called Z DNA because 104 CHAPTER 4 • Basic Molecular Genetic Mechanisms NH NH H O O NH H H HN O O HN HN O O CH 2 H H H a Major groove Minor groove 5 3 3 5 O O O O O O O O O O O O O O O O O O O O O P 3 b CH 2 P CH 2 P CH 2 P CH 2 5 5 5 5 CH 2 O O O O O O O O O O O O O O O P P P CH 2 CH 2 O O O O P 3 CH 3 T A G C A T C G N NH N NH N N HN N HN ▲ FIGURE 4-3 The DNA double helix. a Space-filling model of B DNA the most common form of DNA in cells. The bases light shades project inward from the sugar-phosphate backbones dark red and blue of each strand but their edges are accessible through major and minor grooves. Arrows indicate the 5’n3’ direction of each strand. Hydrogen bonds between the bases are in the center of the structure. The major and minor grooves are lined by potential hydrogen bond donors and acceptors highlighted in yellow. b Chemical structure of DNA double helix. This extended schematic shows the two sugar-phosphate backbones and hydrogen bonding between the Watson-Crick base pairs A T and G C. Part a from R. Wing et al. 1980 Nature 287:755 part b from R. E. Dickerson 1983 Sci. Am. 249:94.

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the bases seem to zigzag when viewed from the side. Some evidence suggests that Z DNA may occur in cells although its function is unknown. Finally a triple-stranded DNA structure is formed when synthetic polymers of polyA and polydeoxyU are mixed in the test tube. In addition ho- mopolymeric stretches of DNA composed of C and T residues in one strand and A and G residues in the other can form a triple-stranded structure by binding matching lengths of synthetic polyC T. Such structures probably do not occur naturally in cells but may prove useful as therapeutic agents. By far the most important modifications in the structure of standard B-form DNA come about as a result of protein binding to specific DNA sequences. Although the multitude of hydrogen and hydrophobic bonds between the bases pro- vide stability to DNA the double helix is flexible about its long axis. Unlike the helix in proteins see Figure 3-3 there are no hydrogen bonds parallel to the axis of the DNA helix. This property allows DNA to bend when complexed with a DNA-binding protein Figure 4-5. Bending of DNA is critical to the dense packing of DNA in chromatin the protein-DNA complex in which nuclear DNA occurs in eu- karyotic cells Chapter 10. DNA Can Undergo Reversible Strand Separation During replication and transcription of DNA the strands of the double helix must separate to allow the internal edges of the bases to pair with the bases of the nucleotides to be poly- merized into new polynucleotide chains. In later sections we describe the cellular mechanisms that separate and subse- quently reassociate DNA strands during replication and transcription. Here we discuss factors influencing the in vitro separation and reassociation of DNA strands. The unwinding and separation of DNA strands referred to as denaturation or “melting” can be induced experimen- tally by increasing the temperature of a solution of DNA. As the thermal energy increases the resulting increase in mo- lecular motion eventually breaks the hydrogen bonds and other forces that stabilize the double helix the strands then separate driven apart by the electrostatic repulsion of the negatively charged deoxyribose-phosphate backbone of each strand. Near the denaturation temperature a small increase in temperature causes a rapid near simultaneous loss of the multiple weak interactions holding the strands together along the entire length of the DNA molecules leading to an abrupt change in the absorption of ultraviolet UV light Figure 4-6a. The melting temperature T m at which DNA strands will separate depends on several factors. Molecules that contain a greater proportion of G·C pairs require higher tempera- tures to denature because the three hydrogen bonds in G·C pairs make these base pairs more stable than A·T pairs which have only two hydrogen bonds. Indeed the percentage of G·C base pairs in a DNA sample can be estimated from its T m Figure 4-6b. The ion concentration also influences the T m because the negatively charged phosphate groups in the 4.1 • Structure of Nucleic Acids 105 a B DNA b A DNA c Z DNA 3.6 nm ▲ FIGURE 4-4 Models of various known DNA structures. The sugar-phosphate backbones of the two strands which are on the outside in all structures are shown in red and blue the bases lighter shades are oriented inward. a The B form of DNA has ≈10.5 base pairs per helical turn. Adjacent stacked base pairs are 0.36 nm apart. b The more compact A form of DNA has 11 base pairs per turn and exhibits a large tilt of the base pairs with respect to the helix axis. c Z DNA is a left-handed double helix. TATA box–binding protein ▲ FIGURE 4-5 Bending of DNA resulting from protein binding. The conserved C-terminal domain of the TATA box– binding protein TBP binds to the minor groove of specific DNA sequences rich in A and T untwisting and sharply bending the double helix. Transcription of most eukaryotic genes requires participation of TBP . Adapted from D. B. Nikolov and S. K. Burley 1997 Proc. Nat’l. Acad. Sci. USA 94:15.

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two strands are shielded by positively charged ions. When the ion concentration is low this shielding is decreased thus increasing the repulsive forces between the strands and re- ducing the T m . Agents that destabilize hydrogen bonds such as formamide or urea also lower the T m . Finally extremes of pH denature DNA at low temperature. At low acid pH the bases become protonated and thus positively charged re- pelling each other. At high alkaline pH the bases lose pro- tons and become negatively charged again repelling each other because of the similar charge. The single-stranded DNA molecules that result from de- naturation form random coils without an organized struc- ture. Lowering the temperature increasing the ion concentration or neutralizing the pH causes the two com- plementary strands to reassociate into a perfect double helix. The extent of such renaturation is dependent on time the DNA concentration and the ionic concentration. Two DNA strands not related in sequence will remain as random coils and will not renature most importantly they will not inhibit complementary DNA partner strands from finding each other and renaturing. Denaturation and renaturation of DNA are the basis of nucleic acid hybridization a powerful technique used to study the relatedness of two DNA sam- ples and to detect and isolate specific DNA molecules in a mixture containing numerous different DNA sequences see Figure 9-16. Many DNA Molecules Are Circular Many prokaryotic genomic DNAs and many viral DNAs are circular molecules. Circular DNA molecules also occur in mitochondria which are present in almost all eukaryotic 106 CHAPTER 4 • Basic Molecular Genetic Mechanisms cells and in chloroplasts which are present in plants and some unicellular eukaryotes. Each of the two strands in a circular DNA molecule forms a closed structure without free ends. Localized un- winding of a circular DNA molecule which occurs during DNA replication induces torsional stress into the remain- ing portion of the molecule because the ends of the strands are not free to rotate. As a result the DNA molecule twists back on itself like a twisted rubber band forming super- coils Figure 4-7b. In other words when part of the DNA helix is underwound the remainder of the molecule be- comes overwound. Bacterial and eukaryotic cells however contain topoisomerase I which can relieve any torsional stress that develops in cellular DNA molecules during repli- cation or other processes. This enzyme binds to DNA at random sites and breaks a phosphodiester bond in one strand. Such a one-strand break in DNA is called a nick. The broken end then winds around the uncut strand lead- ing to loss of supercoils Figure 4-7a. Finally the same en- zyme joins ligates the two ends of the broken strand. Another type of enzyme topoisomerase II makes breaks in both strands of a double-stranded DNA and then religates them. As a result topoisomerase II can both relieve tor- sional stress and link together two circular DNA molecules as in the links of a chain. Although eukaryotic nuclear DNA is linear long loops of DNA are fixed in place within chromosomes Chapter 10. Thus torsional stress and the consequent formation of su- percoils also could occur during replication of nuclear DNA. As in bacterial cells abundant topoisomerase I in eukaryotic nuclei relieves any torsional stress in nuclear DNA that would develop in the absence of this enzyme. Single-stranded DNA Double-stranded DNA 75 80 85 90 Temperature °C T m 1.0 0.75 0.5 Absorption of 260-nm light a 70 90 100 110 80 T m °C 20 40 60 80 100 0 Percentage of G•C pairs b ▲ EXPERIMENTAL FIGURE 4-6 The temperature at which DNA denatures increases with the proportion of G C pairs. a Melting of doubled-stranded DNA can be monitored by the absorption of ultraviolet light at 260 nm. As regions of double- stranded DNA unpair the absorption of light by those regions increases almost twofold. The temperature at which half the bases in a double-stranded DNA sample have denatured is denoted T m for temperature of melting. Light absorption by single-stranded DNA changes much less as the temperature is increased. b The T m is a function of the G C content of the DNA the higher the G+C percentage the greater the T m .

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Different Types of RNA Exhibit Various Conformations Related to Their Functions As noted earlier the primary structure of RNA is generally similar to that of DNA with two exceptions: the sugar com- ponent of RNA ribose has a hydroxyl group at the 2 posi- tion see Figure 2-14b and thymine in DNA is replaced by uracil in RNA. The hydroxyl group on C 2 of ribose makes RNA more chemically labile than DNA and provides a chemically reactive group that takes part in RNA-mediated catalysis. As a result of this lability RNA is cleaved into mononucleotides by alkaline solution whereas DNA is not. Like DNA RNA is a long polynucleotide that can be double- stranded or single-stranded linear or circular. It can also par- ticipate in a hybrid helix composed of one RNA strand and one DNA strand. As noted above RNA-RNA and RNA- DNA double helices have a compact conformation like the A form of DNA see Figure 4-4b. Unlike DNA which exists primarily as a very long dou- ble helix most cellular RNAs are single-stranded and exhibit a variety of conformations Figure 4-8. Differences in the sizes and conformations of the various types of RNA permit them to carry out specific functions in a cell. The simplest secondary structures in single-stranded RNAs are formed by pairing of complementary bases. “Hairpins” are formed by pairing of bases within ≈5–10 nucleotides of each other and “stem-loops” by pairing of bases that are separated by 10 to 4.1 • Structure of Nucleic Acids 107 several hundred nucleotides. These simple folds can cooper- ate to form more complicated tertiary structures one of which is termed a “pseudoknot.” As discussed in detail later tRNA molecules adopt a well- defined three-dimensional architecture in solution that is cru- cial in protein synthesis. Larger rRNA molecules also have locally well-defined three-dimensional structures with more flexible links in between. Secondary and tertiary structures also have been recognized in mRNA particularly near the ends of molecules. Clearly then RNA molecules are like proteins in that they have structured domains connected by less structured flexible stretches. The folded domains of RNA molecules not only are structurally analogous to the helices and strands found in proteins but in some cases also have catalytic capacities. Such catalytic RNAs are called ribozymes. Although ri- bozymes usually are associated with proteins that stabilize the ribozyme structure it is the RNA that acts as a catalyst. Some ribozymes can catalyze splicing a remarkable process in which an internal RNA sequence is cut and removed and the two resulting chains then ligated. This process occurs during formation of the majority of functional mRNA mol- ecules in eukaryotic cells and also occurs in bacteria and ar- chaea. Remarkably some RNAs carry out self-splicing with the catalytic activity residing in the sequence that is removed. The mechanisms of splicing and self-splicing are discussed in detail in Chapter 12. As noted later in this chapter rRNA a Supercoiled b Relaxed circle EXPERIMENTAL FIGURE 4-7 DNA supercoils can be removed by cleavage of one strand. a Electron micrograph of SV40 viral DNA. When the circular DNA of the SV40 virus is isolated and separated from its associated protein the DNA duplex is underwound and assumes the supercoiled configuration. b If a supercoiled DNA is nicked i.e. one strand cleaved the strands can rewind leading to loss of a supercoil. Topoisomerase I catalyzes this reaction and also reseals the broken ends. All the supercoils in isolated SV40 DNA can be removed by the sequential action of this enzyme producing the relaxed-circle conformation. For clarity the shapes of the molecules at the bottom have been simplified.

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plays a catalytic role in the formation of peptide bonds dur- ing protein synthesis. In this chapter we focus on the functions of mRNA tRNA and rRNA in gene expression. In later chapters we will encounter other RNAs often associated with proteins that participate in other cell functions. KEY CONCEPTS OF SECTION 4.1 Structure of Nucleic Acids ■ Deoxyribonucleic acid DNA the genetic material car- ries information to specify the amino acid sequences of proteins. It is transcribed into several types of ribonucleic acid RNA including messenger RNA mRNA transfer RNA tRNA and ribosomal RNA rRNA which func- tion in protein synthesis see Figure 4-1. ■ Both DNA and RNA are long unbranched polymers of nucleotides which consist of a phosphorylated pentose linked to an organic base either a purine or pyrimidine. ■ The purines adenine A and guanine G and the pyrim- idine cytosine C are present in both DNA and RNA. The pyrimidine thymine T present in DNA is replaced by the pyrimidine uracil U in RNA. ■ Adjacent nucleotides in a polynucleotide are linked by phosphodiester bonds. The entire strand has a chemical di- rectionality: the 5 end with a free hydroxyl or phosphate group on the 5 carbon of the sugar and the 3 end with a free hydroxyl group on the 3 carbon of the sugar see Figure 4-2. ■ Natural DNA B DNA contains two complementary an- tiparallel polynucleotide strands wound together into a reg- ular right-handed double helix with the bases on the in- 108 CHAPTER 4 • Basic Molecular Genetic Mechanisms side and the two sugar-phosphate backbones on the out- side see Figure 4-3. Base pairing between the strands and hydrophobic interactions between adjacent bases in the same strand stabilize this native structure. ■ The bases in nucleic acids can interact via hydrogen bonds. The standard Watson-Crick base pairs are G·C A·T in DNA and A·U in RNA. Base pairing stabilizes the native three-dimensional structures of DNA and RNA. ■ Binding of protein to DNA can deform its helical structure causing local bending or unwinding of the DNA molecule. ■ Heat causes the DNA strands to separate denature. The melting temperature T m of DNA increases with the percentage of G·C base pairs. Under suitable condi- tions separated complementary nucleic acid strands will renature. ■ Circular DNA molecules can be twisted on themselves forming supercoils see Figure 4-7. Enzymes called topoi- somerases can relieve torsional stress and remove super- coils from circular DNA molecules. ■ Cellular RNAs are single-stranded polynucleotides some of which form well-defined secondary and tertiary struc- tures see Figure 4-8. Some RNAs called ribozymes have catalytic activity. Transcription of Protein-Coding Genes and Formation of Functional mRNA The simplest definition of a gene is a “unit of DNA that con- tains the information to specify synthesis of a single polypep- tide chain or functional RNA such as a tRNA.” The vast 4.2 a Secondary structure Hairpin Double-helical stem region Stem-loop FIGURE 4-8 RNA secondary and tertiary structures. a Stem-loops hairpins and other secondary structures can form by base pairing between distant complementary segments of an RNA molecule. In stem-loops the single-stranded loop between the base- paired helical stem may be hundreds or even thousands of nucleotides long whereas in hairpins the short turn may contain as few as four nucleotides. b Pseudoknots one type of RNA tertiary structure are formed by interaction of secondary loops through base pairing between complementary bases green and blue. Only base- paired bases are shown. A secondary structure diagram is shown at right. Part b adapted from P . J. A. Michiels et al. 2001 J. Mol. Biol. 310:1109. 5 3 5 3 Stem 1 Loop 1 Loop 2 Stem 2 b Tertiary structure Pseudoknot

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majority of genes carry information to build protein mole- cules and it is the RNA copies of such protein-coding genes that constitute the mRNA molecules of cells. The DNA molecules of small viruses contain only a few genes whereas the single DNA molecule in each of the chromo- somes of higher animals and plants may contain several thousand genes. During synthesis of RNA the four-base language of DNA containing A G C and T is simply copied or transcribed into the four-base language of RNA which is identical except that U replaces T. In contrast during protein synthesis the four-base language of DNA and RNA is translated into the 20–amino acid language of proteins. In this section we focus on formation of functional mRNAs from protein-coding genes see Figure 4-1 step 1. A similar process yields the precursors of rRNAs and tRNAs encoded by rRNA and tRNA genes these precursors are then further modified to yield functional rRNAs and tRNAs Chapter 12. A Template DNA Strand Is Transcribed into a Complementary RNA Chain by RNA Polymerase During transcription of DNA one DNA strand acts as a tem- plate determining the order in which ribonucleoside tri- phosphate rNTP monomers are polymerized to form a complementary RNA chain. Bases in the template DNA strand base-pair with complementary incoming rNTPs which then are joined in a polymerization reaction catalyzed by RNA polymerase. Polymerization involves a nucleophilic attack by the 3 oxygen in the growing RNA chain on the phosphate of the next nucleotide precursor to be added re- sulting in formation of a phosphodiester bond and release of pyrophosphate PP i . As a consequence of this mechanism RNA molecules are always synthesized in the 5 n3 direc- tion Figure 4-9. The energetics of the polymerization reaction strongly fa- vors addition of ribonucleotides to the growing RNA chain because the high-energy bond between the and phos- phate of rNTP monomers is replaced by the lower-energy phosphodiester bond between nucleotides. The equilibrium for the reaction is driven further toward chain elongation by pyrophosphatase an enzyme that catalyzes cleavage of the released PP i into two molecules of inorganic phosphate. Like the two strands in DNA the template DNA strand and the growing RNA strand that is base-paired to it have opposite 5 n3 directionality. By convention the site at which RNA polymerase begins transcription is numbered 1. Downstream denotes the di- rection in which a template DNA strand is transcribed or mRNA translated thus a downstream sequence is toward the 3 end relative to the start site considering the DNA strand with the same polarity as the transcribed RNA. Up- stream denotes the opposite direction. Nucleotide positions in the DNA sequence downstream from a start site are indi- cated by a positive sign those upstream by a negative sign. 4.2 • Transcription of Protein-Coding Genes and Formation of Functional mRNA 109 Stages in Transcription To carry out transcription RNA polymerase performs several distinct functions as depicted in Figure 4-10. During transcription initiation RNA poly- merase recognizes and binds to a specific site called a pro- moter in double-stranded DNA step 1. Nuclear RNA O O − P O O O − P O O O − P O OH H H H H O D N A t e m p l a t e s t r a n d Base Base Base Base 5 3 Base Base Base Base 3 5 Base Base Incoming rNTP 5 3 RNA strand growth α β γ Polymerization O H H H H O − O − O P O O O O H H H H O H H H H O P O O OH OH OH OH OH O − ▲ FIGURE 4-9 Polymerization of ribonucleotides by RNA polymerase during transcription. The ribonucleotide to be added at the 3 end of a growing RNA strand is specified by base pairing between the next base in the template DNA strand and the complementary incoming ribonucleoside triphosphate rNTP. A phosphodiester bond is formed when RNA polymerase catalyzes a reaction between the 3 O of the growing strand and the phosphate of a correctly base-paired rNTP . RNA strands always are synthesized in the 5 n3 direction and are opposite in polarity to their template DNA strands.

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polymerases require various protein factors called general transcription factors to help them locate promoters and ini- tiate transcription. After binding to a promoter RNA poly- merase melts the DNA strands in order to make the bases in the template strand available for base pairing with the bases of the ribonucleoside triphosphates that it will polymerize to- gether. Cellular RNA polymerases melt approximately 14 base pairs of DNA around the transcription start site which is located on the template strand within the promoter region step 2. Transcription initiation is considered complete when the first two ribonucleotides of an RNA chain are linked by a phosphodiester bond step 3. After several ribonucleotides have been polymerized RNA polymerase dissociates from the promoter DNA and general transcription factors. During the stage of strand elon- gation RNA polymerase moves along the template DNA one base at a time opening the double-stranded DNA in front of its direction of movement and hybridizing the strands behind 110 CHAPTER 4 • Basic Molecular Genetic Mechanisms it Figure 4-10 step 4. One ribonucleotide at a time is added to the 3 end of the growing nascent RNA chain during strand elongation by the polymerase. The enzyme maintains a melted region of approximately 14 base pairs called the transcription bubble. Approximately eight nucleotides at the 3 end of the growing RNA strand remain base-paired to the template DNA strand in the transcription bubble. The elon- gation complex comprising RNA polymerase template DNA and the growing nascent RNA strand is extraordi- narily stable. For example RNA polymerase transcribes the longest known mammalian genes containing ≈2 10 6 base pairs without dissociating from the DNA template or releas- ing the nascent RNA. Since RNA synthesis occurs at a rate of about 1000 nucleotides per minute at 37 C the elongation complex must remain intact for more than 24 hours to assure continuous RNA synthesis. During transcription termination the final stage in RNA synthesis the completed RNA molecule or primary transcript Promoter RNA polymerase Start site on template strand Stop site on template strand 5 3 5 3 5 3 5 3 5 5 3 3 Nascent RNA DNA-RNA hybrid region 5 Completed RNA strand INITIATION ELONGATION TERMINATION 5 3 5 3 5 3 5 3 5 3 1 2 3 4 5 Polymerase melts duplex DNA near transcription start site forming a transcription bubble. "Open complex" Polymerase catalyzes phosphodiester linkage of two initial rNTPs. Polymerase advances 3 5 down template strand melting duplex DNA and adding rNTPs to growing RNA. At transcription stop site polymerase releases completed RNA and dissociates from DNA. Initial rNTPs Transcription bubble Polymerase binds to promoter sequence in duplex DNA. "Closed complex" FIGURE 4-10 Three stages in transcription. During initiation of transcription RNA polymerase forms a transcription bubble and begins polymerization of ribonucleotides rNTPs at the start site which is located within the promoter region. Once a DNA region has been transcribed the separated strands reassociate into a double helix displacing the nascent RNA except at its 3 end. The 5’ end of the RNA strand exits the RNA polymerase through a channel in the enzyme. Termination occurs when the polymerase encounters a specific termination sequence stop site. See the text for details. MEDIA CONNECTIONS Focus Animation: Basic Transcriptional Mechanism

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is released from the RNA polymerase and the polymerase dissociates from the template DNA Figure 4-10 step 5. Specific sequences in the template DNA signal the bound RNA polymerase to terminate transcription. Once released an RNA polymerase is free to transcribe the same gene again or another gene. Structure of RNA Polymerases The RNA polymerases of bacteria archaea and eukaryotic cells are fundamentally similar in structure and function. Bacterial RNA polymerases are composed of two related large subunits and two copies of a smaller subunit and one copy of a fifth sub- unit that is not essential for transcription or cell viabil- ity but stabilizes the enzyme and assists in the assembly of its subunits. Archaeal and eukaryotic RNA polymerases have several additional small subunits associated with this core complex which we describe in Chapter 11. Schematic dia- 4.2 • Transcription of Protein-Coding Genes and Formation of Functional mRNA 111 grams of the transcription process generally show RNA poly- merase bound to an unbent DNA molecule as in Figure 4-10. However according to a current model of the interac- tion between bacterial RNA polymerase and promoter DNA the DNA bends sharply following its entry into the enzyme Figure 4-11. Organization of Genes Differs in Prokaryotic and Eukaryotic DNA Having outlined the process of transcription we now briefly consider the large-scale arrangement of information in DNA and how this arrangement dictates the requirements for RNA synthesis so that information transfer goes smoothly. In recent years sequencing of the entire genomes from several organisms has revealed not only large variations in the num- ber of protein-coding genes but also differences in their or- ganization in prokaryotes and eukaryotes. The most common arrangement of protein-coding genes in all prokaryotes has a powerful and appealing logic: genes devoted to a single metabolic goal say the synthesis of the amino acid tryptophan are most often found in a contiguous array in the DNA. Such an arrangement of genes in a func- tional group is called an operon because it operates as a unit from a single promoter. Transcription of an operon produces a continuous strand of mRNA that carries the message for a related series of proteins Figure 4-12a. Each section of the mRNA represents the unit or gene that encodes one of the proteins in the series. In prokaryotic DNA the genes are closely packed with very few noncoding gaps and the DNA is transcribed directly into colinear mRNA which then is translated into protein. This economic clustering of genes devoted to a single metabolic function does not occur in eukaryotes even simple ones like yeasts which can be metabolically similar to bac- teria. Rather eukaryotic genes devoted to a single pathway are most often physically separated in the DNA indeed such genes usually are located on different chromosomes. Each gene is transcribed from its own promoter producing one mRNA which generally is translated to yield a single poly- peptide Figure 4-12b. When researchers first compared the nucleotide se- quences of eukaryotic mRNAs from multicellular organisms with the DNA sequences encoding them they were surprised to find that the uninterrupted protein-coding sequence of a given mRNA was broken up discontinuous in its corre- sponding section of DNA. They concluded that the eukary- otic gene existed in pieces of coding sequence the exons separated by non-protein-coding segments the introns. This astonishing finding implied that the long initial primary tran- script—the RNA copy of the entire transcribed DNA sequence—had to be clipped apart to remove the introns and then carefully stitched back together to produce many eukaryotic mRNAs. Although introns are common in multicellular eukary- otes they are extremely rare in bacteria and archaea and β subunit β subunit α subunit ω subunit +20 +10 −10 −20 −30 ▲ FIGURE 4-11 Current model of bacterial RNA polymerase bound to a promoter. This structure corresponds to the polymerase molecule as schematically shown in step 2 of Figure 4-10. The subunit is in orange is in green. Part of one of the two subunits can be seen in light blue the subunit is in gray. The DNA template and nontemplate strands are shown respectively as gray and pink ribbons. A Mg 2 ion at the active center is shown as a gray sphere. Numbers indicate positions in the DNA sequence relative to the transcription start site with positive numbers in the direction of transcription and negative numbers in the opposite direction. Courtesy of R. H. Ebright Waksman Institute.

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uncommon in many unicellular eukaryotes such as baker’s yeast. However introns are present in the DNA of viruses that infect eukaryotic cells. Indeed the presence of introns was first discovered in such viruses whose DNA is tran- scribed by host-cell enzymes. Eukaryotic Precursor mRNAs Are Processed to Form Functional mRNAs In prokaryotic cells which have no nuclei translation of an mRNA into protein can begin from the 5 end of the mRNA even while the 3 end is still being synthesized by RNA poly- merase. In other words transcription and translation can occur concurrently in prokaryotes. In eukaryotic cells how- ever not only is the nucleus separated from the cytoplasm where translation occurs but also the primary transcripts of protein-coding genes are precursor mRNAs pre-mRNAs that must undergo several modifications collectively termed RNA processing to yield a functional mRNA see Figure 4-1 step 2. This mRNA then must be exported to the 112 CHAPTER 4 • Basic Molecular Genetic Mechanisms cytoplasm before it can be translated into protein. Thus transcription and translation cannot occur concurrently in eukaryotic cells. All eukaryotic pre-mRNAs initially are modified at the two ends and these modifications are retained in mRNAs. As the 5 end of a nascent RNA chain emerges from the sur- face of RNA polymerase II it is immediately acted on by several enzymes that together synthesize the 5 cap a 7-methylguanylate that is connected to the terminal nu- cleotide of the RNA by an unusual 5 5 triphosphate linkage Figure 4-13. The cap protects an mRNA from enzymatic degradation and assists in its export to the cytoplasm. The cap also is bound by a protein factor required to begin trans- lation in the cytoplasm. Processing at the 3 end of a pre-mRNA involves cleav- age by an endonuclease to yield a free 3 -hydroxyl group to which a string of adenylic acid residues is added one at a time by an enzyme called polyA polymerase. The resulting polyA tail contains 100–250 bases being shorter in yeasts and invertebrates than in vertebrates. PolyA polymerase is a Prokaryotes E. coli genome ED CB A trp operon Start site for trp mRNA synthesis Transcription Translation Proteins E D C B A trp mRNA Start sites for protein synthesis 5 3 Yeast chromosomes b Eukaryotes IV V VII XI 1550 580 910 680 TRP1 kb TRP2 TRP4 TRP5 TRP3 Transcription and RNA processing Translation Proteins 325 1 4 trp mRNAs ▲ FIGURE 4-12 Comparison of gene organization transcription and translation in prokaryotes and eukaryotes. a The tryptophan trp operon is a continuous segment of the E. coli chromosome containing five genes blue that encode the enzymes necessary for the stepwise synthesis of tryptophan. The entire operon is transcribed from one promoter into one long continuous trp mRNA red. Translation of this mRNA begins at five different start sites yielding five proteins green. The order of the genes in the bacterial genome parallels the sequential function of the encoded proteins in the tryptophan pathway. b The five genes encoding the enzymes required for tryptophan synthesis in yeast Saccharomyces cerevisiae are carried on four different chromosomes. Each gene is transcribed from its own promoter to yield a primary transcript that is processed into a functional mRNA encoding a single protein. The lengths of the yeast chromosomes are given in kilobases 10 3 bases.

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part of a complex of proteins that can locate and cleave a transcript at a specific site and then add the correct number of A residues in a process that does not require a template. The final step in the processing of many different eu- karyotic mRNA molecules is RNA splicing: the internal cleavage of a transcript to excise the introns followed by lig- ation of the coding exons. Figure 4-14 summarizes the basic steps in eukaryotic mRNA processing using the -globin gene as an example. We examine the cellular machinery for carrying out processing of mRNA as well as tRNA and rRNA in Chapter 12. The functional eukaryotic mRNAs produced by RNA processing retain noncoding regions referred to as 5 and 3 4.2 • Transcription of Protein-Coding Genes and Formation of Functional mRNA 113 untranslated regions UTRs at each end. In mammalian mRNAs the 5 UTR may be a hundred or more nucleotides long and the 3 UTR may be several kilobases in length. Prokaryotic mRNAs also usually have 5 and 3 UTRs but these are much shorter than those in eukaryotic mRNAs generally containing fewer than 10 nucleotides. Alternative RNA Splicing Increases the Number of Proteins Expressed from a Single Eukaryotic Gene In contrast to bacterial and archaeal genes the vast major- ity of genes in higher multicellular eukaryotes contain mul- tiple introns. As noted in Chapter 3 many proteins from HH H O O OH O CH 2 OH P O O 2 1 4 5 3 7-Methylguanylate O OP O O OP O O O OP HH H H O O CH 2 Base 1 OCH 3 CH 3 CH 3 2 1 4 5 3 HH H H O O CH 2 Base 2 O O OP 5 5 linkage N N N O H 2 N HN 1 2 3 4 5 6 7 8 9 ▲ FIGURE 4-13 Structure of the 5 methylated cap of eukaryotic mRNA. The distinguishing chemical features are the 5 n5 linkage of 7-methylguanylate to the initial nucleotide of the mRNA molecule and the methyl group on the 2 hydroxyl of the ribose of the first nucleotide base 1. Both these features occur in all animal cells and in cells of higher plants yeasts lack the methyl group on nucleotide 1. The ribose of the second nucleotide base 2 also is methylated in vertebrates. See A. J. Shatkin 1976 Cell 9:645. β-Globin genomic DNA Primary RNA transcript β-Globin mRNA 131 32 105 106 147 Start site for RNA synthesis 3 cleavage and addition of polyA tail 5 3 1 147 PolyA tail PolyA site A n A n A n Intron excision exon ligation m 7 Gppp Exon Intron UTR ▲ FIGURE 4-14 Overview of RNA processing to produce functional mRNA in eukaryotes. The -globin gene contains three protein-coding exons coding region red and two intervening noncoding introns blue. The introns interrupt the protein-coding sequence between the codons for amino acids 31 and 32 and 105 and 106. Transcription of eukaryotic protein-coding genes starts before the sequence that encodes the first amino acid and extends beyond the sequence encoding the last amino acid resulting in noncoding regions gray at the ends of the primary transcript. These untranslated regions UTRs are retained during processing. The 5 cap m 7 Gppp is added during formation of the primary RNA transcript which extends beyond the polyA site. After cleavage at the polyA site and addition of multiple A residues to the 3 end splicing removes the introns and joins the exons. The small numbers refer to positions in the 147–amino acid sequence of -globin. MEDIA CONNECTIONS Overview Animation: Life Cycle of an mRNA

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higher eukaryotes have a multidomain tertiary structure see Figure 3-8. Individual repeated protein domains often are encoded by one exon or a small number of exons that code for identical or nearly identical amino acid sequences. Such repeated exons are thought to have evolved by the accidental multiple duplication of a length of DNA lying between two sites in adjacent introns resulting in insertion of a string of repeated exons separated by introns between the original two introns. The presence of multiple introns in many eu- karyotic genes permits expression of multiple related pro- teins from a single gene by means of alternative splicing. In higher eukaryotes alternative splicing is an important mech- anism for production of different forms of a protein called isoforms by different types of cells. Fibronectin a multidomain extracellular adhesive pro- tein found in mammals provides a good example of alter- native splicing Figure 4-15. The fibronectin gene contains numerous exons grouped into several regions correspon- ding to specific domains of the protein. Fibroblasts pro- duce fibronectin mRNAs that contain exons EIIIA and EIIIB these exons encode amino acid sequences that bind tightly to proteins in the fibroblast plasma membrane. Consequently this fibronectin isoform adheres fibroblasts to the extracellular matrix. Alternative splicing of the fi- bronectin primary transcript in hepatocytes the major type of cell in the liver yields mRNAs that lack the EIIIA and EIIIB exons. As a result the fibronectin secreted by hepatocytes into the blood does not adhere tightly to fi- broblasts or most other cell types allowing it to circulate. During formation of blood clots however the fibrin- binding domains of hepatocyte fibronectin binds to fibrin one of the principal constituents of clots. The bound fi- bronectin then interacts with integrins on the membranes of passing activated platelets thereby expanding the clot by addition of platelets. More than 20 different isoforms of fibronectin have been identified each encoded by a different alternatively spliced mRNA composed of a unique combination of fibronectin gene exons. Recent sequencing of large numbers of mRNAs 114 CHAPTER 4 • Basic Molecular Genetic Mechanisms isolated from various tissues and comparison of their se- quences with genomic DNA has revealed that nearly 60 per- cent of all human genes are expressed as alternatively spliced mRNAs. Clearly alternative RNA splicing greatly expands the number of proteins encoded by the genomes of higher multicellular organisms. KEY CONCEPTS OF SECTION 4.2 Transcription of Protein-Coding Genes and Formation of Functional mRNA ■ Transcription of DNA is carried out by RNA poly- merase which adds one ribonucleotide at a time to the 3 end of a growing RNA chain see Figure 4-10. The se- quence of the template DNA strand determines the order in which ribonucleotides are polymerized to form an RNA chain. ■ During transcription initiation RNA polymerase binds to a specific site in DNA the promoter locally melts the double-stranded DNA to reveal the unpaired template strand and polymerizes the first two nucleotides. ■ During strand elongation RNA polymerase moves along the DNA melting sequential segments of the DNA and adding nucleotides to the growing RNA strand. ■ When RNA polymerase reaches a termination sequence in the DNA the enzyme stops transcription leading to re- lease of the completed RNA and dissociation of the en- zyme from the template DNA. ■ In prokaryotic DNA several protein-coding genes com- monly are clustered into a functional region an operon which is transcribed from a single promoter into one mRNA encoding multiple proteins with related functions see Figure 4-12a. Translation of a bacterial mRNA can begin before synthesis of the mRNA is complete. ■ In eukaryotic DNA each protein-coding gene is tran- scribed from its own promoter. The initial primary tran- 5 5 3 3 Fibronectin gene EIIIB EIIIA Fibroblast fibronectin mRNA Hepatocyte fibronectin mRNA ▲ FIGURE 4-15 Cell type–specific splicing of fibronectin pre-mRNA in fibroblasts and hepatocytes. The ≈75-kb fibronectin gene top contains multiple exons. The EIIIB and EIIIA exons green encode binding domains for specific proteins on the surface of fibroblasts. The fibronectin mRNA produced in fibroblasts includes the EIIIA and EIIIB exons whereas these exons are spliced out of fibronectin mRNA in hepatocytes. In this diagram introns black lines are not drawn to scale most of them are much longer than any of the exons.

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script very often contains noncoding regions introns in- terspersed among coding regions exons. ■ Eukaryotic primary transcripts must undergo RNA pro- cessing to yield functional RNAs. During processing the ends of nearly all primary transcripts from protein-coding genes are modified by addition of a 5 cap and 3 polyA tail. Transcripts from genes containing introns undergo splicing the removal of the introns and joining of the ex- ons see Figure 4-14. ■ The individual domains of multidomain proteins found in higher eukaryotes are often encoded by individual ex- ons or a small number of exons. Distinct isoforms of such proteins often are expressed in specific cell types as the re- sult of alternative splicing of exons. Control of Gene Expression in Prokaryotes Since the structure and function of a cell are determined by the proteins it contains the control of gene expression is a fundamental aspect of molecular cell biology. Most com- monly the “decision” to initiate transcription of the gene en- coding a particular protein is the major mechanism for controlling production of the encoded protein in a cell. By controlling transcription initiation a cell can regulate which proteins it produces and how rapidly. When transcription of a gene is repressed the corresponding mRNA and encoded protein or proteins are synthesized at low rates. Conversely when transcription of a gene is activated both the mRNA and encoded protein or proteins are produced at much higher rates. In most bacteria and other single-celled organisms gene expression is highly regulated in order to adjust the cell’s en- zymatic machinery and structural components to changes in the nutritional and physical environment. Thus at any given time a bacterial cell normally synthesizes only those proteins of its entire proteome required for survival under the partic- ular conditions. In multicellular organisms control of gene expression is largely directed toward assuring that the right gene is expressed in the right cell at the right time during em- bryological development and tissue differentiation. Here we describe the basic features of transcription control in bacte- ria using the lac operon in E. coli as our primary example. Many of the same processes as well as others are involved in eukaryotic transcription control which is discussed in Chapter 11. In E. coli about half the genes are clustered into oper- ons each of which encodes enzymes involved in a particular metabolic pathway or proteins that interact to form one mul- tisubunit protein. For instance the trp operon mentioned earlier encodes five enzymes needed in the biosynthesis of tryptophan see Figure 4-12. Similarly the lac operon en- codes three enzymes required for the metabolism of lactose a sugar present in milk. Since a bacterial operon is tran- 4.3 4.3 • Control of Gene Expression in Prokaryotes 115 scribed from one start site into a single mRNA all the genes within an operon are coordinately regulated that is they are all activated or repressed to the same extent. Transcription of operons as well as of isolated genes is controlled by an interplay between RNA polymerase and specific repressor and activator proteins. In order to initiate transcription however E. coli RNA polymerase must be as- sociated with one of a small number of sigma factors which function as initiation factors. The most common one in bacterial cells is 70 . Initiation of lac Operon Transcription Can Be Repressed and Activated When E. coli is in an environment that lacks lactose syn- thesis of lac mRNA is repressed so that cellular energy is not wasted synthesizing enzymes the cells cannot use. In an environment containing both lactose and glucose E. coli cells preferentially metabolize glucose the central molecule of carbohydrate metabolism. Lactose is metabolized at a high rate only when lactose is present and glucose is largely depleted from the medium. This metabolic adjustment is achieved by repressing transcription of the lac operon until lactose is present and synthesis of only low levels of lac mRNA until the cytosolic concentration of glucose falls to low levels. Transcription of the lac operon under different conditions is controlled by lac repressor and catabolite ac- tivator protein CAP each of which binds to a specific DNA sequence in the lac transcription-control region Fig- ure 4-16 top. For transcription of the lac operon to begin the 70 sub- unit of the RNA polymerase must bind to the lac promoter which lies just upstream of the start site. When no lactose is present binding of the lac repressor to a sequence called the lac operator which overlaps the transcription start site blocks transcription initiation by the polymerase Figure 4-16a. When lactose is present it binds to specific binding sites in each subunit of the tetrameric lac repressor causing a conformational change in the protein that makes it dissociate from the lac operator. As a result the polymerase can initiate transcription of the lac operon. However when glucose also is present the rate of transcription initiation i.e. the number of times per minute different polymerase molecules initiate transcription is very low resulting in synthesis of only low levels of lac mRNA and the proteins encoded in the lac operon Figure 4-16b. Once glucose is depleted from the media and the intra- cellular glucose concentration falls E. coli cells respond by synthesizing cyclic AMP cAMP see Figure 3-27b. As the concentration of cAMP increases it binds to a site in each subunit of the dimeric CAP protein causing a conforma- tional change that allows the protein to bind to the CAP site in the lac transcription-control region. The bound CAP- cAMP complex interacts with the polymerase bound to the promoter greatly stimulating the rate of transcription initia- tion. This activation leads to synthesis of high levels of lac

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mRNA and subsequently of the enzymes encoded by the lac operon Figure 4-16c. Although the promoters for different E. coli genes exhibit considerable homology their exact sequences differ. The pro- moter sequence determines the intrinsic rate at which an RNA polymerase– complex initiates transcription of a gene in the absence of a repressor or activator protein. Promoters that support a high rate of transcription initiation are called strong promoters. Those that support a low rate of tran- scription initiation are called weak promoters. The lac operon for instance has a weak promoter its low intrinsic 116 CHAPTER 4 • Basic Molecular Genetic Mechanisms rate of initiation is further reduced by the lac repressor and substantially increased by the cAMP-CAP activator. Small Molecules Regulate Expression of Many Bacterial Genes via DNA-Binding Repressors Transcription of most E. coli genes is regulated by processes similar to those described for the lac operon. The general mechanism involves a specific repressor that binds to the op- erator region of a gene or operon thereby blocking tran- scription initiation. A small molecule or molecules called an inducer binds to the repressor controlling its DNA-binding activity and consequently the rate of transcription as appro- priate for the needs of the cell. For example when the tryptophan concentration in the medium and cytosol is high the cell does not synthesize the several enzymes encoded in the trp operon. Binding of tryp- tophan to the trp repressor causes a conformational change that allows the protein to bind to the trp operator thereby repressing expression of the enzymes that synthesize trypto- phan. Conversely when the tryptophan concentration in the medium and cytosol is low tryptophan dissociates from the trp repressor causing a conformational change in the protein that causes it to dissociate from the trp operator allowing transcription of the trp operon. In the case of the lac operon binding of the inducer lactose to the lac repressor reduces binding of the repressor to the operator thereby promoting transcription. Specific activator proteins such as CAP in the lac operon also control transcription of some but not all bacterial genes. These activators bind to DNA together with the RNA poly- merase stimulating transcription from a specific promoter. The DNA-binding activity of an activator is modulated in re- sponse to cellular needs by the binding of specific small mol- ecules e.g. cAMP that alter the conformation of the activator. Transcription by 54 -RNA Polymerase Is Controlled by Activators That Bind Far from the Promoter Most E. coli promoters interact with 70 -RNA polymerase the major form of the bacterial enzyme. Transcription of cer- tain groups of genes however is carried out by E. coli RNA polymerases containing one of several alternative sigma fac- tors that recognize different consensus promoter sequences than 70 does. All but one of these are related to 70 in se- quence. Transcription initiation by RNA polymerases con- taining these 70 -like factors is regulated by repressors and activators that bind to DNA near the region where the poly- merase binds similar to initiation by 70 -RNA polymerase itself. The sequence of one E. coli sigma factor 54 is distinctly different from that of all the 70 -like factors. Transcription of genes by RNA polymerases containing 54 is regulated E. coli lac transcription-control genes lacZ Promoter CAP site Operator +1 transcription start site lacZ a − lactose + glucose low cAMP lacZ b + lactose + glucose low cAMP lacZ c + lactose − glucose high cAMP Pol-σ 70 CAP lac repressor lactose cAMP No mRNA transcription Low transcription High transcription ▲ FIGURE 4-16 Regulation of transcription from the lac operon of E. coli. Top The transcription-control region composed of ≈100 base pairs includes three protein-binding regions: the CAP site which binds catabolite activator protein the lac promoter which binds the RNA polymerase– 70 complex and the lac operator which binds lac repressor. The lacZ gene the first of three genes in the operon is shown to the right. a In the absence of lactose very little lac mRNA is produced because the lac repressor binds to the operator inhibiting transcription initiation by RNA polymerase– 70 . b In the presence of glucose and lactose lac repressor binds lactose and dissociates from the operator allowing RNA polymerase– 70 to initiate transcription at a low rate. c Maximal transcription of the lac operon occurs in the presence of lactose and absence of glucose. In this situation cAMP increases in response to the low glucose concentration and forms the CAP-cAMP complex which binds to the CAP site where it interacts with RNA polymerase to stimulate the rate of transcription initiation.

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solely by activators whose binding sites in DNA referred to as enhancers generally are located 80–160 base pairs up- stream from the start site. Even when enhancers are moved more than a kilobase away from a start site 54 -activators can activate transcription. The best-characterized 54 -activator—the NtrC protein nitrogen regulatory protein C—stimulates transcription from the promoter of the glnA gene. This gene encodes the enzyme glutamine synthetase which synthesizes the amino acid glutamine from glutamic acid and ammonia. The 54 - RNA polymerase binds to the glnA promoter but does not melt the DNA strands and initiate transcription until it is ac- tivated by NtrC a dimeric protein. NtrC in turn is regu- lated by a protein kinase called NtrB. In response to low levels of glutamine NtrB phosphorylates dimeric NtrC which then binds to an enhancer upstream of the glnA pro- 4.3 • Control of Gene Expression in Prokaryotes 117 moter. Enhancer-bound phosphorylated NtrC then stimu- lates the 54 -polymerase bound at the promoter to separate the DNA strands and initiate transcription. Electron mi- croscopy studies have shown that phosphorylated NtrC bound at enhancers and 54 -polymerase bound at the pro- moter directly interact forming a loop in the DNA between the binding sites Figure 4-17. As discussed in Chapter 11 this activation mechanism is somewhat similar to the predominant mechanism of transcriptional activation in eukaryotes. NtrC has ATPase activity and ATP hydrolysis is required for activation of bound 54 -polymerase by phosphorylated NtrC. Evidence for this is that mutants with an NtrC defec- tive in ATP hydrolysis are invariably defective in stimulat- ing the 54 -polymerase to melt the DNA strands at the transcription start site. It is postulated that ATP hydrolysis supplies the energy required for melting the DNA strands. In contrast the 70 -polymerase does not require ATP hy- drolysis to separate the strands at a start site. Many Bacterial Responses Are Controlled by Two-Component Regulatory Systems As we’ve just seen control of the E. coli glnA gene depends on two proteins NtrC and NtrB. Such two-component reg- ulatory systems control many responses of bacteria to changes in their environment. Another example involves the E. coli proteins PhoR and PhoB which regulate transcription in response to the concentration of free phosphate. PhoR is a transmembrane protein located in the inner plasma mem- brane whose periplasmic domain binds phosphate with moderate affinity and whose cytosolic domain has protein kinase activity PhoB is a cytosolic protein. Large protein pores in the E. coli outer membrane allow ions to diffuse freely between the external environment and the periplasmic space. Consequently when the phosphate concentration in the environment falls it also falls in the periplasmic space causing phosphate to dissociate from the PhoR periplasmic domain as depicted in Figure 4-18. This causes a conformational change in the PhoR cytoplasmic do- main that activates its protein kinase activity. The activated PhoR initially transfers a -phosphate from ATP to a histi- dine side chain in the PhoR kinase domain itself. The same phosphate is then transferred to a specific aspartic acid side chain in PhoB converting PhoB from an inactive to an active transcriptional activator. Phosphorylated active PhoB then induces transcription from several genes that help the cell cope with low phosphate conditions. Many other bacterial responses are regulated by two pro- teins with homology to PhoR and PhoB. In each of these reg- ulatory systems one protein called a sensor contains a transmitter domain homologous to the PhoR protein kinase domain. The transmitter domain of the sensor protein is reg- ulated by a second unique protein domain e.g. the periplas- mic domain of PhoR that senses environmental changes. The second protein called a response regulator contains a NtrC 54 polymerase NtrC 54 polymerase b a ▲ EXPERIMENTAL FIGURE 4-17 DNA looping permits interaction of bound NtrC and 54 -polymerase. a Electron micrograph of DNA restriction fragment with phosphorylated NtrC dimer binding to the enhancer region near one end and 54 –RNA polymerase bound to the glnA promoter near the other end. b Electron micrograph of the same fragment preparation showing NtrC dimers and 54 -polymerase binding to each other with the intervening DNA forming a loop between them. From W. Su et al. 1990 Proc. Nat’l. Acad. Sci. USA 87:5505 courtesy of S. Kustu.

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receiver domain homologous to the region of PhoB that is phosphorylated by activated PhoR. The receiver domain of the response regulator is associated with a second domain that determines the protein’s function. The activity of this second functional domain is regulated by phosphorylation of the receiver domain. Although all transmitter domains are homologous as are receiver domains the transmitter do- main of a specific sensor protein will phosphorylate only spe- cific receiver domains of specific response regulators allowing specific responses to different environmental changes. Note that NtrB and NtrC discussed above func- tion as sensor and response regulator proteins respectively in the two-component regulatory system that controls tran- scription of glnA. Similar two-component histidyl-aspartyl phosphorelay regulatory systems are also found in plants. KEY CONCEPTS OF SECTION 4.3 Control of Gene Expression in Prokaryotes ■ Gene expression in both prokaryotes and eukaryotes is regulated primarily by mechanisms that control the initia- tion of transcription. ■ Binding of the subunit in an RNA polymerase to a promoter region is the first step in the initiation of tran- scription in E. coli. 118 CHAPTER 4 • Basic Molecular Genetic Mechanisms ■ The nucleotide sequence of a promoter determines its strength that is how frequently different RNA polymerase molecules can bind and initiate transcription per minute. ■ Repressors are proteins that bind to operator sequences which overlap or lie adjacent to promoters. Binding of a repressor to an operator inhibits transcription initiation. ■ The DNA-binding activity of most bacterial repressors is modulated by small effector molecules inducers. This allows bacterial cells to regulate transcription of specific genes in response to changes in the concentration of vari- ous nutrients in the environment. ■ The lac operon and some other bacterial genes also are regulated by activator proteins that bind next to promot- ers and increase the rate of transcription initiation by RNA polymerase. ■ The major sigma factor in E. coli is 70 but several other less abundant sigma factors are also found each recog- nizing different consensus promoter sequences. ■ Transcription initiation by all E. coli RNA polymerases except those containing 54 can be regulated by repres- sors and activators that bind near the transcription start site see Figure 4-16. ■ Genes transcribed by 54 –RNA polymerase are regulated by activators that bind to enhancers located ≈100 base phoA phoS phoE ugpB P P P H D H D PhoB response regulator active PhoR sensor inactive Outer membrane Inner cytoplasmic membrane Cytoplasm PhoR sensor active PhoB response regulator inactive A Porin Periplasmic space PPP FIGURE 4-18 The PhoR/PhoB two-component regulatory system in E. coli. In response to low phosphate concentrations in the environment and periplasmic space a phosphate ion dissociates from the periplasmic domain of the inactive sensor protein PhoR. This causes a conformational change that activates a protein kinase transmitter domain in the cytosolic region of PhoR. The activated transmitter domain transfers an ATP phosphate to a conserved histidine in the transmitter domain. This phosphate is then transferred to an aspartic acid in the receiver domain of the response regulator PhoB. Several PhoB proteins can be phosphorylated by one activated PhoR. Phosphorylated PhoB proteins then activate transcription from genes encoding proteins that help the cell to respond to low phosphate including phoA phoS phoE and ugpB.

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pairs upstream from the start site. When the activator and 54 –RNA polymerase interact the DNA between their binding sites forms a loop see Figure 4-17. ■ In two-component regulatory systems one protein acts as a sensor monitoring the level of nutrients or other com- ponents in the environment. Under appropriate conditions the -phosphate of an ATP is transferred first to a histi- dine in the sensor protein and then to an aspartic acid in a second protein the response regulator. The phosphory- lated response regulator then binds to DNA regulatory se- quences thereby stimulating or repressing transcription of specific genes see Figure 4-18. The Three Roles of RNA in Translation Although DNA stores the information for protein synthesis and mRNA conveys the instructions encoded in DNA most biological activities are carried out by proteins. As we saw in Chapter 3 the linear order of amino acids in each protein determines its three-dimensional structure and activity. For this reason assembly of amino acids in their correct order as encoded in DNA is critical to production of functional proteins and hence the proper functioning of cells and organisms. Translation is the whole process by which the nucleotide sequence of an mRNA is used to order and to join the amino acids in a polypeptide chain see Figure 4-1 step 3. In eu- karyotic cells protein synthesis occurs in the cytoplasm where three types of RNA molecules come together to per- form different but cooperative functions Figure 4-19: 1. Messenger RNA mRNA carries the genetic information transcribed from DNA in the form of a series of three- nucleotide sequences called codons each of which specifies a particular amino acid. 2. Transfer RNA tRNA is the key to deciphering the codons in mRNA. Each type of amino acid has its own subset of tRNAs which bind the amino acid and carry it to the growing end of a polypeptide chain if the next codon in the mRNA calls for it. The correct tRNA with its attached amino acid is selected at each step because each specific tRNA molecule contains a three-nucleotide sequence an anticodon that can base-pair with its complementary codon in the mRNA. 3. Ribosomal RNA rRNA associates with a set of proteins to form ribosomes. These complex structures which physically move along an mRNA molecule catalyze the assembly of amino acids into polypeptide chains. They also bind tRNAs and various accessory proteins necessary for protein synthesis. Ribosomes are composed of a large and a small subunit each of which contains its own rRNA molecule or molecules. 4.4 4.4 • The Three Roles of RNA in Translation 119 These three types of RNA participate in translation in all cells. Indeed development of three functionally distinct RNAs was probably the molecular key to the origin of life. How the structure of each RNA relates to its specific task is described in this section how the three types work together along with required protein factors to synthesize proteins is detailed in the following section. Since translation is essential for protein synthesis the two processes commonly are re- ferred to interchangeably. However the polypeptide chains resulting from translation undergo post-translational folding and often other changes e.g. chemical modifications asso- ciation with other chains that are required for production of mature functional proteins Chapter 3. Messenger RNA Carries Information from DNA in a Three-Letter Genetic Code As noted above the genetic code used by cells is a triplet code with every three-nucleotide sequence or codon being “read” from a specified starting point in the mRNA. Of the 64 possible codons in the genetic code 61 specify individual amino acids and three are stop codons. Table 4-1 shows that most amino acids are encoded by more than one codon. Only two—methionine and tryptophan—have a single Codon aa 1 Codon aa 2 Codon aa 3 Codon aa 4 Codon aa 5 Codon aa 6 Codon aa 7 Movement of ribosome 5 3 GGG A A A U C GGU C UUU A G C tRNA 4 leaving mRNA aa 7 -tRNA 7 arriving Ribosome CC C H C C R 6 O O N H C H C R 7 O O N H 2 C C R 5 O O H H CAG Growing polypeptide chain aa 4 aa 3 aa 2 aa 1 ▲ FIGURE 4-19 The three roles of RNA in protein synthesis. Messenger RNA mRNA is translated into protein by the joint action of transfer RNA tRNA and the ribosome which is composed of numerous proteins and two major ribosomal RNA rRNA molecules not shown. Note the base pairing between tRNA anticodons and complementary codons in the mRNA. Formation of a peptide bond between the amino group N on the incoming aa-tRNA and the carboxyl-terminal C on the growing protein chain purple is catalyzed by one of the rRNAs. aa amino acid R side group. Adapted from A. J. F . Griffiths et al. 1999 Modern Genetic Analysis W. H. Freeman and Company.

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codon at the other extreme leucine serine and arginine are each specified by six different codons. The different codons for a given amino acid are said to be synonymous. The code itself is termed degenerate meaning that more than one codon can specify the same amino acid. Synthesis of all polypeptide chains in prokaryotic and eu- karyotic cells begins with the amino acid methionine. In most mRNAs the start initiator codon specifying this amino- terminal methionine is AUG. In a few bacterial mRNAs GUG is used as the initiator codon and CUG occasionally is used as an initiator codon for methionine in eukaryotes. The three codons UAA UGA and UAG do not specify amino acids but constitute stop termination codons that mark the carboxyl terminus of polypeptide chains in almost all cells. The sequence of codons that runs from a specific 120 CHAPTER 4 • Basic Molecular Genetic Mechanisms start codon to a stop codon is called a reading frame. This precise linear array of ribonucleotides in groups of three in mRNA specifies the precise linear sequence of amino acids in a polypeptide chain and also signals where synthesis of the chain starts and stops. Because the genetic code is a comma-less non-overlapping triplet code a particular mRNA theoretically could be trans- lated in three different reading frames. Indeed some mRNAs have been shown to contain overlapping information that can be translated in different reading frames yielding different polypeptides Figure 4-20. The vast majority of mRNAs however can be read in only one frame because stop codons encountered in the other two possible reading frames termi- nate translation before a functional protein is produced. An- other unusual coding arrangement occurs because of frame- TABLE 4-1 The Genetic Code RNA to Amino Acids First Third Position Position 5 end 3 end Second Position UCA G Phe Ser Tyr Cys U Phe Ser Tyr Cys C U Leu Ser Stop Stop A Leu Ser Stop Trp G Leu Pro His Arg U Leu Pro His Arg C C Leu Pro Gln Arg A Leu Met Pro Gln Arg G Ile Thr Asn Ser U Ile Thr Asn Ser C A Ile Thr Lys Arg A Met start Thr Lys Arg G Val Ala Asp Gly U Val Ala Asp Gly C G Val Ala Glu Gly A Val Met Ala Glu Gly G AUG is the most common initiator codon GUG usually codes for valine and CUG for leucine but rarely these codons can also code for methionine to initiate a protein chain.

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shifting. In this case the protein-synthesizing machinery may read four nucleotides as one amino acid and then continue reading triplets or it may back up one base and read all suc- ceeding triplets in the new frame until termination of the chain occurs. These frameshifts are not common events but a few dozen such instances are known. The meaning of each codon is the same in most known organisms—a strong argument that life on earth evolved only once. However the genetic code has been found to dif- fer for a few codons in many mitochondria in ciliated pro- tozoans and in Acetabularia a single-celled plant. As shown in Table 4-2 most of these changes involve reading of nor- mal stop codons as amino acids not an exchange of one amino acid for another. These exceptions to the general code probably were later evolutionary developments that is at no single time was the code immutably fixed although massive changes were not tolerated once a general code began to function early in evolution. 4.4 • The Three Roles of RNA in Translation 121 The Folded Structure of tRNA Promotes Its Decoding Functions Translation or decoding of the four-nucleotide language of DNA and mRNA into the 20–amino acid language of pro- teins requires tRNAs and enzymes called aminoacyl-tRNA synthetases. To participate in protein synthesis a tRNA mol- ecule must become chemically linked to a particular amino acid via a high-energy bond forming an aminoacyl-tRNA the anticodon in the tRNA then base-pairs with a codon in mRNA so that the activated amino acid can be added to the growing polypeptide chain Figure 4-21. Some 30–40 different tRNAs have been identified in bacterial cells and as many as 50–100 in animal and plant cells. Thus the number of tRNAs in most cells is more than the number of amino acids used in protein synthesis 20 and also differs from the number of amino acid codons in the genetic code 61. Consequently many amino acids have more than one tRNA to which they can attach explaining how there can be more tRNAs than amino acids in addition many tRNAs can pair with more than one codon explaining how there can be more codons than tRNAs. The function of tRNA molecules which are 70–80 nu- cleotides long depends on their precise three-dimensional structures. In solution all tRNA molecules fold into a simi- lar stem-loop arrangement that resembles a cloverleaf when drawn in two dimensions Figure 4-22a. The four stems are short double helices stabilized by Watson-Crick base pairing three of the four stems have loops containing seven or eight bases at their ends while the remaining unlooped stem con- tains the free 3 and 5 ends of the chain. The three nu- cleotides composing the anticodon are located at the center of the middle loop in an accessible position that facilitates codon-anticodon base pairing. In all tRNAs the 3 end of the unlooped amino acid acceptor stem has the sequence CCA which in most cases is added after synthesis and pro- cessing of the tRNA are complete. Several bases in most tRNAs also are modified after synthesis. Viewed in three GCU UGU UUA CGA AUU Ala Cys Leu Arg Ile 5 Polypeptide mRNA CUU GUU UAC GAA UUA Leu Val Tyr Glu Leu 5 A G Frame 1 Frame 2 ▲ FIGURE 4-20 Example of how the genetic code—a non-overlapping comma-less triplet code—can be read in different frames. If translation of the mRNA sequence shown begins at two different upstream start sites not shown then two overlapping reading frames are possible. In this example the codons are shifted one base to the right in the lower frame. As a result the same nucleotide sequence specifies different amino acids during translation. Although they are rare many instances of such overlaps have been discovered in viral and cellular genes of prokaryotes and eukaryotes. It is theoretically possible for the mRNA to have a third reading frame. TABLE 4-2 Known Deviations from the Universal Genetic Code Universal Unusual Codon Code Code Occurrence UGA Stop Trp Mycoplasma Spiroplasma mitochondria of many species CUG Leu Thr Mitochondria in yeasts UAA UAG Stop Gln Acetabularia Tetrahymena Paramecium etc. UGA Stop Cys Euplotes “Unusual code” is used in nuclear genes of the listed organisms and in mitochondrial genes as indicated. SOURCE: S. Osawa et al. 1992 Microbiol. Rev. 56:229.

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dimensions the folded tRNA molecule has an L shape with the anticodon loop and acceptor stem forming the ends of the two arms Figure 4-22b. Nonstandard Base Pairing Often Occurs Between Codons and Anticodons If perfect Watson-Crick base pairing were demanded between codons and anticodons cells would have to contain exactly 61 different tRNA species one for each codon that specifies an 122 CHAPTER 4 • Basic Molecular Genetic Mechanisms amino acid. As noted above however many cells contain fewer than 61 tRNAs. The explanation for the smaller number lies in the capability of a single tRNA anticodon to recognize more than one but not necessarily every codon corresponding to a given amino acid. This broader recognition can occur be- cause of nonstandard pairing between bases in the so-called wobble position: that is the third 3 base in an mRNA codon and the corresponding first 5 base in its tRNA anticodon. The first and second bases of a codon almost always form standard Watson-Crick base pairs with the third and Amino acid Phe Aminoacyl- tRNA synthetase specific for Phe C C H 2 N CH 2 H O AAA C C H 2 N CH 2 H O O AAA C C H 2 N CH 2 H O O AAA UUU mRNA tRNA Phe binds to the UUU codon 3 5 Linkage of Phe to tRNA Phe Net result: Phe is selected by its codon tRNA specific for Phe tRNA Phe Aminoacyl-tRNA OH ATP AMP + PP i High-energy ester bond OH 1 2 ▲ FIGURE 4-21 Two-step decoding process for translating nucleic acid sequences in mRNA into amino acid sequences in proteins. Step 1: An aminoacyl-tRNA synthetase first couples a specific amino acid via a high-energy ester bond yellow to either the 2 or 3 hydroxyl of the terminal adenosine in the corresponding tRNA. Step 2: A three-base sequence in the tRNA the anticodon then base-pairs with a codon in the mRNA specifying the attached amino acid. If an error occurs in either step the wrong amino acid may be incorporated into a polypeptide chain. Phe phenylalanine. A C C A C C U G C G G G C G U G U U C A G G C C U U A G T A C D D G G G C U C C D C G G G A A G G G G G G G C U U U I C C C C C C U G C G G A C G C 3 5 3 5 mG m 2 G ml T CG loop D loop Variable loop Anticodon loop Anticodon Codon mRNA Acceptor stem 1 1 2 2 3 3 a D I T m dihydrouridine inosine pseudouridine ribothymidine methyl group FIGURE 4-22 Structure of tRNAs. a Although the exact nucleotide sequence varies among tRNAs they all fold into four base-paired stems and three loops. The CCA sequence at the 3 end also is found in all tRNAs. Attachment of an amino acid to the 3 A yields an aminoacyl-tRNA. Some of the A C G and U residues are modified in most tRNAs see key. Dihydrouridine D is nearly always present in the D loop likewise ribothymidine T and pseudouridine are almost always present in the T CG loop. Yeast alanine tRNA represented here also contains other modified bases. The triplet at the tip of the anticodon loop base-pairs with the corresponding codon in mRNA. b Three- dimensional model of the generalized back- bone of all tRNAs. Note the L shape of the molecule. Part a see R. W. Holly et al. 1965 Science 147:1462 part b from J. G. Arnez and D. Moras 1997 Trends Biochem. Sci. 22:211 . b T CG loop D loop Variable loop Anticodon loop Acceptor stem 3 5 C C A

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second bases respectively of the corresponding anticodon but four nonstandard interactions can occur between bases in the wobble position. Particularly important is the G·U base pair which structurally fits almost as well as the stan- dard G·C pair. Thus a given anticodon in tRNA with G in the first wobble position can base-pair with the two corre- sponding codons that have either pyrimidine C or U in the third position Figure 4-23. For example the phenylalanine codons UUU and UUC 5 n3 are both recognized by the tRNA that has GAA 5 n3 as the anticodon. In fact any two codons of the type NNPyr N any base Pyr pyrimidine encode a single amino acid and are decoded by a single tRNA with G in the first wobble position of the anticodon. Although adenine rarely is found in the anticodon wobble position many tRNAs in plants and animals contain inosine 4.4 • The Three Roles of RNA in Translation 123 I a deaminated product of adenine at this position. Ino- sine can form nonstandard base pairs with A C and U. A tRNA with inosine in the wobble position thus can recognize the corresponding mRNA codons with A C or U in the third wobble position see Figure 4-23. For this reason inosine- containing tRNAs are heavily employed in translation of the synonymous codons that specify a single amino acid. For ex- ample four of the six codons for leucine CUA CUC CUU and UUA are all recognized by the same tRNA with the an- ticodon 3 -GAI-5 the inosine in the wobble position forms nonstandard base pairs with the third base in the four codons. In the case of the UUA codon a nonstandard G·U pair also forms between position 3 of the anticodon and position 1 of the codon. Aminoacyl-tRNA Synthetases Activate Amino Acids by Covalently Linking Them to tRNAs Recognition of the codon or codons specifying a given amino acid by a particular tRNA is actually the second step in de- coding the genetic message. The first step attachment of the appropriate amino acid to a tRNA is catalyzed by a specific aminoacyl-tRNA synthetase. Each of the 20 different syn- thetases recognizes one amino acid and all its compatible or cognate tRNAs. These coupling enzymes link an amino acid to the free 2 or 3 hydroxyl of the adenosine at the 3 ter- minus of tRNA molecules by an ATP-requiring reaction. In this reaction the amino acid is linked to the tRNA by a high- energy bond and thus is said to be activated. The energy of this bond subsequently drives formation of the peptide bonds linking adjacent amino acids in a growing polypeptide chain. The equilibrium of the aminoacylation reaction is driven fur- ther toward activation of the amino acid by hydrolysis of the high-energy phosphoanhydride bond in the released pyro- phosphate see Figure 4-21. Because some amino acids are so similar structurally aminoacyl-tRNA synthetases sometimes make mistakes. These are corrected however by the enzymes themselves which have a proofreading activity that checks the fit in their amino acid–binding pocket. If the wrong amino acid be- comes attached to a tRNA the bound synthetase catalyzes removal of the amino acid from the tRNA. This crucial func- tion helps guarantee that a tRNA delivers the correct amino acid to the protein-synthesizing machinery. The overall error rate for translation in E. coli is very low approximately 1 per 50000 codons evidence of the importance of proofreading by aminoacyl-tRNA synthetases. Ribosomes Are Protein-Synthesizing Machines If the many components that participate in translating mRNA had to interact in free solution the likelihood of si- multaneous collisions occurring would be so low that the rate of amino acid polymerization would be very slow. The efficiency of translation is greatly increased by the binding of the mRNA and the individual aminoacyl-tRNAs to the most If these bases are in first or wobble position of anticodon 3 5 3 5 3 2 1 3 2 1 1 2 3 tRNA 3 5 tRNA mRNA 3 5 1 2 3 mRNA then the tRNA may recognize codons in mRNA having these bases in third position C A U C U A G U G CA G U I If these bases are in third or wobble position of codon of an mRNA then the codon may be recognized by a tRNA having these bases in first position of anticodon C U A G I U I G I CA G U ▲ FIGURE 4-23 Nonstandard codon-anticodon base pairing at the wobble position. The base in the third or wobble position of an mRNA codon often forms a nonstandard base pair with the base in the first or wobble position of a tRNA anticodon. Wobble pairing allows a tRNA to recognize more than one mRNA codon top conversely it allows a codon to be recognized by more than one kind of tRNA bottom although each tRNA will bear the same amino acid. Note that a tRNA with I inosine in the wobble position can “read” become paired with three different codons and a tRNA with G or U in the wobble position can read two codons. Although A is theoretically possible in the wobble position of the anticodon it is almost never found in nature.

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abundant RNA-protein complex in the cell the ribosome which directs elongation of a polypeptide at a rate of three to five amino acids added per second. Small proteins of 100–200 amino acids are therefore made in a minute or less. On the other hand it takes 2–3 hours to make the largest known protein titin which is found in muscle and contains about 30000 amino acid residues. The cellular machine that accomplishes this task must be precise and persistent. With the aid of the electron microscope ribosomes were first discovered as small discrete RNA-rich particles in cells that secrete large amounts of protein. However their role in protein synthesis was not recognized until reasonably pure ribosome preparations were obtained. In vitro radiolabeling experiments with such preparations showed that radioactive amino acids first were incorporated into growing polypep- tide chains that were associated with ribosomes before ap- pearing in finished chains. A ribosome is composed of three in bacteria or four in eukaryotes different rRNA molecules and as many as 83 proteins organized into a large subunit and a small subunit Figure 4-24. The ribosomal subunits and the rRNA mole- cules are commonly designated in Svedberg units S a measure of the sedimentation rate of suspended particles cen- 124 CHAPTER 4 • Basic Molecular Genetic Mechanisms trifuged under standard conditions. The small ribosomal subunit contains a single rRNA molecule referred to as small rRNA. The large subunit contains a molecule of large rRNA and one molecule of 5S rRNA plus an additional molecule of 5.8S rRNA in vertebrates. The lengths of the rRNA mol- ecules the quantity of proteins in each subunit and conse- quently the sizes of the subunits differ in bacterial and eukaryotic cells. The assembled ribosome is 70S in bacteria and 80S in vertebrates. But more interesting than these dif- ferences are the great structural and functional similarities between ribosomes from all species. This consistency is an- other reflection of the common evolutionary origin of the most basic constituents of living cells. The sequences of the small and large rRNAs from several thousand organisms are now known. Although the primary nucleotide sequences of these rRNAs vary considerably the same parts of each type of rRNA theoretically can form base- paired stem-loops which would generate a similar three- dimensional structure for each rRNA in all organisms. The actual three-dimensional structures of bacterial rRNAs from Thermus thermopolis recently have been determined by x- ray crystallography of the 70S ribosome. The multiple much smaller ribosomal proteins for the most part are associated 70S Total: 31 5S 120 rNTs + 23S 2900 rNTs 16S 1500 rNTs 28S : 5.8S 4800 rNTs 160 rNTs 28S 5.8S 18S 1900 rNTs rRNA Prokaryotic Proteins Subunits Assembled ribosomes Total: 21 80S Total: 50 5S 120 rNTs + + + Eukaryotic vertebrate Total: 33 50S 30S 60S 40S 5S 23S 5S 28S 5.8S 16S 18S ▲ FIGURE 4-24 The general structure of ribosomes in prokaryotes and eukaryotes. In all cells each ribosome consists of a large and a small subunit. The two subunits contain rRNAs red of different lengths as well as a different set of proteins. All ribosomes contain two major rRNA molecules 23S and 16S rRNA in bacteria 28S and 18S rRNA in vertebrates and a 5S rRNA. The large subunit of vertebrate ribosomes also contains a 5.8S rRNA base-paired to the 28S rRNA. The number of ribonucleotides rNTs in each rRNA type is indicated.

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with the surface of the rRNAs. Although the number of pro- tein molecules in ribosomes greatly exceeds the number of RNA molecules RNA constitutes about 60 percent of the mass of a ribosome. During translation a ribosome moves along an mRNA chain interacting with various protein factors and tRNAs and very likely undergoing large conformational changes. Despite the complexity of the ribosome great progress has been made in determining the overall structure of bacterial ribosomes and in identifying various reactive sites. X-ray crystallographic studies on the T. thermophilus 70S ribo- some for instance not only have revealed the dimensions and overall shape of the ribosomal subunits but also have lo- calized the positions of tRNAs bound to the ribosome during elongation of a growing protein chain. In addition power- ful chemical techniques such as footprinting which is de- scribed in Chapter 11 have been used to identify specific nucleotide sequences in rRNAs that bind to protein or an- other RNA. Some 40 years after the initial discovery of ri- bosomes their overall structure and functioning during protein synthesis are finally becoming clear as we describe in the next section. KEY CONCEPTS OF SECTION 4.4 The Three Roles of RNA in Translation ■ Genetic information is transcribed from DNA into mRNA in the form of a comma-less overlapping degen- erate triplet code. ■ Each amino acid is encoded by one or more three- nucleotide sequences codons in mRNA. Each codon spec- ifies one amino acid but most amino acids are encoded by multiple codons see Table 4-1. ■ The AUG codon for methionine is the most common start codon specifying the amino acid at the NH 2 -terminus of a protein chain. Three codons UAA UAG UGA function as stop codons and specify no amino acids. ■ A reading frame the uninterrupted sequence of codons in mRNA from a specific start codon to a stop codon is translated into the linear sequence of amino acids in a polypeptide chain. ■ Decoding of the nucleotide sequence in mRNA into the amino acid sequence of proteins depends on tRNAs and aminoacyl-tRNA synthetases. ■ All tRNAs have a similar three-dimensional structure that includes an acceptor arm for attachment of a specific amino acid and a stem-loop with a three-base anticodon sequence at its ends see Figure 4-22. The anticodon can base-pair with its corresponding codon in mRNA. ■ Because of nonstandard interactions a tRNA may base- pair with more than one mRNA codon conversely a par- ticular codon may base-pair with multiple tRNAs. In each 4.5 • Stepwise Synthesis of Proteins on Ribosomes 125 case however only the proper amino acid is inserted into a growing polypeptide chain. ■ Each of the 20 aminoacyl-tRNA synthetases recognizes a single amino acid and covalently links it to a cognate tRNA forming an aminoacyl-tRNA see Figure 4-21. This reaction activates the amino acid so it can participate in peptide bond formation. ■ Both prokaryotic and eukaryotic ribosomes—the large ribonucleoprotein complexes on which translation occurs— consist of a small and a large subunit see Figure 4-24. Each subunit contains numerous different proteins and one major rRNA molecule small or large. The large subunit also con- tains one accessory 5S rRNA in bacteria and two accessory rRNAs in eukaryotes 5S and 5.8S in vertebrates. ■ Analogous rRNAs from many different species fold into quite similar three-dimensional structures containing nu- merous stem-loops and binding sites for proteins mRNA and tRNAs. Much smaller ribosomal proteins are associ- ated with the periphery of the rRNAs. Stepwise Synthesis of Proteins on Ribosomes The previous sections have introduced the major participants in protein synthesis—mRNA aminoacylated tRNAs and ri- bosomes containing large and small rRNAs. We now take a detailed look at how these components are brought together to carry out the biochemical events leading to formation of polypeptide chains on ribosomes. Similar to transcription the complex process of translation can be divided into three stages—initiation elongation and termination—which we consider in order. We focus our description on translation in eukaryotic cells but the mechanism of translation is funda- mentally the same in all cells. Methionyl-tRNA i Met Recognizes the AUG Start Codon As noted earlier the AUG codon for methionine functions as the start codon in the vast majority of mRNAs. A critical aspect of translation initiation is to begin protein synthesis at the start codon thereby establishing the correct reading frame for the entire mRNA. Both prokaryotes and eukary- otes contain two different methionine tRNAs: tRNA i Met can initiate protein synthesis and tRNA Met can incorporate me- thionine only into a growing protein chain. The same aminoacyl-tRNA synthetase MetRS charges both tRNAs with methionine. But only Met-tRNA i Met i.e. activated me- thionine attached to tRNA i Met can bind at the appropriate site on the small ribosomal subunit the P site to begin syn- thesis of a polypeptide chain. The regular Met-tRNA Met and all other charged tRNAs bind only to another ribosomal site the A site as described later. 4.5

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Translation Initiation Usually Occurs Near the First AUG Closest to the 5 End of an mRNA During the first stage of translation a ribosome assembles complexed with an mRNA and an activated initiator tRNA which is correctly positioned at the start codon. Large and small ribosomal subunits not actively engaged in translation are kept apart by binding of two initiation factors desig- nated eIF3 and eIF6 in eukaryotes. A translation preinitia- tion complex is formed when the 40S subunit–eIF3 complex is bound by eIF1A and a ternary complex of the Met- tRNA i Met eIF2 and GTP Figure 4-25 step 1. Cells can reg- ulate protein synthesis by phosphorylating a serine residue on the eIF2 bound to GDP the phosphorylated complex is unable to exchange the bound GDP for GTP and cannot bind Met-tRNA i Met thus inhibiting protein synthesis. During translation initiation the 5 cap of an mRNA to be translated is bound by the eIF4E subunit of the eIF4 cap- binding complex. The mRNA-eIF4 complex then associates with the preinitiation complex through an interaction of the eIF4G subunit and eIF3 forming the initiation complex Fig- ure 4-25 step 2. The initiation complex then probably slides along or scans the associated mRNA as the helicase activity of eIF4A uses energy from ATP hydrolysis to unwind the RNA secondary structure. Scanning stops when the tRNA i Met anticodon recognizes the start codon which is the first AUG downstream from the 5 end in most eukaryotic mRNAs step 3. Recognition of the start codon leads to hy- drolysis of the GTP associated with eIF2 an irreversible step that prevents further scanning. Selection of the initiating AUG is facilitated by specific surrounding nucleotides called the Kozak sequence for Marilyn Kozak who defined it: 5 ACCAUGG 3 . The A preceding the AUG underlined and the G immediately following it are the most important nu- cleotides affecting translation initiation efficiency. Once the small ribosomal subunit with its bound Met-tRNA i Met is cor- rectly positioned at the start codon union with the large 60S ribosomal subunit completes formation of an 80S ribosome. This requires the action of another factor eIF5 and hydrolysis 126 CHAPTER 4 • Basic Molecular Genetic Mechanisms 3 1A 1A 4E 3 4G 4A 4B 60S 60S 40S 60S 80S ribosome m 7 Gppp Preinitiation complex eIF2•GTP + Met-tRNA i Met ternary complex eIF1A eIF3 eIF6 40S + 40S 2 Met -GTP Met AUG AAA n Met eIF6 eIF5•GDP + P i 60S subunit-eIF6 eIF5•GTP eIF1A eIF3 eIF4 complex eIF2•GDP + P i ADP + P i ATP P 80S 40S 6 3 3 eIF4 cap-binding complex + mRNA 2 Met -GTP AUG AAA n AUG AAA n Initiation complex 5 3 2 structure unwinding scanning and start site recognition 1 2 3 4 FIGURE 4-25 Initiation of translation in eukaryotes. Inset When a ribosome dissociates at the termination of translation the 40S and 60S subunits associate with initiation factors eIF3 and eIF6 forming complexes that can initiate another round of translation. Steps 1 and 2: Sequential addition of the indicated components to the 40S subunit–eIF3 complex forms the initiation complex. Step 3: Scanning of the mRNA by the associated initiation complex leads to positioning of the small subunit and bound Met-tRNA i Met at the start codon. Step 4 : Association of the large subunit 60S forms an 80S ribosome ready to translate the mRNA. Two initiation factors eIF2 step 1 and eIF5 step 4 are GTP-binding proteins whose bound GTP is hydrolyzed during translation initiation. The precise time at which particular initiation factors are released is not yet well characterized. See the text for details. Adapted from R. Mendez and J. D. Richter 2001 Nature Rev. Mol. Cell Biol. 2:521 .

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of a GTP associated with it step 4. Coupling the joining reaction to GTP hydrolysis makes this an irreversible step so that the ribosomal subunits do not dissociate until the entire mRNA is translated and protein synthesis is terminated. As dis- cussed later during chain elongation the growing polypeptide remains attached to the tRNA at this P site in the ribosome. The eukaryotic protein-synthesizing machinery begins translation of most cellular mRNAs within about 100 nu- cleotides of the 5 capped end as just described. However some cellular mRNAs contain an internal ribosome entry site IRES located far downstream of the 5 end. In addition translation of some viral mRNAs which lack a 5 cap is ini- tiated at IRESs by the host-cell machinery of infected eu- karyotic cells. Some of the same translation initiation factors that assist in ribosome scanning from a 5 cap are required for locating an internal AUG start codon but exactly how an IRES is recognized is less clear. Recent results indicate that some IRESs fold into an RNA structure that binds to a third site on the ribosome the E site thereby positioning a nearby internal AUG start codon in the P site. During Chain Elongation Each Incoming Aminoacyl-tRNA Moves Through Three Ribosomal Sites The correctly positioned eukaryotic 80S ribosome–Met- tRNA i Met complex is now ready to begin the task of stepwise addition of amino acids by the in-frame translation of the mRNA. As is the case with initiation a set of special proteins termed elongation factors EFs are required to carry out this process of chain elongation. The key steps in elongation are entry of each succeeding aminoacyl-tRNA formation of a peptide bond and the movement or translocation of the ribosome one codon at a time along the mRNA. 4.5 • Stepwise Synthesis of Proteins on Ribosomes 127 2 EF1α •GTP EF2•GTP EF2•GDP + P i 80S ribosome 5 EF1α •GDP 3 EF1α •GTP 4 EF1α •GTP Peptide bond formation Ribosome translocation Entry of next aa-tRNA at A site GTP hydrolysis ribosome conformational change + P i P EA 1 2 3 4 1 P EA 1 P EA 1 Met i EF1α GTP 2 2 P EA 1 2 P EA 1 2 • ▲ FIGURE 4-26 Cycle of peptidyl chain elongation during translation in eukaryotes. Once the 80S ribosome with Met-tRNA i Met in the ribosome P site is assembled top a ternary complex bearing the second amino acid aa 2 coded by the mRNA binds to the A site step 1 . Following a conformational change in the ribosome induced by hydrolysis of GTP in EF1 GTP step 2 the large rRNA catalyzes peptide bond formation between Met i and aa 2 step 3 . Hydrolysis of GTP in EF2 GTP causes another conformational change in the ribosome that results in its translocation one codon along the mRNA and shifts the unacylated tRNA i Met to the E site and the tRNA with the bound peptide to the P site step 4 . The cycle can begin again with binding of a ternary complex bearing aa 3 to the now-open A site. In the second and subsequent elongation cycles the tRNA at the E site is ejected during step 2 as a result of the conformational change induced by hydrolysis of GTP in EF1 GTP . See the text for details. Adapted from K. H. Nierhaus et al. 2000 in R. A. Garrett et al. eds. The Ribosome: Structure Function Antibiotics and Cellular Interactions ASM Press p. 319. At the completion of translation initiation as noted al- ready Met-tRNA i Met is bound to the P site on the assembled 80S ribosome Figure 4-26 top. This region of the ribosome is called the P site because the tRNA chemically linked to the growing polypeptide chain is located here. The second MEDIA CONNECTIONS Focus Animation: Protein Synthesis ▲

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aminoacyl-tRNA is brought into the ribosome as a ternary complex in association with EF1 GTP and becomes bound to the A site so named because it is where aminoacylated tRNAs bind step 1. If the anticodon of the incoming sec- ond aminoacyl-tRNA correctly base-pairs with the second codon of the mRNA the GTP in the associated EF1 GTP is hydrolyzed. The hydrolysis of GTP promotes a conforma- tional change in the ribosome that leads to tight binding of the aminoacyl-tRNA in the A site and release of the resulting EF1 GDP complex step 2. This conformational change also positions the aminoacylated 3 end of the tRNA in the A site in close proximity to the 3 end of the Met-tRNA i Met in the P site. GTP hydrolysis and hence tight binding does not occur if the anticodon of the incoming aminoacyl-tRNA can- not base-pair with the codon at the A site. In this case the ternary complex diffuses away leaving an empty A site that can associate with other aminoacyltRNA–EF1 GTP com- plexes until a correctly base-paired tRNA is bound. This phe- nomenon contributes to the fidelity with which the correct aminoacyl-tRNA is loaded into the A site. With the initiating Met-tRNA i Met at the P site and the second aminoacyl-tRNA tightly bound at the A site the amino group of the second amino acid reacts with the “acti- vated” ester-linked methionine on the initiator tRNA forming a peptide bond Figure 4-26 step 3 see Figures 4-19 and 4-21. This peptidyltransferase reaction is catalyzed by the large rRNA which precisely orients the interacting atoms permitting the reaction to proceed. The catalytic abil- ity of the large rRNA in bacteria has been demonstrated by carefully removing the vast majority of the protein from large ribosomal subunits. The nearly pure bacterial 23S rRNA can catalyze a peptidyltransferase reaction between analogs of aminoacylated-tRNA and peptidyl-tRNA. Further support for the catalytic role of large rRNA in protein syn- thesis comes from crystallographic studies showing that no proteins lie near the site of peptide bond synthesis in the crys- tal structure of the bacterial large subunit. Following peptide bond synthesis the ribosome is translocated along the mRNA a distance equal to one codon. This translocation step is promoted by hydroly- sis of the GTP in eukaryotic EF2 GTP. As a result of translocation tRNA i Met now without its activated methi- onine is moved to the E exit site on the ribosome concurrently the second tRNA now covalently bound to a dipeptide a peptidyl-tRNA is moved to the P site Figure 4-26 step 4. Translocation thus returns the ri- bosome conformation to a state in which the A site is open and able to accept another aminoacylated tRNA complexed with EF1 GTP beginning another cycle of chain elongation. Repetition of the elongation cycle depicted in Figure 4-26 adds amino acids one at a time to the C-terminus of the growing polypeptide as directed by the mRNA sequence until a stop codon is encountered. In subsequent cycles the conformational change that occurs in step 2 ejects the 128 CHAPTER 4 • Basic Molecular Genetic Mechanisms unacylated tRNA from the E site. As the nascent polypeptide chain becomes longer it threads through a channel in the large ribosomal subunit exiting at a position opposite the side that interacts with the small subunit Figure 4-27. The locations of tRNAs bound at the A P and E sites are visible in the recently determined crystal structure of the bac- terial ribosome Figure 4-28. Base pairing is also apparent between the tRNAs in the A and P sites with their respective codons in mRNA see Figure 4-28 inset. An RNA-RNA hybrid of only three base pairs is not stable under physio- a 50S 30S 70S ▲ FIGURE 4-27 Low-resolution model of E. coli 70S ribo- some. a Top panels show cryoelectron microscopic images of E. coli 70S ribosomes and 50S and 30S subunits. Bottom panels show computer-derived averages of many dozens of images in the same orientation. b Model of a 70S ribosome based on the computer-derived images and on chemical cross-linking studies. Three tRNAs are superimposed on the A pink P green and E yellow sites. The nascent polypeptide chain is buried in a tunnel in the large ribosomal subunit that begins close to the acceptor stem of the tRNA in the P site. See I. S. Gabashvili et al. 2000 Cell 100:537 courtesy of J. Frank. b 50S Polypeptide 30S mRNA 3 5 E P A

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logical conditions. However multiple interactions between the large and small rRNAs and general domains of tRNAs e.g. the D and T CG loops stabilize the tRNAs in the A and P sites while other RNA-RNA interactions sense correct codon-anticodon base pairing assuring that the genetic code is read properly. Translation Is Terminated by Release Factors When a Stop Codon Is Reached The final stage of translation like initiation and elongation requires highly specific molecular signals that decide the fate of the mRNA–ribosome–tRNA-peptidyl complex. Two types of specific protein release factors RFs have been discovered. Eukaryotic eRF1 whose shape is similar to that of tRNAs apparently acts by binding to the ribosomal A site and rec- ognizing stop codons directly. Like some of the initiation and elongation factors discussed previously the second eukary- otic release factor eRF3 is a GTP-binding protein. The eRF3 GTP acts in concert with eRF1 to promote cleavage of the peptidyl-tRNA thus releasing the completed protein 4.5 • Stepwise Synthesis of Proteins on Ribosomes 129 E P A a 70S b 50S c 30S ▲ FIGURE 4-28 Structure of T. thermophilus 70S ribosome as determined by x-ray crystallography. a Model of the entire ribosome viewed from the side diagrammed in Figure 4-26 with large subunit on top and small subunit below. The tRNAs positioned at the A blue P yellow and E green sites are visible in the interface between the subunits with their anticodon loops pointing down into the small subunit. 16S rRNA is cyan 23S rRNA purple 5S rRNA pink mRNA red small ribosomal proteins dark gray and large ribosomal proteins light gray. Note that the ribosomal proteins are located primarily on the surface of the ribosome and the rRNAs on the inside. b View of the large subunit rotated 90° about the horizontal from the view in a showing the face that interacts with the small subunit. The tRNA anticodon loops point out of the page. In the intact ribosome these extend into the small subunit where the anticodons of the tRNAs in the A and P sites base-pair with codons in the mRNA. c View of the face of the small subunit that interacts with the large subunit in b. Here the tRNA anticodon loops point into the page. The T CG loops and acceptor stems extend out of the page and the 3 CCA ends of the tRNAs in the A and P sites point downward. Note the close opposition of the acceptor stems of tRNAs in the A and P sites which allows the amino group of the acylated tRNA in the A site to react with the carboxyl-terminal C of the peptidyl-tRNA in the P site see Figure 4-19. In the intact ribosome these are located at the peptidyltransferase active site of the large subunit. Adapted from M. M. Yusupov et al. 2001 Science 292:883. chain Figure 4-29. Bacteria have two release factors RF1 and RF2 that are functionally analogous to eRF1 and a GTP-binding factor RF3 that is analogous to eRF3. After its release from the ribosome a newly synthesized protein folds into its native three-dimensional conformation a process facilitated by other proteins called chaperones Chapter 3. Additional release factors then promote dissoci- ation of the ribosome freeing the subunits mRNA and ter- minal tRNA for another round of translation. We can now see that one or more GTP-binding proteins participate in each stage of translation. These proteins be- long to the GTPase superfamily of switch proteins that cycle between a GTP-bound active form and GDP-bound inactive form see Figure 3-29. Hydrolysis of the bound GTP is thought to cause conformational changes in the GTPase itself or other associated proteins that are critical to various com- plex molecular processes. In translation initiation for in- stance hydrolysis of eIF2 GTP to eIF2 GDP prevents further scanning of the mRNA once the start site is encountered and allows binding of the large ribosomal subunit to the small subunit see Figure 4-25 step 3. Similarly hydrolysis of

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disengage from the 3 end of an mRNA. Simultaneous trans- lation of an mRNA by multiple ribosomes is readily observ- able in electron micrographs and by sedimentation analysis revealing mRNA attached to multiple ribosomes bearing nascent growing polypeptide chains. These structures re- ferred to as polyribosomes or polysomes were seen to be cir- cular in electron micrographs of some tissues. Subsequent studies with yeast cells explained the circular shape of poly- ribosomes and suggested the mode by which ribosomes re- cycle efficiently. These studies revealed that multiple copies of a cytosolic protein found in all eukaryotic cells polyA-binding protein PABPI can interact with both an mRNA polyA tail and the 4G subunit of yeast eIF4. Moreover the 4E subunit of yeast eIF4 binds to the 5 end of an mRNA. As a result of these interactions the two ends of an mRNA molecule can be bridged by the intervening proteins forming a “circular” mRNA Figure 4-30. Because the two ends of a polysome are relatively close together ribosomal subunits that disen- gage from the 3 end are positioned near the 5 end facili- tating re-initiation by the interaction of the 40S subunit with eIF4 bound to the 5 cap. The circular pathway depicted in Figure 4-31 which may operate in many eukaryotic cells would enhance ribosome recycling and thus increase the ef- ficiency of protein synthesis. EF2 GTP to EF2 GDP during chain elongation leads to translocation of the ribosome along the mRNA see Figure 4-26 step 4. Polysomes and Rapid Ribosome Recycling Increase the Efficiency of Translation As noted earlier translation of a single eukaryotic mRNA molecule to yield a typical-sized protein takes 30–60 sec- onds. Two phenomena significantly increase the overall rate at which cells can synthesize a protein: the simultaneous translation of a single mRNA molecule by multiple ribo- somes and rapid recycling of ribosomal subunits after they 130 CHAPTER 4 • Basic Molecular Genetic Mechanisms P EA UAA P EA Peptidyl-tRNA cleavage 5 3 eRF1 + eRF3•GTP eRF1 + eRF3•GDP + P i 5 3 eRF1 -GTP eRF3 UAA ▲ FIGURE 4-29 Termination of translation in eukaryotes. When a ribosome bearing a nascent protein chain reaches a stop codon UAA UGA UAG release factor eRF1 enters the ribosomal complex probably at or near the A site together with eRF3 GTP . Hydrolysis of the bound GTP is accompanied by cleavage of the peptide chain from the tRNA in the P site and release of the tRNAs and the two ribosomal subunits. ▲ EXPERIMENTAL FIGURE 4-30 Eukaryotic mRNA forms a circular structure owing to interactions of three proteins. In the presence of purified polyA-binding protein I PABPI eIF4E and eIF4G eukaryotic mRNAs form circular structures visible in this force-field electron micrograph. In these structures protein-protein and protein-mRNA interactions form a bridge between the 5 and 3 ends of the mRNA as diagrammed in Figure 4-31. Courtesy of A. Sachs.

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4.6 • DNA Replication 131 80S 40S 60S elF4E 5 A A A A A A A A m 7 G elF4G mRNA 3 PABPI PABPI ▲ FIGURE 4-31 Model of protein synthesis on circular polysomes and recycling of ribosomal subunits. Multiple individual ribosomes can simultaneously translate a eukaryotic mRNA shown here in circular form stabilized by interactions between proteins bound at the 3 and 5 ends. When a ribosome completes translation and dissociates from the 3 end the separated subunits can rapidly find the nearby 5 cap m 7 G and initiate another round of synthesis. KEY CONCEPTS OF SECTION 4.5 Stepwise Synthesis of Proteins on Ribosomes ■ Of the two methionine tRNAs found in all cells only one tRNA i Met functions in initiation of translation. ■ Each stage of translation—initiation chain elongation and termination—requires specific protein factors includ- ing GTP-binding proteins that hydrolyze their bound GTP to GDP when a step has been completed successfully. ■ During initiation the ribosomal subunits assemble near the translation start site in an mRNA molecule with the tRNA carrying the amino-terminal methionine Met-tRNA i Met base-paired with the start codon Figure 4-25. ■ Chain elongation entails a repetitive four-step cycle: loose binding of an incoming aminoacyl-tRNA to the A site on the ribosome tight binding of the correct aminoacyl-tRNA to the A site accompanied by release of the previously used tRNA from the E site transfer of the growing peptidyl chain to the incoming amino acid catalyzed by large rRNA and translocation of the ribosome to the next codon thereby moving the peptidyl-tRNA in the A site to the P site and the now unacylated tRNA in the P site to the E site see Figure 4-26. ■ In each cycle of chain elongation the ribosome undergoes two conformational changes monitored by GTP-binding proteins. The first permits tight binding of the incoming aminoacyl-tRNA to the A site and ejection of a tRNA from the E site and the second leads to translocation. ■ Termination of translation is carried out by two types of termination factors: those that recognize stop codons and those that promote hydrolysis of peptidyl-tRNA see Figure 4-29. ■ The efficiency of protein synthesis is increased by the si- multaneous translation of a single mRNA by multiple ri- bosomes. In eukaryotic cells protein-mediated interactions MEDIA CONNECTIONS Overview Animation: Life Cycle of an mRNA bring the two ends of a polyribosome close together thereby promoting the rapid recycling of ribosomal sub- units which further increases the efficiency of protein syn- thesis see Figure 4-31. DNA Replication Now that we have seen how genetic information encoded in the nucleotide sequences of DNA is translated into the struc- tures of proteins that perform most cell functions we can ap- preciate the necessity of the precise copying of DNA sequences during DNA replication see Figure 4-1 step 4. The regular pairing of bases in the double-helical DNA struc- ture suggested to Watson and Crick that new DNA strands are synthesized by using the existing parental strands as templates in the formation of new daughter strands comple- mentary to the parental strands. This base-pairing template model theoretically could pro- ceed either by a conservative or a semiconservative mecha- nism. In a conservative mechanism the two daughter strands would form a new double-stranded duplex DNA molecule and the parental duplex would remain intact. In a semicon- servative mechanism the parental strands are permanently separated and each forms a duplex molecule with the daugh- ter strand base-paired to it. Definitive evidence that duplex DNA is replicated by a semiconservative mechanism came from a now classic experiment conducted by M. Meselson and W. F. Stahl outlined in Figure 4-32. Copying of a DNA template strand into a complemen- tary strand thus is a common feature of DNA replication and transcription of DNA into RNA. In both cases the informa- tion in the template is preserved. In some viruses single- stranded RNA molecules function as templates for synthesis of complementary RNA or DNA strands. However the vast preponderance of RNA and DNA in cells is synthesized from preexisting duplex DNA. 4.6 ▲

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DNA Polymerases Require a Primer to Initiate Replication Analogous to RNA DNA is synthesized from deoxynucleo- side 5 -triphosphate precursors dNTPs. Also like RNA syn- thesis DNA synthesis always proceeds in the 5 n3 132 CHAPTER 4 • Basic Molecular Genetic Mechanisms direction because chain growth results from formation of a phosphoester bond between the 3 oxygen of a growing strand and the phosphate of a dNTP see Figure 4-9. As discussed earlier an RNA polymerase can find an appropri- ate transcription start site on duplex DNA and initiate the Parental strands synthesized in 15 N After first doubling in 14 N After second doubling in 14 N H H LHH L LLLHHLL L a Predicted results Conservative mechanism Semiconservative mechanism H H HHL L HHLLLLL L New New Old ▲ EXPERIMENTAL FIGURE 4-32 The Meselson-Stahl experiment showed that DNA replicates by a semiconservative mechanism. In this experiment E. coli cells initially were grown in a medium containing ammonium salts prepared with “heavy” nitrogen 15 N until all the cellular DNA was labeled. After the cells were transferred to a medium containing the normal “light” isotope 14 N samples were removed periodically from the cultures and the DNA in each sample was analyzed by equilibrium density-gradient centrifugation see Figure 5-37. This technique can separate heavy-heavy H-H light-light L-L and heavy-light H-L duplexes into distinct bands. a Expected composition of daughter duplex molecules synthesized from 15 N-labeled DNA after E. coli cells are shifted to 14 N-containing medium if DNA replication occurs by a conservative or semiconservative mechanism. Parental heavy H strands are in red light L strands synthesized after shift to 14 N-containing medium are in blue. Note that the conservative mechanism never generates H-L DNA and that the semiconservative mechanism never generates H-H DNA but does generate H-L DNA during the second and subsequent doublings. With additional replication cycles the 15 N-labeled H strands from the original DNA are diluted so that the vast bulk of the DNA would consist of L-L duplexes with either mechanism. b Actual banding patterns of DNA subjected to equilibrium density-gradient centrifugation before and after shifting 15 N-labeled E. coli cells to 14 N-containing medium. DNA bands were visualized under UV light and photographed. The traces on the left are a measure of the density of the photographic signal and hence the DNA concentration along the length of the centrifuge cells from left to right. The number of generations far left following the shift to 14 N-containing medium was determined by counting the concentration of E. coli cells in the culture. This value corresponds to the number of DNA replication cycles that had occurred at the time each sample was taken. After one generation of growth all the extracted DNA had the density of H-L DNA. After 1.9 generations approximately half the DNA had the density of H-L DNA the other half had the density of L-L DNA. With additional generations a larger and larger fraction of the extracted DNA consisted of L-L duplexes H-H duplexes never appeared. These results match the predicted pattern for the semiconservative replication mechanism depicted in a. The bottom two centrifuge cells contained mixtures of H-H DNA and DNA isolated at 1.9 and 4.1 generations in order to clearly show the positions of H-H H-L and L-L DNA in the density gradient. Part b from M. Meselson and F . W. Stahl 1958 Proc. Nat’l. Acad. Sci. USA 44:671 . b Actual results Density Generation 0 0.7 1.0 1.1 1.5 1.9 2.5 3.0 4.1 0 and 1.9 mixed 0 and 4.1 mixed 0.3 L-L H-L H-H L-L H-L H-H Density

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synthesis of an RNA complementary to the template DNA strand see Figure 4-10. In contrast DNA polymerases can- not initiate chain synthesis de novo instead they require a short preexisting RNA or DNA strand called a primer to begin chain growth. With a primer base-paired to the tem- plate strand a DNA polymerase adds deoxynucleotides to the free hydroxyl group at the 3 end of the primer as di- rected by the sequence of the template strand: 4.6 • DNA Replication 133 Because growth of the lagging strand must occur in the 5 n3 direction copying of its template strand must some- how occur in the opposite direction from the movement of the replication fork. A cell accomplishes this feat by synthe- sizing a new primer every few hundred bases or so on the sec- ond parental strand as more of the strand is exposed by unwinding. Each of these primers base-paired to their tem- plate strand is elongated in the 5 n3 direction forming discontinuous segments called Okazaki fragments after their discoverer Reiji Okazaki see Figure 4-33. The RNA primer of each Okazaki fragment is removed and replaced by DNA chain growth from the neighboring Okazaki fragment finally an enzyme called DNA ligase joins the adjacent fragments. Helicase Primase DNA Polymerases and Other Proteins Participate in DNA Replication Detailed understanding of the eukaryotic proteins that par- ticipate in DNA replication has come largely from studies with small viral DNAs particularly SV40 DNA the circular genome of a small virus that infects monkeys. Figure 4-34 depicts the multiple proteins that coordinate copying of SV40 DNA at a replication fork. The assembled proteins at a replication fork further illustrate the concept of molecular machines introduced in Chapter 3. These multicomponent Parental DNA duplex Direction of fork movement Leading strand Short RNA primer Okazaki fragment Lagging strand Point of joining 3 3 5 5 3 5 Daughterduplex ▲ FIGURE 4-33 Schematic diagram of leading-strand and lagging-strand DNA synthesis at a replication fork. Nucleotides are added by a DNA polymerase to each growing daughter strand in the 5 n3 direction indicated by arrowheads. The leading strand is synthesized continuously from a single RNA primer red at its 5 end. The lagging strand is synthesized discontinuously from multiple RNA primers that are formed periodically as each new region of the parental duplex is unwound. Elongation of these primers initially produces Okazaki fragments. As each growing fragment approaches the previous primer the primer is removed and the fragments are ligated. Repetition of this process eventually results in synthesis of the entire lagging strand. When RNA is the primer the daughter strand that is formed is RNA at the 5 end and DNA at the 3 end. Duplex DNA Is Unwound and Daughter Strands Are Formed at the DNA Replication Fork In order for duplex DNA to function as a template during replication the two intertwined strands must be unwound or melted to make the bases available for base pairing with the bases of the dNTPs that are polymerized into the newly synthesized daughter strands. This unwinding of the parental DNA strands is by specific helicases beginning at unique segments in a DNA molecule called replication ori- gins or simply origins. The nucleotide sequences of origins from different organisms vary greatly although they usually contain A T-rich sequences. Once helicases have unwound the parental DNA at an origin a specialized RNA poly- merase called primase forms a short RNA primer comple- mentary to the unwound template strands. The primer still base-paired to its complementary DNA strand is then elon- gated by a DNA polymerase thereby forming a new daugh- ter strand. The DNA region at which all these proteins come to- gether to carry out synthesis of daughter strands is called the replication fork or growing fork. As replication proceeds the growing fork and associated proteins move away from the origin. As noted earlier local unwinding of duplex DNA produces torsional stress which is relieved by topoisomerase I. In order for DNA polymerases to move along and copy a duplex DNA helicase must sequentially unwind the duplex and topoisomerase must remove the supercoils that form. A major complication in the operation of a DNA repli- cation fork arises from two properties: the two strands of the parental DNA duplex are antiparallel and DNA poly- merases like RNA polymerases can add nucleotides to the growing new strands only in the 5 n3 direction. Synthesis of one daughter strand called the leading strand can pro- ceed continuously from a single RNA primer in the 5 n3 direction the same direction as movement of the replication fork Figure 4-33. The problem comes in synthesis of the other daughter strand called the lagging strand. Primer Template strand 5 5 3 3

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complexes permit the cell to carry out an ordered sequence of events that accomplish essential cell functions. In the molecular machine that replicates SV40 DNA a hexamer of a viral protein called large T-antigen unwinds the parental strands at a replication fork. All other proteins in- volved in SV40 DNA replication are provided by the host cell. Primers for leading and lagging daughter-strand DNA are synthesized by a complex of primase which synthesizes a short RNA primer and DNA polymerase Pol which extends the RNA primer with deoxynucleotides forming a mixed RNA-DNA primer. The primer is extended into daughter-strand DNA by DNA polymerase Pol which is less likely to make errors during copying of the template strand than is Pol . Pol forms a complex with Rfc replication factor C and PCNA proliferating cell nuclear antigen which displaces 134 CHAPTER 4 • Basic Molecular Genetic Mechanisms 5 3 Large T- antigen Primase Pol Direction of fork movement 5 3 PCNA Rfc Pol Lagging strand Primer 3 5 Leading strand Pol Rfc PCNA b PCNA c RPA a SV40 DNA replication fork 1 2 5 3 4 RPA Double- stranded DNA Single- stranded DNA ▲ FIGURE 4-34 Model of an SV40 DNA replication fork and assembled proteins. a A hexamer of large T-antigen 1 a viral protein functions as a helicase to unwind the parental DNA strands. Single-strand regions of the parental template unwound by large T-antigen are bound by multiple copies of the heterotrimeric protein RPA 2 . The leading strand is synthesized by a complex of DNA polymerase Pol PCNA and Rfc 3 . Primers for lagging-strand synthesis red RNA light blue DNA are synthesized by a complex of DNA polymerase Pol and primase 4 . The 3 end of each primer synthesized by Pol –primase is then bound by a PCNA-Rfc–Pol complex which proceeds to extend the primer and synthesize most of each Okazaki fragment 5 . See the text for details. b The three subunits of PCNA shown in different colors form a circular structure with a central hole through which double-stranded DNA passes. A diagram of DNA is shown in the center of a ribbon model of the PCNA trimer. c The large subunit of RPA contains two domains that bind single-stranded DNA. On the left the two DNA-binding domains of RPA are shown perpendicular to the DNA backbone white backbone with blue bases. Note that the single DNA strand is extended with the bases exposed an opti- mal conformation for replication by a DNA polymerase. On the right the view is down the length of the single DNA strand re- vealing how RPA strands wrap around the DNA. Part a adapted from S. J. Flint et al. 2000 Virology: Molecular Biology Pathogenesis and Control ASM Press part b after J. M. Gulbis et al. 1996 Cell 87:297 and part c after A. Bochkarev et al. 1997 Nature 385:176.

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the primase–Pol complex following primer synthesis. As illustrated in Figure 4-34b PCNA is a homotrimeric protein that has a central hole through which the daughter duplex DNA passes thereby preventing the PCNA-Rfc–Pol com- plex from dissociating from the template. After parental DNA is separated into single-stranded templates at the replication fork it is bound by multiple copies of RPA replication protein A a heterotrimeric protein Figure 4-34c. Binding of RPA maintains the tem- plate in a uniform conformation optimal for copying by DNA polymerases. Bound RPA proteins are dislodged from the parental strands by Pol and Pol as they synthesize the complementary strands base-paired with the parental strands. Several eukaryotic proteins that function in DNA repli- cation are not depicted in Figure 4-34. A topoisomerase as- sociates with the parental DNA ahead of the helicase to remove torsional stress introduced by the unwinding of the parental strands. Ribonuclease H and FEN I remove the ri- bonucleotides at the 5 ends of Okazaki fragments these are replaced by deoxynucleotides added by DNA polymerase as it extends the upstream Okazaki fragment. Successive Okazaki fragments are coupled by DNA ligase through stan- dard 5 n3 phosphoester bonds. DNA Replication Generally Occurs Bidirectionally from Each Origin As indicated in Figures 4-33 and 4-34 both parental DNA strands that are exposed by local unwinding at a repli- cation fork are copied into a daughter strand. In theory DNA replication from a single origin could involve one repli- cation fork that moves in one direction. Alternatively two replication forks might assemble at a single origin and then move in opposite directions leading to bidirectional growth of both daughter strands. Several types of experiments in- cluding the one shown in Figure 4-35 provided early evi- dence in support of bidirectional strand growth. The general consensus is that all prokaryotic and eu- karyotic cells employ a bidirectional mechanism of DNA replication. In the case of SV40 DNA replication is initiated by binding of two large T-antigen hexameric helicases to the single SV40 origin and assembly of other proteins to form two replication forks. These then move away from the SV40 origin in opposite directions with leading- and lagging-strand synthesis occurring at both forks. As shown in Figure 4-36 the left replication fork extends DNA synthesis in the left- ward direction similarly the right replication fork extends DNA synthesis in the rightward direction. Unlike SV40 DNA eukaryotic chromosomal DNA mol- ecules contain multiple replication origins separated by tens to hundreds of kilobases. A six-subunit protein called ORC for origin recognition complex binds to each origin and as- sociates with other proteins required to load cellular hexa- meric helicases composed of six homologous MCM proteins. 4.6 • DNA Replication 135 Circular viral chromosome EcoRl Origin Replication bubble Time of replication EcoRl restriction site ▲ EXPERIMENTAL FIGURE 4-35 Electron microscopy of replicating SV40 DNA indicates bidirectional growth of DNA strands from an origin. The replicating viral DNA from SV40- infected cells was cut by the restriction enzyme EcoRI which recognizes one site in the circular DNA. Electron micrographs of treated samples showed a collection of cut molecules with increasingly longer replication “bubbles ” whose centers are a constant distance from each end of the cut molecules. This finding is consistent with chain growth in two directions from a common origin located at the center of a bubble as illustrated in the corresponding diagrams. See G. C. Fareed et al. 1972 J. Virol. 10:484 photographs courtesy of N. P . Salzman. Two opposed MCM helicases separate the parental strands at an origin with RPA proteins binding to the resulting sin- gle-stranded DNA. Synthesis of primers and subsequent steps in replication of cellular DNA are thought to be anal- ogous to those in SV40 DNA replication see Figures 4-34 and 4-36. Replication of cellular DNA and other events leading to proliferation of cells are tightly regulated so that the appro- priate numbers of cells constituting each tissue are produced during development and throughout the life of an organism. As in transcription of most genes control of the initiation

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136 CHAPTER 4 • Basic Molecular Genetic Mechanisms KEY CONCEPTS OF SECTION 4.6 DNA Replication ■ Each strand in a parental duplex DNA acts as a tem- plate for synthesis of a daughter strand and remains base- paired to the new strand forming a daughter duplex semi- conservative mechanism. New strands are formed in the 5 n3 direction. ■ Replication begins at a sequence called an origin. Each eukaryotic chromosomal DNA molecule contains multiple replication origins. ■ DNA polymerases unlike RNA polymerases cannot un- wind the strands of duplex DNA and cannot initiate syn- thesis of new strands complementary to the template strands. ■ At a replication fork one daughter strand the leading strand is elongated continuously. The other daughter strand the lagging strand is formed as a series of discon- tinuous Okazaki fragments from primers synthesized every few hundred nucleotides Figure 4-33. ■ The ribonucleotides at the 5 end of each Okazaki frag- ment are removed and replaced by elongation of the 3 end of the next Okazaki fragment. Finally adjacent Okazaki fragments are joined by DNA ligase. ■ Helicases use energy from ATP hydrolysis to separate the parental template DNA strands. Primase synthesizes 1 2 3 4 5 6 7 Helicases Unwinding Leading-strand primer synthesis Leading-strand extension Unwinding Leading-strand extension Lagging-strand primer synthesis Lagging-strand extension Strand ligation FIGURE 4-36 Bidirectional mechanism of DNA replication. The left replication fork here is comparable to the replication fork diagrammed in Figure 4-34 which also shows proteins other than large T-antigen. Top T wo large T -antigen hexameric helicases first bind at the replication origin in opposite orientations. Step 1: Using energy provided from ATP hydrolysis the helicases move in opposite directions unwinding the parental DNA and generating single-strand templates that are bound by RPA proteins. Step 2: Primase–Pol complexes synthesize short primers base-paired to each of the separated parental strands. Step 3: PCNA-Rfc–Pol complexes replace the primase–Pol complexes and extend the short primers generating the leading strands dark green at each replication fork. Step 4 : The helicases further unwind the parental strands and RPA proteins bind to the newly exposed single-strand regions. Step 5: PCNA-Rfc–Pol complexes extend the leading strands further. Step 6: Primase–Pol complexes synthesize primers for lagging-strand synthesis at each replication fork. Step 7: PCNA-Rfc–Pol complexes displace the primase–Pol complexes and extend the lagging-strand Okazaki fragments light green which eventually are ligated to the 5 ends of the leading strands. The position where ligation occurs is represented by a circle. Replication continues by further unwinding of the parental strands and synthesis of leading and lagging strands as in steps 4 – 7. Although depicted as individual steps for clarity unwinding and synthesis of leading and lagging strands occur concurrently. step is the primary mechanism for regulating cellular DNA replication. Activation of MCM helicase activity which is required to initiate cellular DNA replication is regulated by specific protein kinases called S-phase cyclin-dependent kinases. Other cyclin-dependent kinases regulate additional aspects of cell proliferation including the complex process of mitosis by which a eukaryotic cell divides into two daughter cells. We discuss the various regulatory mechanisms that de- termine the rate of cell division in Chapter 21.

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a short RNA primer which remains base-paired to the tem- plate DNA. This initially is extended at the 3 end by DNA polymerase Pol resulting in a short 5 RNA- 3 DNA daughter strand. ■ Most of the DNA in eukaryotic cells is synthesized by Pol which takes over from Pol and continues elongation of the daughter strand in the 5 n3 direction. Pol remains stably associated with the template by binding to Rfc pro- tein which in turn binds to PCNA a trimeric protein that encircles the daughter duplex DNA see Figure 4-34. ■ DNA replication generally occurs by a bidirectional mechanism in which two replication forks form at an ori- gin and move in opposite directions with both template strands being copied at each fork see Figure 4-36. ■ Synthesis of eukaryotic DNA in vivo is regulated by con- trolling the activity of the MCM helicases that initiate DNA replication at multiple origins spaced along chro- mosomal DNA. Viruses: Parasites of the Cellular Genetic System Viruses cannot reproduce by themselves and must comman- deer a host cell’s machinery to synthesize viral proteins and in some cases to replicate the viral genome. RNA viruses which usually replicate in the host-cell cytoplasm have an RNA genome and DNA viruses which commonly replicate in the host-cell nucleus have a DNA genome see Figure 4-1. Viral genomes may be single- or double-stranded de- pending on the specific type of virus. The entire infectious virus particle called a virion consists of the nucleic acid and an outer shell of protein. The simplest viruses contain only enough RNA or DNA to encode four proteins the most complex can encode 100–200 proteins. In addition to their obvious importance as causes of disease viruses are ex- tremely useful as research tools in the study of basic biolog- ical processes. Most Viral Host Ranges Are Narrow The surface of a virion contains many copies of one type of protein that binds specifically to multiple copies of a receptor protein on a host cell. This interaction determines the host range—the group of cell types that a virus can infect—and begins the infection process. Most viruses have a rather lim- ited host range. A virus that infects only bacteria is called a bacterio- phage or simply a phage. Viruses that infect animal or plant cells are referred to generally as animal viruses or plant viruses. A few viruses can grow in both plants and the insects that feed on them. The highly mobile insects serve as vectors for transferring such viruses between susceptible plant hosts. Wide host ranges are also characteristic of some strictly ani- 4.7 4.7 • Viruses: Parasites of the Cellular Genetic System 137 mal viruses such as vesicular stomatitis virus which grows in insect vectors and in many different types of mammals. Most animal viruses however do not cross phyla and some e.g. poliovirus infect only closely related species such as primates. The host-cell range of some animal viruses is fur- ther restricted to a limited number of cell types because only these cells have appropriate surface receptors to which the virions can attach. Viral Capsids Are Regular Arrays of One or a Few Types of Protein The nucleic acid of a virion is enclosed within a protein coat or capsid composed of multiple copies of one protein or a few different proteins each of which is encoded by a single viral gene. Because of this structure a virus is able to encode all the information for making a relatively large capsid in a small number of genes. This efficient use of genetic informa- tion is important since only a limited amount of RNA or DNA and therefore a limited number of genes can fit into a virion capsid. A capsid plus the enclosed nucleic acid is called a nucleocapsid. Nature has found two basic ways of arranging the mul- tiple capsid protein subunits and the viral genome into a nu- cleocapsid. In some viruses multiple copies of a single coat protein form a helical structure that encloses and protects the viral RNA or DNA which runs in a helical groove within the protein tube. Viruses with such a helical nucleocapsid such as tobacco mosaic virus have a rodlike shape. The other major structural type is based on the icosahedron a solid approximately spherical object built of 20 identical faces each of which is an equilateral triangle. The number and arrangement of coat proteins in icosa- hedral or quasi-spherical viruses differ somewhat depend- ing on their size. In small viruses of this type each of the 20 triangular faces is constructed of three identical capsid pro- tein subunits making a total of 60 subunits per capsid. All the protein subunits are in equivalent contact with one an- other Figure 4-37a. In large quasi-spherical viruses each face of the icosahedron is composed of more than three sub- units. As a result the contacts between subunits not at the vertices are quasi-equivalent Figure 4-37b. Models of sev- eral quasi-spherical viruses based on cryoelectron micro- scopy are shown in Figure 4-37. In the smaller viruses e.g. poliovirus clefts that encircle each of the vertices of the icosahedral structure interact with receptors on the surface of host cells during infection. In the larger viruses e.g. ade- novirus long fiberlike proteins extending from the nucleo- capsid interact with cell-surface receptors on host cells. In many DNA bacteriophages the viral DNA is located within an icosahedral “head” that is attached to a rodlike “tail.” During infection viral proteins at the tip of the tail bind to host-cell receptors and then the viral DNA passes down the tail into the cytoplasm of the host cell. In some viruses the symmetrically arranged nucleocap- sid is covered by an external membrane or envelope which

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consists mainly of a phospholipid bilayer but also contains one or two types of virus-encoded glycoproteins Figure 4-38. The phospholipids in the viral envelope are similar to those in the plasma membrane of an infected host cell. The viral envelope is in fact derived by budding from that mem- brane but contains mainly viral glycoproteins as we discuss shortly. Viruses Can Be Cloned and Counted in Plaque Assays The number of infectious viral particles in a sample can be quantified by a plaque assay. This assay is performed by cul- turing a dilute sample of viral particles on a plate covered with host cells and then counting the number of local le- sions called plaques that develop Figure 4-39. A plaque develops on the plate wherever a single virion initially in- fects a single cell. The virus replicates in this initial host cell and then lyses ruptures the cell releasing many progeny virions that infect the neighboring cells on the plate. After a few such cycles of infection enough cells are lysed to pro- 138 CHAPTER 4 • Basic Molecular Genetic Mechanisms SV40 2 3 4 5 1 1 52 3 4 a Small icosahedral viruses 3 2 15 6 4 1 52 3 b A large icosahedral virus Adenovirus 10 nm CPMV Poliovirus ▲ FIGURE 4-37 Structures of quasi-spherical icosahedral viruses. The actual shape of the protein subunits in these viruses is not a flat triangle as illustrated in the schematic diagrams but the overall effect when the subunits are assembled is of a roughly spherical structure with triangular faces. The three-dimensional models are all shown at the same magnification. a In the simplest and smallest quasi-spherical viruses three identical capsid protein subunits form each triangular face red of the icosahedron schematic. The subunits meet in fivefold symmetry at each vertex. Models of three such viruses are shown: poliovirus a human RNA virus cowpea mosaic virus CPMV a plant RNA virus and simian virus 40 SV40 a monkey DNA virus. b In some larger viruses of this type each triangular face is composed of six subunits. The subunits at the vertices maintain fivefold symmetry but those making up the surfaces in between exhibit sixfold symmetry. A model of adenovirus a human DNA virus illustrates how much larger it is than the viruses in part a and shows the fibers green that bind to receptors on host cells. See P . L. Stewart et al. 1997 EMBO J. 16:1189. Models of CPMV poliovirus and SV40 courtesy of T . S. Baker model of adenovirus courtesy of P . L. Stewart. ▲ EXPERIMENTAL FIGURE 4-38 Viral protein spikes protrude from the surface of an influenza virus virion. Influenza viruses are surrounded by an envelope consisting of a phospholipid bilayer and embedded viral proteins. The large spikes seen in this electron micrograph of a negatively stained influenza virion are composed of neuraminidase a tetrameric protein or hemagglutinin a trimeric protein see Figure 3-7. Inside is the helical nucleocapsid. Courtesy of A. Helenius and J. White.

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duce a visible clear area or plaque in the layer of remaining uninfected cells. Since all the progeny virions in a plaque are derived from a single parental virus they constitute a virus clone. This type of plaque assay is in standard use for bacterial and ani- mal viruses. Plant viruses can be assayed similarly by count- ing local lesions on plant leaves inoculated with viruses. Analysis of viral mutants which are commonly isolated by plaque assays has contributed extensively to current under- standing of molecular cellular processes. The plaque assay also is critical in isolating bacteriophage clones carrying segments of cellular DNA as discussed in Chapter 9. Lytic Viral Growth Cycles Lead to Death of Host Cells Although details vary among different types of viruses those that exhibit a lytic cycle of growth proceed through the following general stages: 1. Adsorption—Virion interacts with a host cell by binding of multiple copies of capsid protein to specific receptors on the cell surface. 2. Penetration—Viral genome crosses the plasma membrane. For animal and plant viruses viral proteins also enter the host cell. 3. Replication—Viral mRNAs are produced with the aid of the host-cell transcription machinery DNA viruses or by viral enzymes RNA viruses. For both types of viruses viral mRNAs are translated by the host-cell translation machinery. Production of multiple copies of the viral 4.7 • Viruses: Parasites of the Cellular Genetic System 139 Add dilute suspension containing virus after infection cover layer of cells with agar incubate Each plaque represents cell lysis initiated by one viral particle agar restricts movement so that virus can infect only contiguous cells a Plaque Confluent layer of susceptible host cells growing on surface of a plate EXPERIMENTAL FIGURE 4-39 Plaque assay determines the number of infectious particles in a viral suspension. a Each lesion or plaque which develops where a single virion initially infected a single cell constitutes a pure viral clone. b Plate illuminated from behind shows plaques formed by bacteriophage plated on E. coli. c Plate showing plaques produced by poliovirus plated on HeLa cells. Part b courtesy of Barbara Morris part c from S. E. Luria et al. 1978 General Virology 3d ed. Wiley p. 26. genome is carried out either by viral proteins alone or with the help of host-cell proteins. 4. Assembly—Viral proteins and replicated genomes associate to form progeny virions. 5. Release—Infected cell either ruptures suddenly lysis releasing all the newly formed virions at once or disinte- grates gradually with slow release of virions. Figure 4-40 illustrates the lytic cycle for T4 bacterio- phage a nonenveloped DNA virus that infects E. coli. Viral capsid proteins generally are made in large amounts because many copies of them are required for the assembly of each progeny virion. In each infected cell about 100–200 T4 progeny virions are produced and released by lysis. The lytic cycle is somewhat more complicated for DNA viruses that infect eukaryotic cells. In most such viruses the DNA genome is transported with some associated proteins into the cell nucleus. Once inside the nucleus the viral DNA is transcribed into RNA by the host’s transcription machin- ery. Processing of the viral RNA primary transcript by host- cell enzymes yields viral mRNA which is transported to the cytoplasm and translated into viral proteins by host-cell ribosomes tRNA and translation factors. The viral proteins are then transported back into the nucleus where some of them either replicate the viral DNA directly or direct cellu- lar proteins to replicate the viral DNA as in the case of SV40 discussed in the last section. Assembly of the capsid proteins with the newly replicated viral DNA occurs in the nucleus yielding hundreds to thousands of progeny virions. Most plant and animal viruses with an RNA genome do not require nuclear functions for lytic replication. In some b Plaque c Plaque

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140 CHAPTER 4 • Basic Molecular Genetic Mechanisms Free virion E. coli chromosome Expression of viral early proteins Replication of viral DNA Expression of viral late proteins Assembly Lysis/release Adsorption/injection T4 DNA Viral proteins 5 4 3 2 1 FIGURE 4-40 Lytic replication cycle of E. coli bacteriophage T4 a nonenveloped virus with a double- stranded DNA genome. After viral coat proteins at the tip of the tail in T4 interact with specific receptor proteins on the exterior of the host cell the viral genome is injected into the host step 1 . Host-cell enzymes then transcribe viral “early” genes into mRNAs and subsequently translate these into viral “early” proteins step 2 . The early proteins replicate the viral DNA and induce expression of viral “late” proteins by host-cell enzymes step 3 . The viral late proteins include capsid and assem- bly proteins and enzymes that degrade the host-cell DNA supplying nucleotides for synthesis of more viral DNA. Progeny virions are assembled in the cell step 4 and released step 5 when viral proteins lyse the cell. Newly liberated viruses initiate another cycle of infection in other host cells. Budding Receptor-binding glycoprotein Lipid bilayer Matrix protein Virus receptor Endocytosis Fusion Nucleocapsid protein Matrix and nucleocapsid synthesis Progeny capsid assembly Association at membrane Fusion Transcription Glycoprotein synthesis Nucleus Viral mRNA Replication Genomic RNA Viral RNA polymerase Adsorption Transport Rabies virus Cytosol ER Golgi Cell membrane Endosome Release 13 1 2 3 4 5 6 10 11 12 9 8 7

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of these viruses a virus-encoded enzyme that enters the host during penetration transcribes the genomic RNA into mRNAs in the cell cytoplasm. The mRNA is directly trans- lated into viral proteins by the host-cell translation machin- ery. One or more of these proteins then produces additional copies of the viral RNA genome. Finally progeny genomes are assembled with newly synthesized capsid proteins into progeny virions in the cytoplasm. After the synthesis of hundreds to thousands of new viri- ons has been completed most infected bacterial cells and some infected plant and animal cells are lysed releasing all the virions at once. In many plant and animal viral infec- tions however no discrete lytic event occurs rather the dead host cell releases the virions as it gradually disintegrates. As noted previously enveloped animal viruses are sur- rounded by an outer phospholipid layer derived from the plasma membrane of host cells and containing abundant viral glycoproteins. The processes of adsorption and release of enveloped viruses differ substantially from these processes in nonenveloped viruses. To illustrate lytic replication of enveloped viruses we consider the rabies virus whose nu- cleocapsid consists of a single-stranded RNA genome sur- rounded by multiple copies of nucleocapsid protein. Like other lytic RNA viruses rabies virions are replicated in the cytoplasm and do not require host-cell nuclear enzymes. As shown in Figure 4-41 a rabies virion is adsorbed by endo- cytosis and release of progeny virions occurs by budding from the host-cell plasma membrane. Budding virions are clearly visible in electron micrographs of infected cells as illustrated in Figure 4-42. Many tens of thousands of prog- eny virions bud from an infected host cell before it dies. Viral DNA Is Integrated into the Host-Cell Genome in Some Nonlytic Viral Growth Cycles Some bacterial viruses called temperate phages can establish a nonlytic association with their host cells that does not kill the cell. For example when bacteriophage infects E. coli the viral DNA may be integrated into the host-cell chromo- some rather than being replicated. The integrated viral DNA called a prophage is replicated as part of the cell’s DNA from one host-cell generation to the next. This phenomenon is referred to as lysogeny. Under certain conditions the prophage DNA is activated leading to its excision from the host-cell chromosome entrance into the lytic cycle and sub- sequent production and release of progeny virions. 4.7 • Viruses: Parasites of the Cellular Genetic System 141 FIGURE 4-41 Lytic replication cycle of rabies virus an enveloped virus with a single-stranded RNA genome. The structural components of this virus are depicted at the top. Note that the nucleocapsid is helical rather than icosahedral. After a virion adsorbs to multiple copies of a specific host membrane protein step 1 the cell engulfs it in an endosome step 2 . A cellular protein in the endosome membrane pumps H ions from the cytosol into the endosome interior. The resulting decrease in endosomal pH induces a conformational change in the viral glycoprotein leading to fusion of the viral envelope with the endosomal lipid bilayer membrane and release of the nucleocapsid into the cytosol steps 3 and 4 . Viral RNA polymerase uses ribonucleoside triphosphates in the cytosol to replicate the viral RNA genome step 5 and to synthesize viral mRNAs step 6. One of the viral mRNAs encodes the viral transmembrane glycoprotein which is inserted into the membrane of the endoplasmic reticulum ER as it is synthesized on ER-bound ribosomes step 7. Carbohydrate is added to the large folded domain inside the ER lumen and is modified as the membrane and the associated glycoprotein pass through the Golgi apparatus step 8. Vesicles with mature glycoprotein fuse with the host plasma membrane depositing viral glycoprotein on the cell surface with the large receptor-binding domain out- side the cell step 9. Meanwhile other viral mRNAs are trans- lated on host-cell ribosomes into nucleocapsid protein matrix protein and viral RNA polymerase step 10. These proteins are assembled with replicated viral genomic RNA bright red into progeny nucleocapsids step 11 which then associate with the cytosolic domain of viral transmembrane glycoproteins in the plasma membrane step 12. The plasma membrane is folded around the nucleocapsid forming a “bud” that eventually is released step 13. ▲ EXPERIMENTAL FIGURE 4-42 Progeny virions of enveloped viruses are released by budding from infected cells. In this transmission electron micrograph of a cell infected with measles virus virion buds are clearly visible protruding from the cell surface. Measles virus is an enveloped RNA virus with a helical nucleocapsid like rabies virus and replicates as illustrated in Figure 4-41. From A. Levine 1991 Viruses Scientific American Library p. 22.

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The genomes of a number of animal viruses also can in- tegrate into the host-cell genome. Probably the most impor- tant are the retroviruses which are enveloped viruses with a genome consisting of two identical strands of RNA. These viruses are known as retroviruses because their RNA genome acts as a template for formation of a DNA molecule—the opposite flow of genetic information compared with the more common transcription of DNA into RNA. In the retro- viral life cycle Figure 4-43 a viral enzyme called reverse transcriptase initially copies the viral RNA genome into single- stranded DNA complementary to the virion RNA the same enzyme then catalyzes synthesis of a complementary DNA strand. This complex reaction is detailed in Chapter 10 when we consider closely related intracellular parasites called retrotransposons. The resulting double-stranded DNA is in- tegrated into the chromosomal DNA of the infected cell. Fi- nally the integrated DNA called a provirus is transcribed by the cell’s own machinery into RNA which either is trans- lated into viral proteins or is packaged within virion coat proteins to form progeny virions that are released by bud- ding from the host-cell membrane. Because most retroviruses do not kill their host cells infected cells can replicate pro- 142 CHAPTER 4 • Basic Molecular Genetic Mechanisms ducing daughter cells with integrated proviral DNA. These daughter cells continue to transcribe the proviral DNA and bud progeny virions. Some retroviruses contain cancer-causing genes oncogenes and cells infected by such retroviruses are oncogenically transformed into tumor cells. Studies of oncogenic retroviruses mostly viruses of birds and mice have revealed a great deal about the processes that lead to transformation of a normal cell into a cancer cell Chapter 23. Among the known human retroviruses are human T-cell lymphotrophic virus HTLV which causes a form of leukemia and human immunodeficiency virus HIV which causes acquired immune deficiency syndrome AIDS. Both of these viruses can infect only specific cell types primarily certain cells of the immune system and in the case of HIV some central nervous system neurons and glial cells. Only these cells have cell-surface receptors that interact with viral envelope proteins accounting for the host-cell specificity of these viruses. Unlike most other retroviruses HIV eventually kills its host cells. The eventual death of large numbers of Reverse transcriptase Reverse transcription Host-cell chromosomal DNA Transcription Retrovirus proteins Provirus Transport to nucleus and integration Fusion Budding Genomic ssRNA Viral DNA Nucleocapsid 5 1 2 4 3 ▲ FIGURE 4-43 Retroviral life cycle. Retroviruses have a genome of two identical copies of single-stranded RNA and an outer envelope. Step 1: After viral glycoproteins in the envelope interact with a specific host-cell membrane protein the retroviral envelope fuses directly with the plasma membrane allowing entry of the nucleocapsid into the cytoplasm of the cell. Step 2 : Viral reverse transcriptase and other proteins copy the viral ssRNA genome into a double-stranded DNA. Step 3: The viral dsDNA is transported into the nucleus and integrated into one of many possible sites in the host-cell chromosomal DNA. For simplicity only one host-cell chromosome is depicted. Step 4 : The integrated viral DNA provirus is transcribed by the host-cell RNA polymerase generating mRNAs dark red and genomic RNA molecules bright red. The host-cell machinery translates the viral mRNAs into glycoproteins and nucleocapsid proteins. Step 5: Progeny virions then assemble and are released by budding as illustrated in Figure 4-41. MEDIA CONNECTIONS Overview Animation: Life Cycle of a Retrovirus

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immune-system cells results in the defective immune re- sponse characteristic of AIDS. Some DNA viruses also can integrate into a host-cell chromosome. One example is the human papillomaviruses HPVs which most commonly cause warts and other be- nign skin lesions. The genomes of certain HPV serotypes however occasionally integrate into the chromosomal DNA of infected cervical epithelial cells initiating development of cervical cancer. Routine Pap smears can detect cells in the early stages of the transformation process initiated by HPV integration permitting effective treatment. ❚ KEY CONCEPTS OF SECTION 4.7 Viruses: Parasites of the Cellular Genetic System ■ Viruses are small parasites that can replicate only in host cells. Viral genomes may be either DNA DNA viruses or RNA RNA viruses and either single- or double-stranded. ■ The capsid which surrounds the viral genome is com- posed of multiple copies of one or a small number of virus- encoded proteins. Some viruses also have an outer enve- lope which is similar to the plasma membrane but contains viral transmembrane proteins. ■ Most animal and plant DNA viruses require host-cell nuclear enzymes to carry out transcription of the viral genome into mRNA and production of progeny genomes. In contrast most RNA viruses encode enzymes that can transcribe the RNA genome into viral mRNA and produce new copies of the RNA genome. ■ Host-cell ribosomes tRNAs and translation factors are used in the synthesis of all viral proteins in infected cells. ■ Lytic viral infection entails adsorption penetration syn- thesis of viral proteins and progeny genomes replication assembly of progeny virions and release of hundreds to thou- sands of virions leading to death of the host cell see Fig- ure 4-40. Release of enveloped viruses occurs by budding through the host-cell plasma membrane see Figure 4-41. ■ Nonlytic infection occurs when the viral genome is in- tegrated into the host-cell DNA and generally does not lead to cell death. ■ Retroviruses are enveloped animal viruses containing a single-stranded RNA genome. After a host cell is pene- trated reverse transcriptase a viral enzyme carried in the virion converts the viral RNA genome into double- stranded DNA which integrates into chromosomal DNA see Figure 4-43. ■ Unlike infection by other retroviruses HIV infection eventually kills host cells causing the defects in the im- mune response characteristic of AIDS. ■ Tumor viruses which contain oncogenes may have an RNA genome e.g. human T-cell lymphotrophic virus or a DNA genome e.g. human papillomaviruses. In the case Perspectives for the Future 143 of these viruses integration of the viral genome into a host- cell chromosome can cause transformation of the cell into a tumor cell. PERSPECTIVES FOR THE FUTURE In this chapter we first reviewed the basic structure of DNA and RNA and then described fundamental aspects of the transcription of DNA by RNA polymerases. Eukaryotic RNA polymerases are discussed in greater detail in Chapter 11 along with additional factors required for transcription initiation in eukaryotic cells and interactions with regulatory transcription factors that control transcription initiation. Next we discussed the genetic code and the participation of tRNA and the protein-synthesizing machine the ribosome in decoding the information in mRNA to allow accurate as- sembly of protein chains. Mechanisms that regulate protein synthesis are considered further in Chapter 12. Finally we considered the molecular details underlying the accurate replication of DNA required for cell division. Chapter 21 covers the mechanisms that regulate when a cell replicates its DNA and that coordinate DNA replication with the complex process of mitosis that distributes the daughter DNA mole- cules equally to each daughter cell. These basic cellular processes form the foundation of mo- lecular cell biology. Our current understanding of these processes is grounded in a wealth of experimental results and is not likely to change. However the depth of our under- standing will continue to increase as additional details of the structures and interactions of the macromolecular machines involved are uncovered. The determination in recent years of the three-dimensional structures of RNA polymerases ribo- somal subunits and DNA replication proteins has allowed researchers to design ever more penetrating experimental ap- proaches for revealing how these macromolecules operate at the molecular level. The detailed level of understanding that results may allow the design of new and more effective drugs for treating human illnesses. For example the recent high- resolution structures of ribosomes are providing insights into the mechanism by which antibiotics inhibit bacterial protein synthesis without affecting the function of mammalian ribo- somes. This new knowledge may allow the design of even more effective antibiotics. Similarly detailed understanding of the mechanisms regulating transcription of specific human genes may lead to therapeutic strategies that can reduce or prevent inappropriate immune responses that lead to multi- ple sclerosis and arthritis the inappropriate cell division that is the hallmark of cancer and other pathological processes. Much of current biological research is focused on discovering how molecular interactions endow cells with decision-making capacity and their special properties. For this reason several of the following chapters describe current knowledge about how such interactions regulate transcrip- tion and protein synthesis in multicellular organisms and how such regulation endows cells with the capacity to become

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specialized and grow into complicated organs. Other chapters deal with how protein-protein interactions underlie the con- struction of specialized organelles in cells and how they de- termine cell shape and movement. The rapid advances in molecular cell biology in recent years hold promise that in the not too distant future we will understand how the regulation of specialized cell function shape and mobility coupled with regulated cell replication and cell death apoptosis lead to the growth of complex organisms like trees and human beings. KEY TERMS anticodon 119 codons 119 complementary 104 DNA polymerases 133 double helix 103 envelope viral 137 exons 111 genetic code 119 introns 111 lagging strand 133 leading strand 133 messenger RNA mRNA 119 Okazaki fragments 133 operon 111 phosphodiester bond 103 REVIEW THE CONCEPTS 1. What are Watson-Crick base pairs Why are they important 2. TATA box–binding protein binds to the minor groove of DNA resulting in the bending of the DNA helix see Figure 4-5. What property of DNA allows the TATA box–binding protein to recognize the DNA helix 3. Preparing plasmid double-stranded circular DNA for sequencing involves annealing a complementary short single-stranded oligonucleotide DNA primer to one strand of the plasmid template. This is routinely accomplished by heat- ing the plasmid DNA and primer to 90 C and then slowly bringing the temperature down to 25 C. Why does this pro- tocol work 4. What difference between RNA and DNA helps to ex- plain the greater stability of DNA What implications does this have for the function of DNA 5. What are the major differences in the synthesis and structure of prokaryotic and eukaryotic mRNAs 144 CHAPTER 4 • Basic Molecular Genetic Mechanisms 6. While investigating the function of a specific growth fac- tor receptor gene from humans it was found that two types of proteins are synthesized from this gene. A larger protein containing a membrane-spanning domain functions to rec- ognize growth factors at the cell surface stimulating a spe- cific downstream signaling pathway. In contrast a related smaller protein is secreted from the cell and functions to bind available growth factor circulating in the blood thus in- hibiting the downstream signaling pathway. Speculate on how the cell synthesizes these disparate proteins. 7. Describe the molecular events that occur at the lac operon when E. coli cells are shifted from a glucose-containing medium to a lactose-containing medium. 8. The concentration of free phosphate affects transcrip- tion of some E. coli genes. Describe the mechanism for this. 9. Contrast how selection of the translational start site oc- curs on bacterial eukaryotic and poliovirus mRNAs. 10. What is the evidence that the 23S rRNA in the large rRNA subunit has a peptidyl transferase activity 11. How would a mutation in the polyA-binding protein I gene affect translation How would an electron micrograph of polyribosomes from such a mutant differ from the nor- mal pattern 12. What characteristic of DNA results in the requirement that some DNA synthesis is discontinuous How are Okazaki fragments and DNA ligase utilized by the cell 13. What gene is unique to retroviruses Why is the protein encoded by this gene absolutely necessary for maintaining the retroviral life cycle but not that of other viruses ANALYZE THE DATA NASA has identified a new microbe present on Mars and re- quests that you determine the genetic code of this organism. To accomplish this goal you isolate an extract from this mi- crobe that contains all the components necessary for protein synthesis except mRNA. Synthetic mRNAs are added to this extract and the resulting polypeptides are analyzed: Synthetic mRNA Resulting Polypeptides AAAAAAAAAAAAAAAA Lysine-Lysine-Lysine etc. CACACACACACACACA Threonine-Histidine- Threonine-Histidine etc. AACAACAACAACAACA Threonine-Threonine- Threonine etc. Glutamine-Glutamine- Glutamine etc. Asparagine-Asparagine- Asparagine etc. plaque assay 138 polyribosomes 130 primary transcript 110 primer 133 promoter 109 reading frame 120 replication fork 133 reverse transcriptase 142 ribosomal RNA rRNA 119 ribosomes 119 RNA polymerase 109 transcription 101 transfer RNA tRNA 119 translation 101 Watson-Crick base pairs 103

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References 145 From these data what specifics can you conclude about the microbe’s genetic code What is the sequence of the anticodon loop of a tRNA carrying a threonine If you found that this microbe contained 61 different tRNAs what could you speculate about the fidelity of translation in this organism REFERENCES Structure of Nucleic Acids Dickerson R. E. 1983. The DNA helix and how it is read. Sci. Am. 249:94–111. Doudna J. A. and T. R. Cech. 2002. The chemical repertoire of natural ribozymes. Nature 418:222–228. Kornberg A. and T. A. Baker. 1992. DNA Replication 2d ed. W. H. Freeman and Company chap. 1. A good summary of the prin- ciples of DNA structure. Wang J. C. 1980. Superhelical DNA. Trends Biochem. Sci. 5:219–221. Transcription of Protein-Coding Genes and Formation of Functional mRNA Brenner S. F. Jacob and M. Meselson. 1961. An unstable in- termediate carrying information from genes to ribosomes for protein synthesis. Nature 190:576–581. Young B. A. T. M. Gruber and C. A. Gross. 2002. Views of transcription initiation. Cell 109:417–420. Control of Gene Expression in Prokaryotes Bell C. E. and M. Lewis. 2001. The Lac repressor: a second generation of structural and functional studies. Curr. Opin. Struc. Biol. 11:19–25. Busby S. and R. H. Ebright. 1999. Transcription activation by catabolite activator protein CAP. J. Mol. Biol. 293:199–213. Darst S. A. 2001. Bacterial RNA polymerase. Curr. Opin. Struc. Biol. 11:155–162. Muller-Hill B. 1998. Some repressors of bacterial transcription. Curr. Opin. Microbiol. 1:145–151. The Three Roles of RNA in Translation Alexander R. W. and P. Schimmel. 2001. Domain-domain com- munication in aminoacyl-tRNA synthetases. Prog. Nucleic Acid Res. Mol. Biol. 69:317–349. Bjork G. R. et al. 1987. Transfer RNA modification. Ann. Rev. Biochem. 56:263–287. Garrett R. A. et al. eds. 2000. The Ribosome: Structure Func- tion Antibiotics and Cellular Interactions. ASM Press. Hatfield D. L. and V. N. Gladyshev. 2002. How selenium has altered our understanding of the genetic code. Mol. Cell Biol. 22:3565–3576. Hoagland M. B. et al. 1958. A soluble ribonucleic acid inter- mediate in protein synthesis. J. Biol. Chem. 231:241–257. Holley R. W. et al. 1965. Structure of a ribonucleic acid. Sci- ence 147:1462–1465. Ibba M. and D. Soll. 2001.The renaissance of aminoacyl-tRNA synthesis. EMBO Rep. 2:382–387. Khorana G. H. et al. 1966. Polynucleotide synthesis and the genetic code. Cold Spring Harbor Symp. Quant. Biol. 31:39–49. Maguire B. A. and R. A. Zimmermann. 2001. The ribosome in focus. Cell 104:813–816. Nirenberg M. et al. 1966. The RNA code in protein synthesis. Cold Spring Harbor Symp. Quant. Biol. 31:11–24. Ramakrishnan V. 2002. Ribosome structure and the mechanism of translation. Cell 108:557–572. Rich A. and S.-H. Kim. 1978. The three-dimensional structure of transfer RNA. Sci. Am. 2401:52–62 offprint 1377. Stepwise Synthesis of Proteins on Ribosomes Gingras A. C. R. Raught and N. Sonenberg. 1999. eIF4 initi- ation factors: effectors of mRNA recruitment to ribosomes and reg- ulators of translation. Ann. Rev. Biochem. 68:913–963. Green R. 2000. Ribosomal translocation: EF-G turns the crank. Curr. Biol. 10:R369–R373. Hellen C. U. and P. Sarnow. 2001. Internal ribosome entry sites in eukaryotic mRNA molecules. Genet. Devel. 15:1593–1612. Kisselev L. L. and R. H. Buckingham. 2000. Translational ter- mination comes of age. Trends Biochem. Sci. 25:561–566. Kozak M. 1999. Initiation of translation in prokaryotes and eukaryotes. Gene 234:187–208. Noller H. F. et al. 2002. Translocation of tRNA during protein synthesis. FEBS Lett. 514:11–16. Pestova T. V. et al. 2001. Molecular mechanisms of translation initiation in eukaryotes. Proc. Nat’l. Acad. Sci. USA 98:7029–7036. Poole E. and W. Tate. 2000. Release factors and their role as decoding proteins: specificity and fidelity for termination of protein synthesis. Biochim. Biophys. Acta 1493:1–11. Ramakrishnan V. 2002. Ribosome structure and the mechanism of translation. Cell 108:557–572. Sonenberg N. J. W. B. Hershey and M. B. Mathews eds. 2000. Translational Control of Gene Expression. Cold Spring Harbor Lab- oratory Press. DNA Replication Bullock P. A. 1997. The initiation of simian virus 40 DNA repli- cation in vitro. Crit. Rev. Biochem. Mol. Biol. 32:503–568. Kornberg A. and T. A. Baker. 1992. DNA Replication 2d ed. W. H. Freeman and Company Waga S. and B. Stillman. 1998. The DNA replication fork in eukaryotic cells. Ann. Rev. Biochem. 67:721–751. Viruses: Parasites of the Cellular Genetic System Flint S. J. et al. 2000. Principles of Virology: Molecular Biol- ogy Pathogenesis and Control. ASM Press. Hull R. 2002. Mathews’ Plant Virology. Academic Press. Knipe D. M. and P . M. Howley eds. 2001. Fields Virology. Lip- pincott Williams Wilkins. Kornberg A. and T. A. Baker. 1992. DNA Replication 2d ed. W. H. Freeman and Company. Good summary of bacteriophage molecular biology.

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Although the basic architecture of all eukaryotic cells is constructed from membranes organelles and the cytosol each type of cell exhibits a distinctive design defined by the shape of the cell and the location of its organelles. The struc- tural basis of the unique design of each cell type lies in the cytoskeleton a dense network of three classes of protein fila- ments that permeate the cytosol and mechanically support cel- lular membranes. Cytoskeletal proteins are among the most abundant proteins in a cell and the enormous surface area of the cytoskeleton see Figure 5-1 constitutes a scaffold to which particular sets of proteins and membranes are bound. We begin our examination of cell architecture by consid- ering the basic structure of biomembranes. The lipid com- ponents of membranes not only affect their shape and 5 Atomic force microscopy reveals sphyingomyelin rafts orange protruding from a dioleoylphosphatidylcholine background black in a mica-supported lipid bilayer. Placental alkaline phosphatase yellow peaks a glycosylphosphatidylinositol- anchored protein is shown to be almost exclusively raft associated. From D. E. Saslowsky et al. 2002 J. Biol. Chem. 277:26966–26970. BIOMEMBRANES AND CELL ARCHITECTURE P rokaryotes which represent the simplest and smallest cells about 1–2 m in length are surrounded by a plasma membrane but contain no internal membrane- limited subcompartments see Figure 1-2a. Although DNA is concentrated in the center of these unicellular organisms most enzymes and metabolites are thought to diffuse freely within the single internal aqueous compartment. Certain metabolic reactions including protein synthesis and anaerobic glycolysis take place there others such as the replication of DNA and the production of ATP take place at the plasma membrane. In the larger cells of eukaryotes however the rates of chemical reactions would be limited by the diffusion of small molecules if a cell were not partitioned into smaller subcom- partments termed organelles. Each organelle is surrounded by one or more biomembranes and each type of organelle contains a unique complement of proteins—some embedded in its membranes others in its aqueous interior space or lumen. These proteins enable each organelle to carry out its characteristic cellular functions. The cytoplasm is the part of the cell outside the largest organelle the nucleus. The cytosol the aqueous part of the cytoplasm outside all of the organelles also contains its own distinctive proteins. All biomembranes form closed structures separating the lumen on the inside from the outside and are based on a sim- ilar bilayer structure. They control the movement of mole- cules between the inside and the outside of a cell and into and out of the organelles of eukaryotic cells. In accord with the importance of internal membranes to cell function the total surface area of these membranes is roughly tenfold as great as that of the plasma membrane Figure 5-1. 147 OUTLINE 5.1 Biomembranes: Lipid Composition and Structural Organization 5.2 Biomembranes: Protein Components and Basic Functions 5.3 Organelles of the Eukaryotic Cell 5.4 The Cytoskeleton: Components and Structural Functions 5.5 Purification of Cells and Their Parts 5.6 Visualizing Cell Architecture

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function but also play important roles in anchoring proteins to the membrane modifying membrane protein activities and transducing signals to the cytoplasm. We then consider the general structure of membrane proteins and how they can relate to different membranes. The unique function of each membrane is determined largely by the complement of proteins within and adjacent to it. The theme of membrane- limited compartments is continued with a review of the func- tions of various organelles. We then introduce the structure and function of the cytoskeleton which is intimately associ- ated with all biomembranes changes in the organization of this filamentous network affect the structure and function of the attached membranes. In the remainder of the chapter we describe common methods for isolating particular types of cells and subcellular structures and various microscopic techniques for studying cell structure and function. 148 CHAPTER 5 • Biomembranes and Cell Architecture Plasma membrane 700 µ m 2 Internal membranes 7000 µ m 2 Cytoskeleton 94000 µ m 2 Nucleus ER Golgi Mitochondrion ▲ FIGURE 5-1 Schematic overview of the major components of eukaryotic cell architecture. The plasma membrane red defines the exterior of the cell and controls the movement of molecules between the cytosol and the extracellular medium. Different types of organelles and smaller vesicles enclosed within their own distinctive membranes black carry out special functions such as gene expression energy production membrane synthesis and intracellular transport. Fibers of the cytoskeleton green provide structural support for the cell and its internal compartments. The internal membranes of organelles and vesicles possess more surface area than that of the plasma membrane but less area than that of the cytoskeleton as schematically represented by the red black and green boxes. The enormous surface area of the cytoskeleton allows it to function as a scaffold on which cellular reactions can take place. Membrane bilayer Exterior a b Polar head groups Hydrophobic tails Polar head groups Cytosol FIGURE 5-2 The bilayer structure of biomembranes. a Electron micrograph of a thin section through an erythrocyte membrane stained with osmium tetroxide. The characteristic “railroad track” appearance of the membrane indicates the presence of two polar layers consistent with the bilayer structure for phospholipid membranes. b Schematic interpretation of the phospholipid bilayer in which polar groups face outward to shield the hydrophobic fatty acyl tails from water. The hydrophobic effect and van der Waals interactions between the fatty acyl tails drive the assembly of the bilayer Chapter 2. Part a courtesy of J. D. Robertson.

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Biomembranes: Lipid Composition and Structural Organization Phospholipids of the composition present in cells sponta- neously form sheetlike phospholipid bilayers which are two molecules thick. The hydrocarbon chains of the phospho- lipids in each layer or leaflet form a hydrophobic core that is 3–4 nm thick in most biomembranes. Electron microscopy of thin membrane sections stained with osmium tetroxide which binds strongly to the polar head groups of phospho- lipids reveals the bilayer structure Figure 5-2. A cross sec- tion of all single membranes stained with osmium tetroxide looks like a railroad track: two thin dark lines the stain– head group complexes with a uniform light space of about 2 nm the hydrophobic tails between them. The lipid bilayer has two important properties. First the hydrophobic core is an impermeable barrier that prevents the diffusion of water-soluble hydrophilic solutes across the membrane. Importantly this simple barrier function is mod- ulated by the presence of membrane proteins that mediate the transport of specific molecules across this otherwise im- permeable bilayer. The second property of the bilayer is its stability. The bilayer structure is maintained by hydropho- bic and van der Waals interactions between the lipid chains. Even though the exterior aqueous environment can vary widely in ionic strength and pH the bilayer has the strength to retain its characteristic architecture. Natural membranes from different cell types exhibit a va- riety of shapes which complement a cell’s function Figure 5-3. The smooth flexible surface of the erythrocyte plasma membrane allows the cell to squeeze through narrow blood capillaries. Some cells have a long slender extension of the plasma membrane called a cilium or flagellum which beats in a whiplike manner. This motion causes fluid to flow across the surface of an epithelium or a sperm cell to swim through the medium. The axons of many neurons are encased by multiple layers of modified plasma membrane called the myelin sheath. This membranous structure is elaborated by 5.1 5.1 • Biomembranes: Lipid Composition and Structural Organization 149 Myelin sheath 0.3 m SN AX FIGURE 5-3 Variation in biomembranes in different cell types. a A smooth flexible membrane covers the surface of the discoid erythrocyte cell. b Tufts of cilia Ci project from the ependymal cells that line the brain ventricles. c Many nerve axons are enveloped in a myelin sheath composed of multiple layers of modified plasma membrane. The individual myelin layers can be seen in this electron micrograph of a cross section of an axon AX. The myelin sheath is formed by an adjacent supportive glial cell SC. Parts a and b from R. G. Kessel and R. H. Kardon 1979 Tissues and Organs: A Text-Atlas of Scanning Electron Microscopy W. H. Freeman and Company. Part c from P . C. Cross and K. L. Mercer 1993 Cell and Tissue Ultrastructure: A Functional Perspective W. H. Freeman and Company p. 137 . 10 m b a c

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an adjacent supportive cell and facilitates the conduction of nerve impulses over long distances Chapter 7. Despite their diverse shapes and functions these biomembranes and all other biomembranes have a common bilayer structure. Because all cellular membranes enclose an entire cell or an internal compartment they have an internal face the sur- face oriented toward the interior of the compartment and an external face the surface presented to the environment. More commonly the surfaces of a cellular membrane are designated as the cytosolic face and the exoplasmic face. This nomenclature is useful in highlighting the topological equiv- alence of the faces in different membranes as diagrammed in Figure 5-4. For example the exoplasmic face of the plasma membrane is directed away from the cytosol toward the ex- tracellular space or external environment and defines the outer limit of the cell. For organelles and vesicles surrounded by a single membrane however the face directed away from the cytosol—the exoplasmic face—is on the inside in con- tact with an internal aqueous space equivalent to the extra- cellular space. This equivalence is most easily understood for vesicles that arise by invagination of the plasma membrane this process results in the external face of the plasma mem- brane becoming the internal face of the vesicle membrane. Three organelles—the nucleus mitochondrion and chloro- plast—are surrounded by two membranes the exoplasmic surface of each membrane faces the space between the two membranes. Three Classes of Lipids Are Found in Biomembranes A typical biomembrane is assembled from phosphoglyc- erides sphingolipids and steroids. All three classes of lipids are amphipathic molecules having a polar hydrophilic head group and hydrophobic tail. The hydrophobic effect and van der Waals interactions discussed in Chapter 2 cause the tail groups to self-associate into a bilayer with the polar head groups oriented toward water see Figure 5-2. Although the common membrane lipids have this amphipathic character in common they differ in their chemical structures abundance and functions in the membrane. Phosphoglycerides the most abundant class of lipids in most membranes are derivatives of glycerol 3-phosphate Figure 5-5a. A typical phosphoglyceride molecule consists of a hydrophobic tail composed of two fatty acyl chains es- terified to the two hydroxyl groups in glycerol phosphate and a polar head group attached to the phosphate group. The two fatty acyl chains may differ in the number of car- bons that they contain commonly 16 or 18 and their degree of saturation 0 1 or 2 double bonds. A phosphogyceride is 150 CHAPTER 5 • Biomembranes and Cell Architecture Endoplasmic reticulum Nucleus Cytosol Golgi Plasma membrane Exoplasmic face Cytosolic face Outer Inner Mitochondrial membranes Matrix Intermembrane space Inner Outer Nuclear membranes Intermembrane space Exterior Mitochondrion Vesicle Lysosome FIGURE 5-4 The faces of cellular membranes. The plasma membrane a single bilayer membrane encloses the cell. In this highly schematic representation internal cytosol green stipple and external environment purple define the cytosolic red and exoplasmic black faces of the bilayer. Vesicles and some organelles have a single membrane and their internal aqueous space purple is topologically equivalent to the outside of the cell. Three organelles—the nucleus mitochondrion and chloroplast which is not shown—are enclosed by two membranes separated by a small intermembrane space. The exoplasmic faces of the inner and outer membranes around these organelles border the intermembrane space between them. For simplicity the hydrophobic membrane interior is not indicated in this diagram.

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classified according to the nature of its head group. In phos- phatidylcholines the most abundant phospholipids in the plasma membrane the head group consists of choline a pos- itively charged alcohol esterified to the negatively charged phosphate. In other phosphoglycerides an OH-containing molecule such as ethanolamine serine and the sugar deriv- ative inositol is linked to the phosphate group. The nega- tively charged phosphate group and the positively charged groups or the hydroxyl groups on the head group interact strongly with water. The plasmalogens are a group of phosphoglycerides that contain one fatty acyl chain attached to glycerol by an ester linkage and one long hydrocarbon chain attached to glyc- erol by an ether linkage COOOC. These molecules con- stitute about 20 percent of the total phosphoglyceride content in humans. Their abundance varies among tissues and species but is especially high in human brain and heart tissue. The additional chemical stability of the ether linkage in plasmalogens or the subtle differences in their three- dimensional structure compared with that of other phos- phoglycerides may have as-yet unrecognized physiologic significance. A second class of membrane lipid is the sphingolipids. All of these compounds are derived from sphingosine an amino alcohol with a long hydrocarbon chain and contain a long-chain fatty acid attached to the sphingosine amino group. In sphingomyelin the most abundant sphingolipid phosphocholine is attached to the terminal hydroxyl group of sphingosine Figure 5-5b. Thus sphingomyelin is a phos- pholipid and its overall structure is quite similar to that of phosphatidylcholine. Other sphingolipids are amphipathic glycolipids whose polar head groups are sugars. Glucosyl- cerebroside the simplest glycosphingolipid contains a single glucose unit attached to sphingosine. In the complex gly- cosphingolipids called gangliosides one or two branched sugar chains containing sialic acid groups are attached to 5.1 • Biomembranes: Lipid Composition and Structural Organization 151 a Phosphoglycerides b Sphingolipids c Cholesterol Head group Hydrophobic tail OH OH CH 3 CH 3 O P O O O − N + O NH CH 3 2 3 4 5 1 GlcCer SM OH O HO O OH OH PI HO O OH OH OH OH CH 3 CH 3 O P OO O O − N + O O O CH 3 H H O N + OO − H PC PS H H O N + H PE 3 2 1 6 FIGURE 5-5 Three classes of membrane lipids. a Most phosphoglycerides are derivatives of glycerol 3-phosphate red containing two esterified fatty acyl chains constituting the hydrophobic “tail” and a polar “head group” esterified to the phosphate. The fatty acids can vary in length and be saturated no double bonds or unsaturated one two or three double bonds. In phosphatidylcholine PC the head group is choline. Also shown are the molecules attached to the phosphate group in three other common phosphoglycerides: phosphatidylethanolamine PE phosphatidyl- serine PS and phosphatidylinositol PI. b Sphingolipids are derivatives of sphingosine red an amino alcohol with a long hydrocarbon chain. Various fatty acyl chains are connected to sphingosine by an amide bond. The sphingomyelins SM which contain a phosphocholine head group are phospholipids. Other sphingolipids are glycolipids in which a single sugar residue or branched oligosaccharide is attached to the sphingosine backbone. For instance the simple glycolipid glucosylcerebroside GlcCer has a glucose head group. c Like other membrane lipids the steroid cholesterol is amphipathic. Its single hydroxyl group is equivalent to the polar head group in other lipids the conjugated ring and short hydrocarbon chain form the hydrophobic tail. See H. Sprong et al. 2001 Nature Rev. Mol. Cell Biol. 2:504.

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sphingosine. Glycolipids constitute 2–10 percent of the total lipid in plasma membranes they are most abundant in nerv- ous tissue. Cholesterol and its derivatives constitute the third im- portant class of membrane lipids the steroids. The basic structure of steroids is a four-ring hydrocarbon. Cholesterol the major steroidal constituent of animal tissues has a hy- droxyl substituent on one ring Figure 5-5c. Although cho- lesterol is almost entirely hydrocarbon in composition it is amphipathic because its hydroxyl group can interact with water. Cholesterol is especially abundant in the plasma mem- branes of mammalian cells but is absent from most prokary- otic cells. As much as 30–50 percent of the lipids in plant plasma membranes consist of certain steroids unique to plants. At neutral pH some phosphoglycerides e.g. phos- phatidylcholine and phosphatidylethanolamine carry no net electric charge whereas others e.g. phosphatidylinositol and phosphatidylserine carry a single net negative charge. Nonetheless the polar head groups in all phospholipids can pack together into the characteristic bilayer structure. Sphin- gomyelins are similar in shape to phosphoglycerides and can form mixed bilayers with them. Cholesterol and other steroids are too hydrophobic to form a bilayer structure un- less they are mixed with phospholipids. Most Lipids and Many Proteins Are Laterally Mobile in Biomembranes In the two-dimensional plane of a bilayer thermal motion per- mits lipid molecules to rotate freely around their long axes and to diffuse laterally within each leaflet. Because such move- ments are lateral or rotational the fatty acyl chains remain in the hydrophobic interior of the bilayer . In both natural and ar- 152 CHAPTER 5 • Biomembranes and Cell Architecture a b Bleach with laser Fluorescence recovery Time s 100 150 50 3000 2000 1000 Fluorescence intensity arb. units Fluorescence before bleaching 50 immobile 50 mobile Bleach Label Bleached area Membrane protein Fluorescent reagent Cell 1 2 3 ▲ EXPERIMENTAL FIGURE 5-6 Fluorescence recovery after photobleaching FRAP experiments can quantify the lateral movement of proteins and lipids within the plasma membrane. a Experimental protocol. Step : Cells are first labeled with a fluorescent reagent that binds uniformly to a specific membrane lipid or protein. Step : A laser light is then focused on a small area of the surface irreversibly bleaching the bound reagent and thus reducing the fluorescence in the illuminated area. Step : In time the fluorescence of the bleached patch increases as unbleached fluorescent surface molecules diffuse into it and bleached ones diffuse outward. The extent of recovery of fluorescence in the bleached patch is 3 2 1 proportional to the fraction of labeled molecules that are mobile in the membrane. b Results of FRAP experiment with human hepatoma cells treated with a fluorescent antibody specific for the asialoglycoprotein receptor protein. The finding that 50 percent of the fluorescence returned to the bleached area indicates that 50 percent of the receptor molecules in the illuminated membrane patch were mobile and 50 percent were immobile. Because the rate of fluorescence recovery is proportional to the rate at which labeled molecules move into the bleached region the diffusion coefficient of a protein or lipid in the membrane can be calculated from such data. See Y. I. Henis et al. 1990 J. Cell Biol. 111:1409.

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tificial membranes a typical lipid molecule exchanges places with its neighbors in a leaflet about 10 7 times per second and diffuses several micrometers per second at 37 C. These diffu- sion rates indicate that the viscosity of the bilayer is 100 times as great as that of water—about the same as the viscosity of olive oil. Even though lipids diffuse more slowly in the bilayer than in an aqueous solvent a membrane lipid could diffuse the length of a typical bacterial cell 1 m in only 1 second and the length of an animal cell in about 20 seconds. The lateral movements of specific plasma-membrane pro- teins and lipids can be quantified by a technique called fluo- rescence recovery after photobleaching FRAP. With this method described in Figure 5-6 the rate at which membrane lipid or protein molecules move—the diffusion coefficient— can be determined as well as the proportion of the molecules that are laterally mobile. The results of FRAP studies with fluorescence-labeled phospholipids have shown that in fibroblast plasma mem- branes all the phospholipids are freely mobile over distances of about 0.5 m but most cannot diffuse over much longer distances. These findings suggest that protein-rich regions of the plasma membrane about 1 m in diameter separate lipid-rich regions containing the bulk of the membrane phospholipid. Phospholipids are free to diffuse within such a region but not from one lipid-rich region to an adjacent one. Furthermore the rate of lateral diffusion of lipids in the plasma membrane is nearly an order of magnitude slower than in pure phospholipid bilayers: diffusion constants of 10 8 cm 2 /s and 10 7 cm 2 /s are characteristic of the plasma membrane and a lipid bilayer respectively. This difference suggests that lipids may be tightly but not irreversibly bound to certain integral proteins in some membranes. Lipid Composition Influences the Physical Properties of Membranes A typical cell contains myriad types of membranes each with unique properties bestowed by its particular mix of lipids and proteins. The data in Table 5-1 illustrate the variation in lipid composition among different biomembranes. Several phenomena contribute to these differences. For instance dif- ferences between membranes in the endoplasmic reticulum ER and the Golgi are largely explained by the fact that phospholipids are synthesized in the ER whereas sphin- golipids are synthesized in the Golgi. As a result the propor- tion of sphingomyelin as a percentage of total membrane lipid phosphorus is about six times as high in Golgi mem- branes as it is in ER membranes. In other cases the translo- cation of membranes from one cellular compartment to another can selectively enrich membranes in certain lipids. Differences in lipid composition may also correspond to specialization of membrane function. For example the plasma membrane of absorptive epithelial cells lining the in- testine exhibits two distinct regions: the apical surface faces the lumen of the gut and is exposed to widely varying exter- nal conditions the basolateral surface interacts with other epithelial cells and with underlying extracellular structures see Figure 6-5. In these polarized cells the ratio of sphin- golipid to phosphoglyceride to cholesterol in the basolateral membrane is 0.5:1.5:1 roughly equivalent to that in the plasma membrane of a typical unpolarized cell subjected to mild stress. In contrast the apical membrane of intestinal cells which is subjected to considerable stress exhibits a 1:1:1 ratio of these lipids. The relatively high concentration of sphingolipid in this membrane may increase its stability 5.1 • Biomembranes: Lipid Composition and Structural Organization 153 TABLE 5-1 Major Lipid Components of Selected Biomembranes Composition mol Source/Location PC PE PS SM Cholesterol Plasma membrane human erythrocytes 21 29 21 26 Myelin membrane human neurons 16 37 13 34 Plasma membrane E. coli0 85 0 0 Endoplasmic reticulum membrane rat 54 26 5 7 Golgi membrane rat 45 20 13 13 Inner mitochondrial membrane rat 45 45 2 7 Outer mitochondrial membrane rat 34 46 2 11 Primary leaflet location Exoplasmic Cytosolic Exoplasmic Both PC phosphatidylcholine PE phosphatidylethanolamine PS phosphatidylserine SM sphingomyelin. SOURCE:W. Dowhan and M. Bogdanov 2002 in D. E. Vance and J. E. Vance eds. Biochemistry of Lipids Lipoproteins and Membranes Elsevier.

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because of extensive hydrogen bonding by the free OOH group in the sphingosine moiety see Figure 5-5. The ability of lipids to diffuse laterally in a bilayer indicates that it can act as a fluid. The degree of bilayer fluidity depends on the lipid composition structure of the phospholipid hy- drophobic tails and temperature. As already noted van der Waals interactions and the hydrophobic effect cause the non- polar tails of phospholipids to aggregate. Long saturated fatty acyl chains have the greatest tendency to aggregate packing tightly together into a gel-like state. Phospholipids with short fatty acyl chains which have less surface area for interaction form more fluid bilayers. Likewise the kinks in unsaturated fatty acyl chains result in their forming less stable van der Waals interactions with other lipids than do saturated chains and hence more fluid bilayers. When a highly ordered gel-like bilayer is heated the increased molecular motions of the fatty acyl tails cause it to undergo a transition to a more fluid dis- ordered state Figure 5-7. At usual physiologic temperatures the hydrophobic in- terior of natural membranes generally has a low viscosity and a fluidlike rather than gel-like consistency. Choles- terol is important in maintaining the fluidity of natural membranes which appears to be essential for normal cell growth and reproduction. As noted previously cholesterol cannot form a sheetlike bilayer on its own. At concentra- tions found in natural membranes cholesterol is interca- lated inserted among phospholipids. Cholesterol restricts the random movement of phospholipid head groups at the outer surfaces of the leaflets but its effect on the movement of long phospholipid tails depends on concentration. At the usual cholesterol concentrations the interaction of the steroid ring with the long hydrophobic tails of phospho- lipids tends to immobilize these lipids and thus decrease biomembrane fluidity. At lower cholesterol concentrations however the steroid ring separates and disperses phospho- lipid tails causing the inner regions of the membrane to be- come slightly more fluid. The lipid composition of a bilayer also influences its thick- ness which in turn may play a role in localizing proteins to a particular membrane. The results of studies on artificial mem- branes demonstrate that sphingomyelin associates into a 154 CHAPTER 5 • Biomembranes and Cell Architecture Gel-like consistency Fluidlike consistency Heat ▲ FIGURE 5-7 Gel and fluid forms of the phospholipid bilayer. Top Depiction of gel-to-fluid transition. Phospholipids with long saturated fatty acyl chains tend to assemble into a highly ordered gel-like bilayer in which there is little overlap of the nonpolar tails in the two leaflets. Heat disorders the nonpolar tails and induces a transition from a gel to a fluid within a temperature range of only a few degrees. As the chains become disordered the bilayer also decreases in thickness. Bottom Molecular models of phospholipid monolayers in gel and fluid states as determined by molecular dynamics calculations. Bottom based on H. Heller et al. 1993 J. Phys. Chem. 97:8343. PC PC and cholesterol SM SM and cholesterol a 4.6–5.6 nm 4.0 nm 3.5 nm b PC PE c ▲ FIGURE 5-8 Effect of lipid composition on bilayer thickness and curvature. a A pure sphingomyelin SM bilayer is thicker than one formed from a phosphoglyceride such as phosphatidylcholine PC. Cholesterol has a lipid-ordering effect on phosphoglyceride bilayers that increases their thickness but does not affect the thickness of the more ordered SM bilayer. b Phospholipids such as PC have a cylindrical shape and form more or less flat monolayers whereas those with smaller head groups such as phosphatidylethanolamine PE have a conical shape. c A bilayer enriched with PC in the exoplasmic leaflet and with PE in the cytosolic face as in many plasma membranes would have a natural curvature. Adapted from H. Sprong et al. 2001 Nature Rev. Mol. Cell Biol. 2:504.

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more gel-like and thicker bilayer than phospholipids do Fig- ure 5-8a. Similarly cholesterol and other molecules that de- crease membrane fluidity increase membrane thickness. Because sphingomyelin tails are already optimally stabilized the addition of cholesterol has no effect on the thickness of a sphingomyelin bilayer. Another property dependent on the lipid composition of a bilayer is its local curvature which depends on the rel- ative sizes of the polar head groups and nonpolar tails of its constituent phospholipids. Lipids with long tails and large head groups are cylindrical in shape those with small head groups are cone shaped Figure 5-8b. As a result bi- layers composed of cylindrical lipids are relatively flat whereas those containing large amounts of cone-shaped lipids form curved bilayers Figure 5-8c. This effect of lipid composition on bilayer curvature may play a role in the for- mation of highly curved membrane pits and blebs internal membrane vesicles and specialized membrane structures such as microvilli. Membrane Lipids Are Usually Distributed Unequally in the Exoplasmic and Cytosolic Leaflets A characteristic of all membranes is an asymmetry in lipid composition across the bilayer. Although most phospho- lipids are present in both membrane leaflets they are com- monly more abundant in one or the other leaflet. For instance in plasma membranes from human erythrocytes and certain canine kidney cells grown in culture almost all the sphingomyelin and phosphatidylcholine both of which form less fluid bilayers are found in the exoplasmic leaflet. In contrast phosphatidylethanolamine phosphatidylserine and phosphatidylinositol which form more fluid bilayers are preferentially located in the cytosolic leaflet. This segre- gation of lipids across the bilayer may influence membrane curvature see Figure 5-8c. Unlike phospholipids choles- terol is relatively evenly distributed in both leaflets of cellu- lar membranes. The relative abundance of a particular phospholipid in the two leaflets of a plasma membrane can be determined on the basis of its susceptibility to hydrolysis by phospho- lipases enzymes that cleave various bonds in the hy- drophilic ends of phospholipids Figure 5-9. Phospholipids in the cytosolic leaflet are resistant to hydrolysis by phos- pholipases added to the external medium because the en- zymes cannot penetrate to the cytosolic face of the plasma membrane. How the asymmetric distribution of phospholipids in membrane leaflets arises is still unclear. In pure bilayers phospholipids do not spontaneously migrate or flip-flop from one leaflet to the other. Energetically such flip-flopping is extremely unfavorable because it entails movement of the polar phospholipid head group through the hydrophobic in- terior of the membrane. To a first approximation the asym- metry in phospholipid distribution results from the vectorial synthesis of lipids in the endoplasmic reticulum and Golgi. Sphingomyelin is synthesized on the luminal exoplasmic face of the Golgi which becomes the exoplasmic face of the plasma membrane. In contrast phosphoglycerides are syn- thesized on the cytosolic face of the ER membrane which is topologically identical with the cytosolic face of the plasma membrane see Figure 5-4. Clearly this explanation does not account for the preferential location of phosphatidyl- choline in the exoplasmic leaflet. Movement of this phos- phoglyceride and perhaps others from one leaflet to the other in some natural membranes is catalyzed by certain ATP- powered transport proteins called flippases discussed in Chapters 7 and 18. The preferential location of lipids to one face of the bi- layer is necessary for a variety of membrane-based functions. For example the head groups of all phosphorylated forms of phosphatidylinositol face the cytosol. Certain of them are cleaved by phospholipase C located in the cytosol this en- zyme in turn is activated as a result of cell stimulation by many hormones. These cleavages generate cytosol-soluble phosphoinositols and membrane-soluble diacylglycerol. As we see in later chapters these molecules participate in intra- cellular signaling pathways that affect many aspects of cel- lular metabolism. Phosphatidylserine also is normally most abundant in the cytosolic leaflet of the plasma membrane. In the initial stages of platelet stimulation by serum phos- phatidylserine is briefly translocated to the exoplasmic face presumably by a flippase enzyme where it activates enzymes participating in blood clotting. 5.1 • Biomembranes: Lipid Composition and Structural Organization 155 P O R O O O O CH 2 Polar head group C O OC O CH CH 2 A 2 A 1 CH 2 n CH 2 n CH 3 CH 3 D C 1 2 3 ▲ FIGURE 5-9 Specificity of phospholipases. Each type of phospholipase cleaves one of the susceptible bonds shown in red. The glycerol carbon atoms are indicated by small numbers. In intact cells only phospholipids in the exoplasmic leaflet of the plasma membrane are cleaved by phospholipases in the surrounding medium. Phospholipase C a cytosolic enzyme cleaves certain phospholipids in the cytosolic leaflet of the plasma membrane.

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Cholesterol and Sphingolipids Cluster with Specific Proteins in Membrane Microdomains The results of recent studies have challenged the long-held belief that lipids are randomly mixed in each leaflet of a bi- layer. The first hint that lipids may be organized within the leaflets was the discovery that the residues remaining after the extraction of plasma membranes with detergents contain two lipids: cholesterol and sphingomyelin. Because these two lipids are found in more ordered less fluid bilayers re- searchers hypothesized that they form microdomains termed lipid rafts surrounded by other more fluid phospholipids that are easily extracted by detergents. Biochemical and microscopic evidence supports the exis- tence of lipid rafts in natural membranes. For instance flu- orescence microscopy reveals aggregates of lipids and raft-specific proteins in the membrane Figure 5-10. The rafts are heterogeneous in size but are typically 50 nm in diameter. Rafts can be disrupted by methyl- -cyclodextrin which depletes the membrane of cholesterol or by antibi- otics such as filipin that sequester cholesterol such find- ings indicate the importance of cholesterol in maintaining the integrity of these rafts. Besides their enrichment by choles- terol and sphingolipids lipid rafts are enriched for many types of cell-surface receptor proteins as well as many sig- naling proteins that bind to the receptors and are activated by them. These lipid–protein complexes can form only in the two-dimensional environment of a hydrophobic bilayer and as discussed in later chapters they are thought to facilitate the detection of chemical signals from the external environ- ment and the subsequent activation of cytosolic events. KEY CONCEPTS OF SECTION 5.1 Biomembranes: Lipid Composition and Structural Organization ■ The eukaryotic cell is demarcated from the external en- vironment by the plasma membrane and organized into membrane-limited internal compartments organelles and vesicles. ■ The total surface area of internal membranes far exceeds that of the plasma membrane. 156 CHAPTER 5 • Biomembranes and Cell Architecture Y Y Y Y Y Y Cholera toxin GM1 PLAP Cholera toxin a b Raft Copatch Separate patches Antibodies Antibodies TfR ▲ EXPERIMENTAL FIGURE 5-10 Some membrane lipids and proteins colocalize in lipid rafts. The results of biochemical studies suggested that GM1 a glycosphingolipid and placental alkaline phosphatase PLAP a lipid-anchored membrane protein aggregate together into lipid rafts whereas the transferrin receptor TfR which traverses the entire membrane does not. To locate these components in the intact plasma membrane cells were treated with fluorescence-labeled cholera toxin green which cross-links closely spaced GM1 molecules and with fluorescence-labeled antibodies red specific for either PLAP or TfR. Each antibody can cross-link closely spaced molecules of the protein that it recognizes. Cross-linking causes the proteins or lipids to form larger patches that can be detected by fluorescence microscopy see Figure 5-42. a Micrograph of a cell treated with toxin and with anti-PLAP antibody shows GM1 and PLAP colocalized in the same patches yellow. This copatching suggests that both GM1 and PLAP are present in lipid rafts that coalesce in the presence of the cross-linking reagents. b Micrograph of a cell treated with toxin and with anti-TfR antibody shows that GM1 and TfR reside in separate patches i.e. red and green indicating that TfR is not a raft-resident protein. Micrographs from T. Harder et al. 1998 J. Cell Biol. 141:929.

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■ The phospholipid bilayer the basic structural unit of all biomembranes is a two-dimensional lipid sheet with hy- drophilic faces and a hydrophobic core which is imperme- able to water-soluble molecules and ions see Figure 5-2. ■ Certain proteins present in biomembranes make them selectively permeable to water-soluble molecules and ions. ■ The primary lipid components of biomembranes are phos- phoglycerides sphingolipids and steroids see Figure 5-5. ■ Most lipids and many proteins are laterally mobile in biomembranes. ■ Different cellular membranes vary in lipid composition see Table 5-1. Phospholipids and sphingolipids are asym- metrically distributed in the two leaflets of the bilayer whereas cholesterol is fairly evenly distributed in both leaflets. ■ Natural biomembranes generally have a fluidlike con- sistency. In general membrane fluidity is decreased by sphingolipids and cholesterol and increased by phospho- glycerides. The lipid composition of a membrane also in- fluences its thickness and curvature see Figure 5-8. ■ Lipid rafts are microdomains containing cholesterol sphingolipids and certain membrane proteins that form in the plane of the bilayer. These aggregates are sites for sig- naling across the plasma membrane. Biomembranes: Protein Components and Basic Functions Membrane proteins are defined by their location within or at the surface of a phospholipid bilayer. Although every bio- logical membrane has the same basic bilayer structure the proteins associated with a particular membrane are respon- sible for its distinctive activities. The density and comple- ment of proteins associated with biomembranes vary depending on cell type and subcellular location. For exam- ple the inner mitochondrial membrane is 76 percent protein the myelin membrane only 18 percent. The high phospho- lipid content of myelin allows it to electrically insulate a nerve cell from its environment. The importance of mem- brane proteins is suggested from the finding that approxi- mately a third of all yeast genes encode a membrane protein. The relative abundance of genes for membrane proteins is even greater in multicellular organisms in which membrane proteins have additional functions in cell adhesion. The lipid bilayer presents a unique two-dimensional hy- drophobic environment for membrane proteins. Some pro- teins are buried within the lipid-rich bilayer other proteins are associated with the exoplasmic or cytosolic leaflet of the bilayer. Protein domains on the extracellular surface of the plasma membrane generally bind to other molecules includ- ing external signaling proteins ions and small metabolites e.g. glucose fatty acids and to adhesion molecules on 5.2 other cells or in the external environment. Domains within the plasma membrane particularly those that form channels and pores move molecules in and out of cells. Domains lying along the cytosolic face of the plasma membrane have a wide range of functions from anchoring cytoskeletal proteins to the membrane to triggering intracellular signaling pathways. In many cases the function of a membrane protein and the topology of its polypeptide chain in the membrane can be predicted on the basis of its homology with another well- characterized protein. In this section we examine the char- acteristic structural features of membrane proteins and some of their basic functions. More complete characterization of the structure and function of various types of membrane pro- teins is presented in several later chapters the synthesis and processing of this large diverse group of proteins are dis- cussed in Chapters 16 and 17. Proteins Interact with Membranes in Three Different Ways Membrane proteins can be classified into three categories— integral lipid-anchored and peripheral—on the basis of the nature of the membrane–protein interactions Figure 5-11. Integral membrane proteins also called transmembrane proteins span a phospholipid bilayer and are built of three segments. The cytosolic and exoplasmic domains have hy- drophilic exterior surfaces that interact with the aqueous solutions on the cytosolic and exoplasmic faces of the mem- brane. These domains resemble other water-soluble proteins in their amino acid composition and structure. In contrast the 3-nm-thick membrane-spanning domain contains many hydrophobic amino acids whose side chains protrude out- ward and interact with the hydrocarbon core of the phos- pholipid bilayer. In all transmembrane proteins examined to date the membrane-spanning domains consist of one or more helices or of multiple strands. In addition most trans- membrane proteins are glycosylated with a complex branched sugar group attached to one or several amino acid side chains. Invariably these sugar chains are localized to the exoplasmic domains. Lipid-anchored membrane proteins are bound covalently to one or more lipid molecules. The hydrophobic carbon chain of the attached lipid is embedded in one leaflet of the membrane and anchors the protein to the membrane. The polypeptide chain itself does not enter the phospholipid bilayer. Peripheral membrane proteins do not interact with the hydrophobic core of the phospholipid bilayer. Instead they are usually bound to the membrane indirectly by interactions with integral membrane proteins or directly by interactions with lipid head groups. Peripheral proteins are localized to either the cytosolic or the exoplasmic face of the plasma membrane. In addition to these proteins which are closely associated with the bilayer cytoskeletal filaments are more loosely as- sociated with the cytosolic face usually through one or more 5.2 • Biomembranes: Protein Components and Basic Functions 157

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peripheral adapter proteins see Figure 5-11. Such associa- tions with the cytoskeleton provide support for various cel- lular membranes see Section 5.4 they also play a role in the two-way communication between the cell interior and the cell exterior as we learn in Chapter 6. Finally peripheral proteins on the outer surface of the plasma membrane and the exoplasmic domains of integral membrane proteins are often attached to components of the extracellular matrix or to the cell wall surrounding bacterial and plant cells. Membrane-Embedded Helices Are the Primary Secondary Structures in Most Transmembrane Proteins Soluble proteins exhibit hundreds of distinct localized folded structures or motifs see Figure 3-6. In comparison the repertoire of folded structures in integral membrane proteins is quite limited with the hydrophobic helix predominating. Integral proteins containing membrane-spanning -helical domains are embedded in membranes by hydrophobic inter- actions with specific lipids and probably also by ionic inter- actions with the polar head groups of the phospholipids. Glycophorin A the major protein in the erythrocyte plasma membrane is a representative single-pass transmem- brane protein which contains only one membrane-spanning helix Figure 5-12. Typically a membrane-embedded helix is composed of 20–25 hydrophobic uncharged amino acids see Figure 2-13. The predicted length of such a helix 3.75 nm is just sufficient to span the hydrocarbon core of a phospholipid bilayer. The hydrophobic side chains pro- trude outward from the helix and form van der Waals in- teractions with the fatty acyl chains in the bilayer. In con- trast the carbonyl CUO and imino NH groups taking part in the formation of backbone peptide bonds through hydrogen bonding are in the interior of the helix see Figure 3-3 thus these polar groups are shielded from the hydrophobic interior of the membrane. The transmem- brane helix of one glycophorin A molecule associates with the helix in another to form a coiled-coil dimer see Figure 5-12b. Such interaction of membrane-spanning helices is a common mechanism for creating dimeric membrane pro- teins. Many cell-surface receptors for instance are activated by dimerization. A large and important family of integral proteins is de- fined by the presence of seven membrane-spanning he- lices. Among the more than 150 such “seven spanning” multipass proteins that have been identified are the G pro- tein–coupled receptors described in Chapter 13. The struc- ture of bacteriorhodopsin a protein found in the membrane of certain photosynthetic bacteria illustrates the general structure of all these proteins Figure 5-13. Absorption of light by the retinal group covalently attached to bacteri- orhodopsin causes a conformational change in the protein that results in the pumping of protons from the cytosol across the bacterial membrane to the extracellular space. The proton concentration gradient thus generated across the membrane is used to synthesize ATP Chapter 8. In the high-resolution structure of bacteriorhodopsin now avail- able the positions of all the individual amino acids retinal and the surrounding lipids are determined. As might be ex- pected virtually all of the amino acids on the exterior of the membrane-spanning segments of bacteriorhodopsin are hydrophobic and interact with the hydrocarbon core of the surrounding lipid bilayer. Ion channels compose a second large and important fam- ily of multipass transmembrane proteins. As revealed by the crystal structure of a resting K channel ion channels are typically tetrameric proteins. Each of the four subunits has a pair of membrane-spanning helices that bundle with helices 158 CHAPTER 5 • Biomembranes and Cell Architecture Integral Peripheral Peripheral Lipid anchored Cytoskeleton Extracellular matrix Exterior Cytosol Peripheral Integral FIGURE 5-11 Diagram of how various classes of proteins associate with the lipid bilayer. Integral transmembrane proteins span the bilayer. Lipid-anchored proteins are tethered to one leaflet by a long covalently attached hydrocarbon chain. Peripheral proteins associate with the membrane primarily by specific noncovalent interactions with integral proteins or membrane lipids. Farther from the membrane are membrane- associated proteins including the cytoskeleton extracellular matrix in animal cells and cell wall in plant and bacterial cells not depicted. Carbohydrate chains are attached to many extracellular proteins and to the exoplasmic domains of many transmembrane proteins.

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of other subunits forming a central channel see Figure 7-15. Polar and hydrophobic residues lining the center of the bundle form a channel in the membrane but as with bac- teriorhodopsin virtually all of the amino acids on the exterior of the membrane-spanning domain are hydrophobic. In many ion channels external factors e.g. a ligand voltage or mechanical strain regulate ion flow across the bilayer by reorienting the helices. Details of ion channels and their structures are discussed in Chapter 7. 5.2 • Biomembranes: Protein Components and Basic Functions 159 73 96 N N C C Extracellular domain Cytosolic domain Membrane- spanning helices a b ▲ FIGURE 5-12 Structure of glycophorin A a typical single- pass transmembrane protein. a Diagram of dimeric glycophorin showing major sequence features and its relation to the membrane. The single 23-residue membrane-spanning helix in each monomer is composed of amino acids with hydrophobic uncharged side chains red spheres. By binding negatively charged phospholipid head groups the positively charged arginine and lysine residues blue spheres near the cytosolic side of the helix help anchor glycophorin in the membrane. Both the extracellular and the cytosolic domains are rich in charged residues and polar uncharged residues the extracellular domain is heavily glycosylated with the carbohydrate side chains green diamonds attached to specific serine threonine and asparagine residues. b Molecular model of the transmembrane domain of dimeric glycophorin corresponding to residues 73–96. The side chains of the helix in one monomer are shown in red those in the other monomer in gray. Residues depicted as space-filling structures participate in intermonomer van der Waals interactions that stabilize the coiled-coil dimer. Part b adapted from K. R. MacKenzie et al. 1997 Science 276:131 . Exterior Cytosol FIGURE 5-13 Structural model of bacteriorhodopsin a multipass transmembrane protein that functions as a photoreceptor in certain bacteria. The seven hydrophobic helices in bacteriorhodopsin traverse the lipid bilayer. A retinal molecule red covalently attached to one helix absorbs light. The large class of G protein–coupled receptors in eukaryotic cells also has seven membrane-spanning helices their three-dimensional structure is similar to that of bacteriorhodopsin. After H. Luecke et al. 1999 J. Mol. Biol. 291:899.

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Multiple Strands in Porins Form Membrane-Spanning “Barrels” The porins are a class of transmembrane proteins whose structure differs radically from that of other integral pro- teins. Several types of porin are found in the outer membrane of gram-negative bacteria such as E. coli and in the outer membranes of mitochondria and chloroplasts. The outer membrane protects an intestinal bacterium from harmful agents e.g. antibiotics bile salts and proteases but per- mits the uptake and disposal of small hydrophilic molecules including nutrients and waste products. The porins in the outer membrane of an E. coli cell provide channels for the passage of disaccharides and other small molecules as well as phosphate. The amino acid sequences of porins are predominantly polar and contain no long hydrophobic segments typical of integral proteins with -helical membrane-spanning do- mains. X-ray crystallography has revealed that porins are trimers of identical subunits. In each subunit 16 strands form a barrel-shaped structure with a pore in the center Fig- ure 5-14. Unlike a typical water-soluble globular protein a porin has a hydrophilic inside and a hydrophobic exterior in this sense porins are inside-out. In a porin monomer the outward-facing side groups on each of the strands are hy- drophobic and form a nonpolar ribbonlike band that encir- cles the outside of the barrel. This hydrophobic band interacts with the fatty acyl groups of the membrane lipids or with other porin monomers. The side groups facing the in- side of a porin monomer are predominantly hydrophilic they line the pore through which small water-soluble mole- cules cross the membrane. As discussed in Chapter 7 the plasma membranes of ani- mal cells contain a water channel called aquaporin. Like most other integral proteins aquaporin contains multiple trans- membrane helices. Thus despite its name aquaporin differs structurally from the porins as well as functionally in that it mediates transport of a single molecule—namely water. Covalently Attached Hydrocarbon Chains Anchor Some Proteins to Membranes In eukaryotic cells several types of covalently attached lipids anchor some proteins to one or the other leaflet of the plasma membrane and certain other cellular membranes. In these lipid-anchored proteins the lipid hydrocarbon chains are embedded in the bilayer but the protein itself does not enter the bilayer. A group of cytosolic proteins are anchored to the cytoso- lic face of a membrane by a fatty acyl group e.g. myristate or palmitate attached to the N-terminal glycine residue Fig- ure 5-15a. Retention of such proteins at the membrane by the N-terminal acyl anchor may play an important role in a membrane-associated function. For example v-Src a mutant form of a cellular tyrosine kinase is oncogenic and can trans- form cells only when it has a myristylated N-terminus. A second group of cytosolic proteins are anchored to membranes by an unsaturated fatty acyl group attached to a cysteine residue at or near the C-terminus Figure 5-15b. In these proteins a farnesyl or geranylgeranyl group is bound through a thioether bond to the OSH group of a C-terminal cysteine residue. These prenyl anchors are built from isoprene units C 5 which are also used in the synthesis of cholesterol Chapter 18. In some cases a second geranylgeranyl group or a palmitate group is linked to a nearby cysteine residue. The additional anchor is thought to reinforce the attachment of the protein to the membrane. Ras a GTPase superfamily protein that functions in intracellular signaling is localized to the cytosolic face of the plasma membrane by such a double anchor. Rab proteins which also belong to the GTPase su- perfamily are similarly bound to the cytosolic surface of in- tracellular vesicles by prenyl-type anchors these proteins are required for the fusion of vesicles with their target membranes Chapter 17. Some cell-surface proteins and heavily glycosylated pro- teoglycans of the extracellular matrix are bound to the exo- 160 CHAPTER 5 • Biomembranes and Cell Architecture Exterior Periplasm ▲ FIGURE 5-14 Structural model of one subunit of OmpX a porin found in the E. coli outer membrane. All porins are trimeric transmembrane proteins. Each subunit is barrel shaped with strands forming the wall and a transmembrane pore in the center. A band of aliphatic noncyclic side chains yellow and a border of aromatic ring-containing side chains red position the protein in the bilayer. After G. E. Schulz 2000 Curr. Opin. Struc. Biol. 10:443.

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plasmic face of the plasma membrane by a third type of an- chor group glycosylphosphatidylinositol GPI. The exact structures of GPI anchors vary greatly in different cell types but they always contain phosphatidylinositol PI whose two fatty acyl chains extend into the lipid bilayer phospho- ethanolamine which covalently links the anchor to the C-terminus of a protein and several sugar residues Figure 5-15c. Various experiments have shown that the GPI anchor is both necessary and sufficient for binding proteins to the membrane. For instance the enzyme phospholipase C cleaves the phosphate–glycerol bond in phospholipids and in GPI an- chors see Figure 5-9. Treatment of cells with phospholipase C releases GPI-anchored proteins such as Thy-1 and placental alkaline phosphatase PLAP from the cell surface. As already discussed PLAP is concentrated in lipid rafts the more ordered bilayer microdomains that are enriched in sphingolipids and cholesterol see Figure 5-10. Although PLAP and other GPI-anchored proteins lie in the opposite membrane leaflet from acyl-anchored proteins both types of membrane proteins are concentrated in lipid rafts. In con- trast prenylated proteins are not found in lipid rafts. All Transmembrane Proteins and Glycolipids Are Asymmetrically Oriented in the Bilayer Lipid-anchored proteins are just one example of membrane proteins that are asymmetrically located with respect to the faces of cellular membranes. Each type of transmembrane protein also has a specific orientation with respect to the membrane faces. In particular the same parts of a particu- lar protein always faces the cytosol whereas other parts face the exoplasmic space. This asymmetry in protein orientation confers different properties on the two membrane faces. We describe how the orientation of different types of transmem- brane proteins is established during their synthesis in Chap- ter 16. Membrane proteins have never been observed to flip-flop across a membrane such movement requiring a transient movement of hydrophilic amino acid residues through the hydrophobic interior of the membrane would be energetically unfavorable. Accordingly the asymmetry of a transmembrane protein which is established during its biosynthesis and insertion into a membrane is maintained throughout the protein’s lifetime. Many transmembrane proteins contain carbohydrate chains covalently linked to serine threonine or asparagine side chains of the polypeptide. Such transmembrane glyco- proteins are always oriented so that the carbohydrate chains are in the exoplasmic domain see Figures 5-11 and 5-12. Likewise glycolipids in which a carbohydrate chain is at- tached to the glycerol or sphingosine backbone are always located in the exoplasmic leaflet with the carbohydrate chain protruding from the membrane surface. Both glycoproteins and glycolipids are especially abundant in the plasma mem- branes of eukaryotic cells they are absent from the inner mi- tochondrial membrane chloroplast lamellae and several other intracellular membranes. Because the carbohydrate chains of glycoproteins and glycolipids in the plasma mem- brane extend into the extracellular space they are available to interact with components of the extracellular matrix as well as lectins growth factors and antibodies. One important consequence of such interactions is illustrated by the A B and O blood-group antigens. These three structurally related oligo- saccharide components of certain glycoproteins and gly- colipids are expressed on the surfaces of human erythrocytes and many other cell types Figure 5-16. All humans have the enzymes for synthesizing O antigen. Persons with type A blood also have a glycosyltransferase that adds an extra 5.2 • Biomembranes: Protein Components and Basic Functions 161 Cytosol Exterior c GPI anchor + H 3 N b Prenylation Gly NH 3 + a Acylation Cys COO − ▲ FIGURE 5-15 Anchoring of plasma-membrane proteins to the bilayer by covalently linked hydrocarbon groups. a Cytosolic proteins such as v-Src are associated with the plasma membrane through a single fatty acyl chain attached to the N-terminal glycine Gly residue of the polypeptide. Myristate C 14 and palmitate C 16 are common acyl anchors. b Other cytosolic proteins e.g. Ras and Rab proteins are anchored to the membrane by prenylation of one or two cysteine Cys residues at or near the C-terminus. The anchors are farnesyl C 15 and geranylgeranyl C 20 groups both of which are unsaturated. c The lipid anchor on the exoplasmic surface of the plasma membrane is glycosylphosphatidylinositol GPI. The phosphatidylinositol part red of this anchor contains two fatty acyl chains that extend into the bilayer. The phosphoethanolamine unit purple in the anchor links it to the protein. The two green hexagons represent sugar units which vary in number and arrangement in different GPI anchors. The complete structure of a yeast GPI anchor is shown in Figure 16-14. Adapted from H. Sprong et al. 2001 Nature Rev. Mol. Cell Biol. 2:504.

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N-acetylgalactosamine to O antigen to form A antigen. Those with type B blood have a different transferase that adds an extra galactose to O antigen to form B antigen. Peo- ple with both transferases produce both A and B antigen AB blood type those who lack these transferases produce O antigen only O blood type. Persons whose erythrocytes lack the A antigen B anti- gen or both on their surface normally have antibodies against the missing antigens in their serum. Thus if a type A or O person receives a transfusion of type B blood anti- bodies against the B epitope will bind to the introduced red cells and trigger their destruction. To prevent such harmful reactions blood-group typing and appropriate matching of blood donors and recipients are required in all transfusions Table 5-2. ❚ Interactions with the Cytoskeleton Impede the Mobility of Integral Membrane Proteins The results of experiments like the one depicted in Figure 5-6 and other types of studies have shown that many trans- membrane proteins and lipid-anchored proteins like phos- pholipids float quite freely within the plane of a natural membrane. From 30 to 90 percent of all integral proteins in the plasma membrane are freely mobile depending on the cell type. The lateral diffusion rate of a mobile protein in a pure phospholipid bilayer or isolated plasma membrane is similar to that of lipids. However the diffusion rate of a protein in the plasma membrane of intact cells is generally 10–30 times lower than that of the same protein embedded in synthetic spherical bilayer structures liposomes. These findings suggest that the mobility of integral proteins in the plasma membrane of living cells is restricted by interactions with the rigid submembrane cytoskeleton. Some integral proteins are permanently linked to the underlying cyto- skeleton these proteins are completely immobile in the membrane. In regard to mobile proteins such interactions are broken and remade as the proteins diffuse laterally in the plasma membrane slowing down their rate of diffusion. We consider the nature and functional consequences of link- ages between integral membrane proteins and the cyto- skeleton in Chapter 6. Lipid-Binding Motifs Help Target Peripheral Proteins to the Membrane Until the past decade or so the interaction of peripheral proteins with integral proteins was thought to be the major 162 CHAPTER 5 • Biomembranes and Cell Architecture GlcNAc Glc Fuc O antigen A antigen GalNAc B antigen Gal Lipid or protein GalNAc transferase Gal transferase Glc Glucose Gal Galactose GlcNAc N-Acetylglucosamine GalNAc N-Acetylgalactosamine Fuc Fucose Fuc Fuc Gal Gal Glc GlcNAc Gal Gal Gal Gal GlcNAc Glc Lipid or protein Lipid or protein ▲ FIGURE 5-16 Human ABO blood-group antigens. These antigens are oligosaccharide chains covalently attached to glycolipids or glycoproteins in the plasma membrane. The terminal oligosaccharide sugars distinguish the three antigens. The presence or absence of the glycosyltransferases that add galactose Gal or N-acetylgalactosamine GalNAc to O antigen determine a person’s blood type. TABLE 5-2 ABO Blood Groups Blood-Group Antigens on Can Receive Type RBCs Serum Antibodies Blood Types AA Anti-A A and O BB Anti-B B and O AB A and B None All OO Anti-A and anti-B O See Figure 5-16 for antigen structures.

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mechanism by which peripheral proteins were bound to membranes. The results of more recent research indicate that protein–lipid interactions are equally important in localizing peripheral proteins to cellular membranes see Figure 5-11. Analyses of genome sequences have revealed several widely distributed lipid-binding motifs in proteins Table 5-3. For instance the pleckstrin homology PH domain which binds two types of phosphorylated phosphatidyli- nositols is the eleventh most common protein domain en- coded in the human genome. This domain was initially recognized in pleckstrin a protein found in platelets. The high frequency of the PH domain indicates that proteins localized to membrane surfaces carry out many important functions. Other common lipid-binding motifs include the C2 domain the ankyrin-repeat domain and the FERM domain. Originally discovered in protein kinase C the C2 domain is a membrane-targeting domain for various kinases phosphatases and phospholipases. The phospholipases are representative of those water- soluble enzymes that associate with the polar head groups of membrane phospholipids to carry out their catalytic func- tions. As noted earlier phospholipases hydrolyze various bonds in the head groups of phospholipids see Figure 5-9. These enzymes have an important role in the degradation of damaged or aged cell membranes and are active mole- cules in many snake venoms. The mechanism of action of phospholipase A 2 illustrates how such water-soluble en- zymes can reversibly interact with membranes and catalyze reactions at the interface of an aqueous solution and lipid surface. When this enzyme is in aqueous solution its Ca 2 - containing active site is buried in a channel lined with hy- drophobic amino acids. The enzyme binds with greatest affinity to bilayers composed of negatively charged phos- pholipids e.g. phosphotidylethanolamine. This finding suggests that a rim of positively charged lysine and arginine residues around the entrance catalytic channel is particularly important in interfacial binding Figure 5-17a. Binding 5.2 • Biomembranes: Protein Components and Basic Functions 163 TABLE 5-3 Selected Lipid-Binding Motifs Motif Ligand Selected Proteins with Motif PH PIP 2 PIP 3 Phospholipase C 1 protein kinase B pleckstrin C2 Acidic phospholipids Protein kinase C PI-3 kinase phospholipase PTEN phosphatase Ankyrin repeat PS Ankyrin † FERM PIP 2 Band 4.1 protein ezrin radixin moesin ERM † PIP 2 PIP 3 and PI-3P phosphatidylinositol derivatives with additional phosphate groups on the inositol ring see Figure 14-26 PH pleckstrin homology PS phosphatidylserine. † These proteins have roles in linking the actin cytoskeleton to the plasma membrane. a Active site b Ca 2+ P O CH 2 CH NH 3 CH 2 CH 2 . . O − O O CH 2 O C R 1 O CO O . . . R 2 Ca 2+ ▲ FIGURE 5-17 Interfacial binding surface and mechanism of action of phospholipase A 2 . a A structural model of the enzyme showing the surface that interacts with a membrane. This interfacial binding surface contains a rim of positively charged arginine and lysine residues shown in blue surrounding the cavity of the catalytic active site in which a substrate lipid red stick structure is bound. b Diagram of catalysis by phospholipase A 2 . When docked on a model lipid membrane positively charged residues of the interfacial binding site bind to negatively charged polar groups at the membrane surface. This binding triggers a small conformational change opening a channel lined with hydrophobic amino acids that leads from the bilayer to the catalytic site. As a phospholipid moves into the channel an enzyme-bound Ca 2+ ion green binds to the head group positioning the ester bond to be cleaved next to the catalytic site. Part a adapted from M. H. Gelb et al. 1999 Curr. Opin. Struc. Biol. 9:428. Part b see D. Blow 1991 Nature 351:444.

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induces a small conformational change in phospholipase A 2 that fixes the protein to the phospholipid heads and opens the hydrophobic channel. As a phospholipid molecule dif- fuses from the bilayer into the channel the enzyme-bound Ca 2 binds to the phosphate in the head group thereby po- sitioning the ester bond to be cleaved next to the catalytic site Figure 5-17b. The Plasma Membrane Has Many Common Functions in All Cells Although the lipid composition of a membrane largely de- termines its physical characteristics its complement of pro- teins is primarily responsible for a membrane’s functional properties. We have alluded to many functions of the plasma membrane in the preceding discussion and briefly consider its major functions here. In all cells the plasma membrane acts as a permeability barrier that prevents the entry of unwanted materials from the extracellular milieu and the exit of needed metabolites. Specific membrane transport proteins in the plasma mem- brane permit the passage of nutrients into the cell and meta- bolic wastes out of it others function to maintain the proper ionic composition and pH ≈7.2 of the cytosol. The struc- ture and function of proteins that make the plasma mem- brane selectively permeable to different molecules are discussed in Chapter 7. The plasma membrane is highly permeable to water but poorly permeable to salts and small molecules such as sug- ars and amino acids. Owing to osmosis water moves across such a semipermeable membrane from a solution of low solute high water concentration to one of high solute low water concentration until the total solute concentrations and thus the water concentrations on both sides are equal. Figure 5-18 illustrates the effect on animal cells of different external ion concentrations. When most animal cells are placed in an isotonic solution i.e. one with total concen- tration of solutes equal to that of the cell interior there is no net movement of water into or out of cells. However when cells are placed in a hypotonic solution i.e. one with a lower solute concentration than that of the cell interior water flows into the cells causing them to swell. Conversely in a hypertonic solution i.e. one with a higher solute con- centration than that of the cell interior water flows out of cells causing them to shrink. Under normal in vivo con- ditions ion channels in the plasma membrane control the movement of ions into and out of cells so that there is no net movement of water and the usual cell volume is maintained. Unlike animal cells bacterial fungal and plant cells are surrounded by a rigid cell wall and lack the extracellu- lar matrix found in animal tissues. The plasma membrane is intimately engaged in the assembly of cell walls which in plants are built primarily of cellulose. The cell wall prevents the swelling or shrinking of a cell that would oth- erwise occur when it is placed in a hypotonic or hyper- tonic medium respectively. For this reason cells surrounded by a wall can grow in media having an osmotic strength much less than that of the cytosol. The properties func- tion and formation of the plant cell wall are covered in Chapter 6. In addition to these universal functions the plasma membrane has other crucial roles in multicellular organ- isms. Few of the cells in multicellular plants and animals exist as isolated entities rather groups of cells with related specializations combine to form tissues. In animal cells spe- cialized areas of the plasma membrane contain proteins and glycolipids that form specific junctions between cells to strengthen tissues and to allow the exchange of metabolites 164 CHAPTER 5 • Biomembranes and Cell Architecture a Isotonic medium b Hypotonic medium c Hypertonic medium 0.15 M KCl 0.075 M NaCl 0.15 M KCl 0.15 M NaCl 0.15 M KCl 0.30 M NaCl ▲ FIGURE 5-18 Effect of external ion concentration on water flow across the plasma membrane of an animal cell. Sodium potassium and chloride ions do not move freely across the plasma membrane but water channels aquaporins in the membrane permit the flow of water in the direction dictated by the ion concentration of the surrounding medium. a When the medium is isotonic there is no net flux of water into or out of the cell. b When the medium is hypotonic water flows into the cell red arrow until the ion concentration inside and outside the cell is the same. Because of the influx of water the cell volume increases. c When the medium is hypertonic water flows out of the cell until the ion concentration inside and outside the cell is the same. Because water is lost the cell volume decreases.

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between cells. Certain plasma-membrane proteins anchor cells to components of the extracellular matrix the mixture of fibrous proteins and polysaccharides that provides a bed- ding on which most sheets of epithelial cells or small glands lie. We examine both of these membrane functions in Chap- ter 6. Still other proteins in the plasma membrane act as an- choring points for many of the cytoskeletal fibers that permeate the cytosol imparting shape and strength to cells see Section 5.4. The plasma membranes of many types of eukaryotic cells also contain receptor proteins that bind specific signaling molecules e.g. hormones growth factors neurotransmit- ters leading to various cellular responses. These proteins which are critical for cell development and functioning are described in several later chapters. Finally peripheral cy- tosolic proteins that are recruited to the membrane surface function as enzymes intracellular signal transducers and structural proteins for stabilizing the membrane. Like the plasma membrane the membrane surround- ing each organelle in eukaryotic cells contains a unique set of proteins essential for its proper functioning. In the next section we provide a brief overview of the main eukaryotic organelles. KEY CONCEPTS OF SECTION 5.2 Biomembranes: Protein Components and Basic Functions ■ Biological membranes usually contain both integral trans- membrane and peripheral membrane proteins which do not enter the hydrophobic core of the bilayer see Figure 5-11. ■ Most integral membrane proteins contain one or more membrane-spanning hydrophobic helices and hydro- philic domains that extend from the cytosolic and exo- plasmic faces of the membrane see Figure 5-12. ■ The porins unlike other integral proteins contain membrane- spanning sheets that form a barrel-like channel through the bilayer . ■ Long-chain lipids attached to certain amino acids an- chor some proteins to one or the other membrane leaflet see Figure 5-15. ■ Some peripheral proteins associate with the membrane by interactions with integral proteins. Lipid-binding mo- tifs in other peripheral proteins interact with the polar head groups of membrane phospholipids see Table 5-3. ■ The binding of a water-soluble enzyme e.g. a phos- pholipase kinase or phosphatase to a membrane surface brings the enzyme close to its substrate and in some cases activates it. Such interfacial binding is due to the attrac- tion between positive charges on basic residues in the pro- tein and negative charges on phospholipid head groups in the bilayer. Organelles of the Eukaryotic Cell The cell is in a dynamic flux. In the light microscope a live cell exhibits myriad movements ranging from the transloca- tion of chromosomes and vesicles to the changes in shape as- sociated with cell crawling and swimming. Investigation of intracellular structures begins with micrographs of fixed sec- tioned cells in which all cell movements are frozen. Such static pictures of the cell reveal the organization of the cyto- plasm into compartments and the stereotypic location of each type of organelle within the cell. In this section we de- scribe the basic structures and functions of the major or- ganelles in animal and plant cells Figure 5-19. Plant and fungal cells contain most of the organelles found in an ani- mal cell but lack lysosomes. Instead they contain a large cen- tral vacuole that subserves many of the functions of a lysosome. A plant cell also contains chloroplasts and its membrane is strengthened by a rigid cell wall. Unique pro- teins in the interior and membranes of each type of organelle largely determine its specific functional characteristics which are examined in more detail in later chapters. Those organelles bounded by a single membrane are covered first followed by the three types that have a double membrane— the nucleus mitochondrion and chloroplast. Endosomes Take Up Soluble Macromolecules from the Cell Exterior Although transport proteins in the plasma membrane medi- ate the movement of ions and small molecules across the lipid bilayer proteins and some other soluble macromole- cules in the extracellular milieu are internalized by endocy- tosis. In this process a segment of the plasma membrane invaginates into a “coated pit” whose cytosolic face is lined by a specific set of proteins including clathrin. The pit pinches from the membrane into a small membrane-bounded vesicle that contains extracellular material and is delivered to an early endosome a sorting station of membrane-limited tubules and vesicles Figure 5-20a b. From this compartment some membrane proteins are recycled back to the plasma membrane other membrane proteins are transported to a late endosome where further sorting takes place. The endo- cytic pathway ends when a late endosome delivers its mem- brane and internal contents to lysosomes for degradation. The entire endocytic pathway is described in some detail in Chapter 17. Lysosomes Are Acidic Organelles That Contain a Battery of Degradative Enzymes Lysosomes provide an excellent example of the ability of in- tracellular membranes to form closed compartments in which the composition of the lumen the aqueous interior of the compartment differs substantially from that of the surrounding cytosol. Found exclusively in animal cells 5.3 5.3 • Organelles of the Eukaryotic Cell 165

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▲ FIGURE 5-19 Schematic overview of a “typical” animal cell and plant cell and their major substructures. Not every cell will contain all the organelles granules and fibrous structures shown here and other substructures can be present in some. Cells also differ considerably in shape and in the prominence of various organelles and substructures. 166 Plasma membrane controls movement of molecules in and out of the cell and functions in cell-cell signaling and cell adhesion. Mitochondria which are surrounded by a double membrane generate ATP by oxidation of glucose and fatty acids. Lysosomes which have an acidic lumen degrade material internalized by the cell and worn-out cellular membranes and organelles. Nuclear envelope a double membrane encloses the contents of the nucleus the outer nuclear membrane is continuous with the rough ER. Nucleolus is a nuclear subcompartment where most of the cells rRNA is synthesized. Nucleus is filled with chromatin composed of DNA and proteins in dividing cells is site of mRNA and tRNA synthesis. Smooth endoplasmic reticulum ER synthesizes lipids and detoxifies certain hydrophobic compounds. Rough endoplasmic reticulum ER functions in the synthesis processing and sorting of secreted proteins lysosomal proteins and certain membrane. Golgi complex processes and sorts secreted proteins lysosomal proteins and membrane proteins synthesized on the rough ER. Secretory vesicles store secreted proteins and fuse with the plasma membrane to release their contents. Peroxisomes detoxify various molecules and also break down fatty acids to produce acetyl groups for biosynthesis. Cytoskeletal fibers form networks and bundles that support cellular membranes help organize organelles and participate in cell movement. Microvilli increase surface area for absorption of nutrients from surrounding medium. Cell wall composed largely of cellulose helps maintain the cells shape and provides protection against mechanical stress. Vacuole stores water ions and nutrients degrades macromolecules and functions in cell elongation during growth. Chloroplasts which carry out photosynthesis are surrounded by a double membrane and contain a network of internal membrane-bounded sacs. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1 2 3 4 7 8 9 10 11 12 13 14 16 5 6 15

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lysosomes are responsible for degrading certain components that have become obsolete for the cell or organism. The process by which an aged organelle is degraded in a lysosome is called autophagy “eating oneself”. Materials taken into a cell by endocytosis or phagocytosis also may be degraded in lysosomes see Figure 5-20a. In phagocytosis large insolu- ble particles e.g. bacteria are enveloped by the plasma membrane and internalized. Lysosomes contain a group of enzymes that degrade polymers into their monomeric subunits. For example nu- cleases degrade RNA and DNA into their mononucleotide building blocks proteases degrade a variety of proteins and peptides phosphatases remove phosphate groups from mononucleotides phospholipids and other compounds still other enzymes degrade complex polysaccharides and glycol- ipids into smaller units. All the lysosomal enzymes work most efficiently at acid pH values and collectively are termed acid hydrolases. Two types of transport proteins in the lyso- somal membrane work together to pump H and Cl ions HCl from the cytosol across the membrane thereby acidi- fying the lumen see Figure 7-10b. The acid pH helps to de- nature proteins making them accessible to the action of the lysosomal hydrolases which themselves are resistant to acid denaturation. Lysosomal enzymes are poorly active at the neutral pH of cells and most extracellular fluids. Thus if a lysosome releases its enzymes into the cytosol where the pH is between 7.0 and 7.3 they cause little degradation of cy- tosolic components. Cytosolic and nuclear proteins generally are not degraded in lysosomes but rather in proteasomes large multiprotein complexes in the cytosol see Figure 3-13. Lysosomes vary in size and shape and several hundred may be present in a typical animal cell. In effect they func- tion as sites where various materials to be degraded collect. Primary lysosomes are roughly spherical and do not contain obvious particulate or membrane debris. Secondary lyso- somes which are larger and irregularly shaped appear to re- sult from the fusion of primary lysosomes with other membrane-bounded organelles and vesicles. They contain particles or membranes in the process of being digested Figure 5-20c. Tay-Sachs disease is caused by a defect in one en- zyme catalyzing a step in the lysosomal breakdown of gangliosides. The resulting accumulation of these glycolipids especially in nerve cells has devastating consequences. The symptoms of this inherited disease are usually evident before the age of 1. Affected children com- monly become demented and blind by age 2 and die before their third birthday. Nerve cells from such children are greatly enlarged with swollen lipid-filled lysosomes. ❚ 5.3 • Organelles of the Eukaryotic Cell 167 1 2 3 Phagosome Bacterium Phagocytosis Early endosome ER Endocytosis Autophagy Mitochondrion Autophagosome Primary lysosome Primary lysosome Plasma membrane Primary lysosome Late endosome Secondary lysosome a c 1 µ m M P 0.1 µ m b SL ▲ FIGURE 5-20 Cellular structures that participate in delivering materials to lysosomes. a Schematic overview of three pathways by which materials are moved to lysosomes. Soluble macromolecules are taken into the cell by invagination of coated pits in the plasma membrane and delivered to lysosomes through the endocytic pathway . Whole cells and other large insoluble particles move from the cell surface to lysosomes through the phagocytic pathway . Worn-out organelles and bulk cytoplasm are delivered to lysosomes through the autophagic pathway . Within the acidic lumen of lysosomes 3 2 1 hydrolytic enzymes degrade proteins nucleic acids and other large molecules. b An electron micrograph of a section of a cultured mammalian cell that had taken up small gold particles coated with the egg protein ovalbumin. Gold-labeled ovalbumin black spots is found in early endosomes EE and late endosomes LE but very little is present in autophagosomes AV. c Electron micrograph of a section of a rat liver cell showing a secondary lysosome containing fragments of a mitochondrion M and a peroxisome P. Part b from T. E. Tjelle et al. 1996 J. Cell Sci. 109:2905. Part c courtesy of D. Friend.

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Peroxisomes Degrade Fatty Acids and Toxic Compounds All animal cells except erythrocytes and many plant cells contain peroxisomes a class of roughly spherical organelles 0.2–1.0 m in diameter Figure 5-21. Peroxisomes contain several oxidases—enzymes that use molecular oxygen to ox- idize organic substances in the process forming hydrogen peroxide H 2 O 2 a corrosive substance. Peroxisomes also contain copious amounts of the enzyme catalase which de- grades hydrogen peroxide to yield water and oxygen: Catalase 2 H 2 O 2 _____ → 2 H 2 O + O 2 In contrast with the oxidation of fatty acids in mito- chondria which produces CO 2 and is coupled to the gener- ation of ATP peroxisomal oxidation of fatty acids yields acetyl groups and is not linked to ATP formation see Fig- ure 8-11. The energy released during peroxisomal oxidation is converted into heat and the acetyl groups are transported into the cytosol where they are used in the synthesis of cho- lesterol and other metabolites. In most eukaryotic cells the peroxisome is the principal organelle in which fatty acids are oxidized thereby generating precursors for important biosynthetic pathways. Particularly in liver and kidney cells various toxic molecules that enter the bloodstream also are degraded in peroxisomes producing harmless products. In the human genetic disease X-linked adreno- leukodystrophy ADL peroxisomal oxidation of very long chain fatty acids is defective. The ADL gene encodes the peroxisomal membrane protein that trans- ports into peroxisomes an enzyme required for the oxidation of these fatty acids. Persons with the severe form of ADL are unaffected until midchildhood when severe neurological dis- orders appear followed by death within a few years. ❚ Plant seeds contain glyoxisomes small organelles that oxidize stored lipids as a source of carbon and energy for growth. They are similar to peroxi- somes and contain many of the same types of enzymes as well as additional ones used to convert fatty acids into glu- cose precursors. ❚ The Endoplasmic Reticulum Is a Network of Interconnected Internal Membranes Generally the largest membrane in a eukaryotic cell encloses the endoplasmic reticulum ER—an extensive network of closed flattened membrane-bounded sacs called cisternae see Figure 5-19. The endoplasmic reticulum has a number of functions in the cell but is particularly important in the synthesis of lipids membrane proteins and secreted pro- teins. The smooth endoplasmic reticulum is smooth because it lacks ribosomes. In contrast the cytosolic face of the rough endoplasmic reticulum is studded with ribosomes. The Smooth Endoplasmic Reticulum The synthesis of fatty acids and phospholipids takes place in the smooth ER. Al- though many cells have very little smooth ER this organelle is abundant in hepatocytes. Enzymes in the smooth ER of the liver also modify or detoxify hydrophobic chemicals such as pesticides and carcinogens by chemically converting them into more water-soluble conjugated products that can be ex- creted from the body. High doses of such compounds result in a large proliferation of the smooth ER in liver cells. The Rough Endoplasmic Reticulum Ribosomes bound to the rough ER synthesize certain membrane and organelle proteins and virtually all proteins to be secreted from the cell Chapter 16. A ribosome that fabricates such a protein is bound to the rough ER by the nascent polypeptide chain of the protein. As the growing polypeptide emerges from the ribosome it passes through the rough ER membrane with the help of specific proteins in the membrane. Newly made membrane proteins remain associated with the rough ER membrane and proteins to be secreted accumulate in the lumen of the organelle. All eukaryotic cells contain a discernible amount of rough ER because it is needed for the synthesis of plasma- membrane proteins and proteins of the extracellular matrix. Rough ER is particularly abundant in specialized cells that produce an abundance of specific proteins to be secreted. For example plasma cells produce antibodies pancreatic acinar cells synthesize digestive enzymes and cells in the pancre- atic islets of Langerhans produce the polypeptide hormones insulin and glucagon. In these secretory cells and others a large part of the cytosol is filled with rough ER and secretory vesicles Figure 5-22. 168 CHAPTER 5 • Biomembranes and Cell Architecture Smooth ER P P M M Rough ER Glycogen 1 m ▲ FIGURE 5-21 Electron micrograph showing various organelles in a rat liver cell. Two peroxisomes P lie in close proximity to mitochondria M and the rough and smooth endoplasmic reticulum ER. Also visible are accumulations of glycogen a polysaccharide that is the primary glucose-storage molecule in animals. Courtesy of P . Lazarow.

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The Golgi Complex Processes and Sorts Secreted and Membrane Proteins Several minutes after proteins are synthesized in the rough ER most of them leave the organelle within small membrane- bounded transport vesicles. These vesicles which bud from regions of the rough ER not coated with ribosomes carry the proteins to another membrane-limited organelle the Golgi complex see Figure 5-22. Three-dimensional reconstructions from serial sections of a Golgi complex reveal this organelle to be a series of flat- tened membrane vesicles or sacs cisternae surrounded by a number of more or less spherical membrane-limited vesi- cles Figure 5-23. The stack of Golgi cisternae has three de- fined regions—the cis the medial and the trans. Transport vesicles from the rough ER fuse with the cis region of the Golgi complex where they deposit their protein contents. As detailed in Chapter 17 these proteins then progress from the cis to the medial to the trans region. Within each region are different enzymes that modify proteins to be secreted and membrane proteins differently depending on their structures and their final destinations. After proteins to be secreted and membrane proteins are modified in the Golgi complex they are transported out of the complex by a second set of vesicles which seem to bud from the trans side of the Golgi complex. Some vesicles carry membrane proteins destined for the plasma membrane or soluble proteins to be released from the cell surface others 5.3 • Organelles of the Eukaryotic Cell 169 Intercellular space Plasma membrane Endoplasmic reticulum Golgi vesicles Secretory vesicle Nuclear membrane Nucleus Mitochondrion a 2 µ m Secretory vesicle Golgi vesicles Rough ER Nucleus Secreted protein b 1 2 3 4 ▲ FIGURE 5-22 Charateristic features of cells specialized to secrete large amounts of particular proteins e.g. hormones antibodies. a Electron micrograph of a thin section of a hormone-secreting cell from the rat pituitary. One end of the cell top is filled with abundant rough ER and Golgi sacs where polypeptide hormones are synthesized and packaged. At the opposite end of the cell bottom are numerous secretory vesicles which contain recently made hormones eventually to be secreted. b Diagram of a typical secretory cell tracing the pathway followed by a protein small red dots to be secreted. Immediately after their synthesis on ribosomes blue dots of the rough ER secreted proteins are found in the lumen of the rough ER. Transport vesicles bud off and carry these proteins to the Golgi complex where the proteins are concentrated and packaged into immature secretory vesicles . These vesicles then coalesce to form larger mature secretory vesicles that lose water to the cytosol leaving an almost crystalline mixture of secreted proteins in the lumen . After these vesicles accumulate under the apical surface they fuse with the plasma membrane and release their contents exocytosis in response to appropriate hormonal or nerve stimulation . Part a courtesy of Biophoto Associates. 4 3 2 1 MEDIA CONNECTIONS Overview Animation: Protein Secretion

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carry soluble or membrane proteins to lysosomes or other or- ganelles. How intracellular transport vesicles “know” with which membranes to fuse and where to deliver their contents is also discussed in Chapter 17. Plant Vacuoles Store Small Molecules and Enable a Cell to Elongate Rapidly Most plant cells contain at least one membrane- limited internal vacuole. The number and size of vacuoles depend on both the type of cell and its stage of development a single vacuole may occupy as much as 80 percent of a mature plant cell Figure 5-24. A variety of transport proteins in the vacuolar membrane allow plant cells to accumulate and store water ions and nutrients e.g. sucrose amino acids within vacuoles Chapter 7. Like a lysosome the lumen of a vacuole contains a battery of degradative enzymes and has an acidic pH which is main- tained by similar transport proteins in the vacuolar mem- brane. Thus plant vacuoles may also have a degradative function similar to that of lysosomes in animal cells. Similar storage vacuoles are found in green algae and many mi- croorganisms such as fungi. Like most cellular membranes the vacuolar membrane is permeable to water but is poorly permeable to the small mol- ecules stored within it. Because the solute concentration is much higher in the vacuole lumen than in the cytosol or ex- tracellular fluids water tends to move by osmotic flow into vacuoles just as it moves into cells placed in a hypotonic medium see Figure 5-18. This influx of water causes both the vacuole to expand and water to move into the cell cre- ating hydrostatic pressure or turgor inside the cell. This pressure is balanced by the mechanical resistance of the cellulose-containing cell walls that surround plant cells. Most plant cells have a turgor of 5–20 atmospheres atm their cell walls must be strong enough to react to this pressure in a controlled way. Unlike animal cells plant cells can elongate extremely rapidly at rates of 20–75 m/h. This elongation 170 CHAPTER 5 • Biomembranes and Cell Architecture ▲ FIGURE 5-23 Model of the Golgi complex based on three-dimensional reconstruction of electron microscopy images. Transport vesicles white spheres that have budded off the rough ER fuse with the cis membranes light blue of the Golgi complex. By mechanisms described in Chapter 17 proteins move from the cis region to the medial region and finally to the trans region of the Golgi complex. Eventually vesicles bud off the trans-Golgi membranes orange and red some move to the cell surface and others move to lysosomes. The Golgi complex like the rough endoplasmic reticulum is especially prominent in secretory cells. From B. J. Marsh et al. 2001 Proc Nat’l. Acad. Sci USA 98:2399. Vacuole a Chloroplast Granum Cell wall 2 m ▲ FIGURE 5-24 Electron micrograph of a thin section of a leaf cell. In this cell a single large vacuole occupies much of the cell volume. Parts of five chloroplasts and the cell wall also are visible. Note the internal subcompartments in the chloroplasts. Courtesy of Biophoto Associates/Myron C. Ledbetter/Brookhaven National Laboratory. MEDIA CONNECTIONS Video: Three-Dimensional Model of a Golgi Complex

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which usually accompanies plant growth occurs when a seg- ment of the somewhat elastic cell wall stretches under the pressure created by water taken into the vacuole. ❚ The Nucleus Contains the DNA Genome RNA Synthetic Apparatus and a Fibrous Matrix The nucleus the largest organelle in animal cells is sur- rounded by two membranes each one a phospholipid bilayer containing many different types of proteins. The inner nu- clear membrane defines the nucleus itself. In most cells the outer nuclear membrane is continuous with the rough endo- plasmic reticulum and the space between the inner and outer nuclear membranes is continuous with the lumen of the rough endoplasmic reticulum see Figure 5-19. The two nu- clear membranes appear to fuse at nuclear pores the ringlike complexes composed of specific membrane proteins through which material moves between the nucleus and the cytosol. The structure of nuclear pores and the regulated transport of material through them are detailed in Chapter 12. In a growing or differentiating cell the nucleus is meta- bolically active replicating DNA and synthesizing rRNA tRNA and mRNA. Within the nucleus mRNA binds to spe- cific proteins forming ribonucleoprotein particles. Most of the cell’s ribosomal RNA is synthesized in the nucleolus a subcompartment of the nucleus that is not bounded by a phospholipid membrane Figure 5-25. Some ribosomal pro- teins are added to ribosomal RNAs within the nucleolus as well. The finished or partly finished ribosomal subunits as well as tRNAs and mRNA-containing particles pass through a nuclear pore into the cytosol for use in protein synthesis Chapter 4. In mature erythrocytes from nonmammalian vertebrates and other types of “resting” cells the nucleus is inactive or dormant and minimal synthesis of DNA and RNA takes place. How nuclear DNA is packaged into chromosomes is de- scribed in Chapter 10. In a nucleus that is not dividing the chromosomes are dispersed and not dense enough to be ob- served in the light microscope. Only during cell division are individual chromosomes visible by light microscopy. In the electron microscope the nonnucleolar regions of the nucleus called the nucleoplasm can be seen to have dark- and light- staining areas. The dark areas which are often closely asso- ciated with the nuclear membrane contain condensed concentrated DNA called heterochromatin see Figure 5-25. Fibrous proteins called lamins form a two-dimensional network along the inner surface of the inner membrane giv- ing it shape and apparently binding DNA to it. The break- down of this network occurs early in cell division as we detail in Chapter 21. Mitochondria Are the Principal Sites of ATP Production in Aerobic Cells Most eukaryotic cells contain many mitochondria which oc- cupy up to 25 percent of the volume of the cytoplasm. These complex organelles the main sites of ATP production during aerobic metabolism are generally exceeded in size only by the nucleus vacuoles and chloroplasts. The two membranes that bound a mitochondrion differ in composition and function. The outer membrane com- posed of about half lipid and half protein contains porins see Figure 5-14 that render the membrane permeable to molecules having molecular weights as high as 10000. In this respect the outer membrane is similar to the outer mem- brane of gram-negative bacteria. The inner membrane which is much less permeable is about 20 percent lipid and 80 percent protein—a higher proportion of protein than ex- ists in other cellular membranes. The surface area of the inner membrane is greatly increased by a large number of infoldings or cristae that protrude into the matrix or cen- tral space Figure 5-26. In nonphotosynthetic cells the principal fuels for ATP synthesis are fatty acids and glucose. The complete aerobic degradation of glucose to CO 2 and H 2 O is coupled to the synthesis of as many as 30 molecules of ATP. In eukaryotic cells the initial stages of glucose degradation take place in 5.3 • Organelles of the Eukaryotic Cell 171 Hetero- chromatin N n 1 m ▲ FIGURE 5-25 Electron micrograph of a thin section of a bone marrow stem cell. The nucleolus n is a subcompartment of the nucleus N and is not surrounded by a membrane. Most ribosomal RNA is produced in the nucleolus. Darkly staining areas in the nucleus outside the nucleolus are regions of heterochromatin. From P . C. Cross and K. L. Mercer 1993 Cell and Tissue Ultrastructure W. H. Freeman and Company p. 165.

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the cytosol where 2 ATP molecules per glucose molecule are generated. The terminal stages of oxidation and the coupled synthesis of ATP are carried out by enzymes in the mito- chondrial matrix and inner membrane Chapter 8. As many as 28 ATP molecules per glucose molecule are generated in mitochondria. Similarly virtually all the ATP formed in the oxidation of fatty acids to CO 2 is generated in mitochondria. Thus mitochondria can be regarded as the “power plants” of the cell. Chloroplasts Contain Internal Compartments in Which Photosynthesis Takes Place Except for vacuoles chloroplasts are the largest and the most characteristic organelles in the cells of plants and green algae. They can be as long as 10 m and are typically 0.5–2 m thick but they vary in size and shape in different cells especially among the algae. In ad- dition to the double membrane that bounds a chloroplast this organelle also contains an extensive internal system of inter- connected membrane-limited sacs called thylakoids which are flattened to form disks Figure 5-27. Thylakoids often form stacks called grana and are embedded in a matrix the stroma. The thylakoid membranes contain green pigments chlorophylls and other pigments that absorb light as well as enzymes that generate ATP during photosynthesis. Some of the ATP is used to convert CO 2 into three-carbon intermedi- ates by enzymes located in the stroma the intermediates are then exported to the cytosol and converted into sugars. ❚ The molecular mechanisms by which ATP is formed in mitochondria and chloroplasts are very similar as explained in Chapter 8. Chloroplasts and mitochondria have other fea- tures in common: both often migrate from place to place within cells and they contain their own DNA which en- codes some of the key organellar proteins Chapter 10. The proteins encoded by mitochondrial or chloroplast DNA are synthesized on ribosomes within the organelles. However most of the proteins in each organelle are encoded in nuclear DNA and are synthesized in the cytosol these proteins are then incorporated into the organelles by processes described in Chapter 16. KEY CONCEPTS OF SECTION 5.3 Organelles of the Eukaryotic Cell ■ All eukaryotic cells contain a nucleus and numerous other organelles in their cytosols see Figure 5-19. 172 CHAPTER 5 • Biomembranes and Cell Architecture ▲ FIGURE 5-26 Electron micrograph of a mitochondrion. Most ATP production in nonphotosynthetic cells takes place in mitochondria. The inner membrane which surrounds the matrix space has many infoldings called cristae. Small calcium-containing matrix granules also are evident. From D. W. Fawcett 1981 The Cell 2d ed. Saunders p. 421. Plasma membrane Grana Chloroplast membranes outer and inner Thylakoid membrane Stroma Starch granule 1 m ▲ FIGURE 5-27 Electron micrograph of a plant chloroplast. The internal membrane vesicles thylakoids are fused into stacks grana which reside in a matrix the stroma. All the chlorophyll in the cell is contained in the thylakoid membranes where the light- induced production of ATP takes place during photosynthesis. Courtesy of Biophoto Associates/M. C. Ledbetter/Brookhaven National Laboratory. MEDIA CONNECTIONS Video: Three-Dimensional Model of a Mitochondrion Inner membrane Cristae Matrix Intermembrane space Outer membrane Matrix granules

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■ The nucleus mitochondrion and chloroplast are bounded by two bilayer membranes separated by an in- termembrane space. All other organelles are surrounded by a single membrane. ■ Endosomes internalize plasma-membrane proteins and soluble materials from the extracellular medium and they sort them back to the membranes or to lysosomes for degradation. ■ Lysosomes have an acidic interior and contain various hydrolases that degrade worn-out or unneeded cellular components and some ingested materials see Figure 5-20. ■ Peroxisomes are small organelles containing enzymes that oxidize various organic compounds without the pro- duction of ATP. By-products of oxidation are used in biosynthetic reactions. ■ Secreted proteins and membrane proteins are synthesized on the rough endoplasmic reticulum a network of flat- tened membrane-bounded sacs studded with ribosomes. ■ Proteins synthesized on the rough ER first move to the Golgi complex where they are processed and sorted for trans- port to the cell surface or other destination see Figure 5-22. ■ Plant cells contain one or more large vacuoles which are storage sites for ions and nutrients. Osmotic flow of water into vacuoles generates turgor pressure that pushes the plasma membrane against the cell wall. ■ The nucleus houses the genome of a cell. The inner and outer nuclear membranes are fused at numerous nuclear pores through which materials pass between the nucleus and the cytosol. The outer nuclear membrane is continu- ous with that of the rough endoplasmic reticulum. ■ Mitochondria have a highly permeable outer membrane and a protein-enriched inner membrane that is extensively folded. Enzymes in the inner mitochondrial membrane and central matrix carry out the terminal stages of sugar and lipid oxidation coupled to ATP synthesis. ■ Chloroplasts contain a complex system of thylakoid membranes in their interiors. These membranes contain the pigments and enzymes that absorb light and produce ATP during photosynthesis. The Cytoskeleton: Components and Structural Functions The cytosol is a major site of cellular metabolism and con- tains a large number of different enzymes. Proteins constitute about 20–30 percent of the cytosol by weight and from a quarter to half of the total protein within cells is in the cy- tosol. Estimates of the protein concentration in the cytosol range from 200 to 400 mg/ml. Because of the high concen- tration of cytosolic proteins complexes of proteins can form even if the energy that stabilizes them is weak. Many inves- 5.4 tigators believe that the cytosol is highly organized with most soluble proteins either bound to filaments or otherwise localized in specific regions. In an electron micrograph of a typical animal cell soluble proteins packing the cell interior conceal much of the internal structure. If a cell is pretreated with a nonionic detergent e.g. Triton X-100 which per- meabilizes the membrane soluble cytosolic proteins diffuse away. In micrographs of detergent-extracted animal cells two types of structures stand out—membrane-limited or- ganelles and the filaments of the cytoskeleton which fill the cytosol Figure 5-28. 5.4 • The Cytoskeleton: Components and Structural Functions 173 Membrane- microfilament linkages Core actin filaments Actin filaments rootlets Spectrin connecting fibers Keratin intermediate filaments ▲ FIGURE 5-28 Electron micrograph of the apical part of a detergent-extracted intestinal epithelial cell. Microvilli fingerlike projections of the plasma membrane cover the apical surface of an intestinal epithelial cell. A bundle of microfilaments in the core of each microvillus stabilizes the structure. The plasma membrane surrounding a microvillus is attached to the sides of the bundle by evenly spaced membrane–microfilament linkages yellow. The bundle continues into the cell as a short rootlet. The rootlets of multiple microvilli are cross-braced by connecting fibers red composed of an intestinal isoform of spectrin. This fibrous actin-binding protein is found in a narrow band just below the plasma membrane in many animal cells. The bases of the rootlets are attached to keratin intermediate filaments. These numerous connections anchor the rootlets in a meshwork of filaments and thereby support the upright orientation of the microvilli. Courtesy of N. Hirokawa.

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In this section we introduce the protein filaments that compose the cytoskeleton and then describe how they sup- port the plasma and nuclear membranes and organize the contents of the cell. Later chapters will deal with the dynamic properties of the cytoskeleton—its assembly and disassembly and its role in cellular movements. Three Types of Filaments Compose the Cytoskeleton The cytosol of a eukaryotic cell contains three types of fila- ments that can be distinguished on the bases of their diameter type of subunit and subunit arrangment Figure 5-29. Actin filaments also called microfilaments are 8–9 nm in diame- ter and have a twisted two-stranded structure. Microtubules are hollow tubelike structures 24 nm in diameter whose walls are formed by adjacent protofilaments. Intermediate filaments IFs have the structure of a 10-nm-diameter rope. Each type of cytoskeletal filament is a polymer of pro- tein subunits Table 5-4. Monomeric actin subunits assem- ble into microfilaments dimeric subunits composed of - and -tubulin polymerize into microtubules. Unlike mi- crofilaments and microtubules which are assembled from one or two proteins intermediate filaments are assembled from a large diverse family of proteins. The most common intermediate filaments found in the nucleus are composed of lamins. Intermediate filaments constructed from other proteins are expressed preferentially in certain tissues: for ex- ample keratin-containing filaments in epithelial cells desmin-containing filaments in muscle cells and vimentin- containing filaments in mesenchymal cells. Most eukaryotic cells contain all three types of cy- toskeletal filaments often concentrated in distinct loca- tions. For example in the absorptive epithelial cells that line the lumen of the intestine actin microfilaments are abundant in the apical region where they are associated with cell–cell junctions and support a dense carpet of mi- crovilli Figure 5-30a. Actin filaments are also present in a narrow zone adjacent to the plasma membrane in the lat- eral regions of these cells. Keratin intermediate filaments 174 CHAPTER 5 • Biomembranes and Cell Architecture TABLE 5-4 Protein Subunits in Cytoskeletal Filaments Protein Subunits MW Expression Function MICROFILAMENTS Actin 42000 Fungi plant animal Structural support motility MreB 36000 Rod-shaped bacteria Width control MICROTUBULES Tubulin and 58000 Fungi plant animal Structural support motility cell polarity FtsZ 58000 Bacteria Cell division INTERMEDIATE FILAMENTS Lamins Various Plant animal Support for nuclear membrane Desmin keratin vimentin others Various Animal Cell adhesion OTHER MSP 50000 Nematode sperm Motility FIGURE 5-29 Comparison of the three types of filaments that form the cytoskeleton. a Diagram of the basic structures of an actin filament AF intermediate filament IF and microtubule MT. The beadlike structure of an actin filament shows the packing of actin subunits. Intermediate filament subunits pack to form ropes in which the individual subunits are difficult to distinguish. The walls of microtubules are formed from protofilaments of tubulin subunits. b Micrograph of a mixture of actin filaments microtubules and vimentin intermediate filaments showing the differences in their shape size and flexibility. Purified preparations of actin tubulin and vimentin subunits were separately polymerized in a test tube to form the corresponding filaments. A mixture of the filaments was applied to a carbon film on a microscope grid and then rinsed with a dilute solution of uranyl acetate UC which surrounds but does not penetrate the protein c. Because uranyl acetate is a heavy metal that easily scatters electrons areas of the microscope grid occupied by protein produce a “negative” image in metal film when projected onto a photographic plate as seen in part b. Part b courtesy of G. Waller and P . Matsudaira.

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forming a meshwork connect microvilli and are tethered to junctions between cells. Lamin intermediate filaments sup- port the inner nuclear membrane. Finally microtubules aligned with the long axis of the cell are in close proxim- ity to major cell organelles such as the endoplasmic reticu- lum Golgi complex and vesicles. The cytoskeleton has been highly conserved in evolu- tion. A comparison of gene sequences shows only a small percentage of differences in sequence between yeast actin and tubulin and human actin and tubulin. This structural conservation is explained by the variety of critical functions that depend on the cytoskeleton. A mutation in a cy- toskeleton protein subunit could disrupt the assembly of fil- aments and their binding to other proteins. Analyses of gene sequences and protein structures have identified bac- terial homologs of actin and tubulin. The absence of IF-like proteins in bacteria and unicellular eukaryotes is evidence that intermediate filaments appeared later in the evolution of the cytoskeletal system. The first IF protein to arise was most likely a nuclear lamin from which cytosolic IF pro- teins later evolved. The simple bacterial cytoskeleton controls cell length width and the site of cell division. The FtsZ protein a bac- terial homolog of tubulin is localized around the neck of di- viding bacterial cells suggesting that FtsZ participates in cell division Figure 5-30b. The results of biochemical experi- ments with purified FtsZ demonstrate that it can polymer- ize into protofilaments but these protofilaments do not assemble into intact microtubules. Another bacterial protein MreB has been found to be similar to actin in atomic struc- ture and filament structure—strong evidence that actin evolved from MreB. Clues to the function of MreB include its localization in a filament that girdles rod-shaped bacterial cells its absence from spherical bacteria and the finding that mutant cells lacking MreB become wider but not longer. These observations suggest MreB controls the width of rod- shaped bacteria. 5.4 • The Cytoskeleton: Components and Structural Functions 175 MT IF AF a b c AF IF MT Carbon film Actin MreB MreB FtsZ MTs IFs a b ▲ FIGURE 5-30 Schematic depiction of the distribution of cytoskeletal filaments in eukaryotic cells and bacterial cells. a In absorptive epithelial cells actin filaments red are concentrated in the apical region and in a narrow band in the basolateral region. Microtubules blue are oriented with the long axis of the cell and intermediate filaments green are concentrated along the cell periphery especially at specialized junctions with neighboring cells and lining the nuclear membrane. b In a rod-shaped bacterial cell filaments of MreB the bacterial actin homolog ring the cell and constrict its width. The bacterial tubulin homolog FtsZ forms filaments at the site of cell division.

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Cytoskeletal Filaments Are Organized into Bundles and Networks On first looking at micrographs of a cell one is struck by the dense seemingly disorganized mat of filaments present in the cytosol. However a keen eye will start to pick out areas—generally where the membrane protrudes from the cell surface or where a cell adheres to the surface or another cell—in which the filaments are concentrated into bundles. From these bundles the filaments continue into the cell in- terior where they fan out and become part of a network of filaments. These two structures bundles and networks are the most common arrangements of cytoskeletal filaments in a cell. Structurally bundles differ from networks mainly in the organization of the filaments. In bundles the filaments are closely packed in parallel arrays. In a network the fila- ments crisscross often at right angles and are loosely packed. Networks can be further subdivided. One type as- sociated with the nuclear and plasma membranes is pla- nar two-dimensional like a net or a web the other type present within the cell is three-dimensional giving the cytosol gel-like properties. In all bundles and networks the filaments are held together by various cross-linking proteins. We will consider various cytoskeletal cross-linking proteins and their functions in Chapters 19 and 20. Microfilaments and Membrane-Binding Proteins Form a Skeleton Underlying the Plasma Membrane The distinctive shape of a cell depends on the organization of actin filaments and proteins that connect microfilaments to the membrane. These proteins called membrane–microfilament binding proteins act as spot welds that tack the actin cytoskeleton framework to the overlying membrane. When attached to a bundle of filaments the membrane acquires the fingerlike shape of a microvillus or similar projection see Fig- ure 5-28. When attached to a planar network of filaments the membrane is held flat like the red blood cell membrane. The simplest membrane–cytoskeleton connections entail the bind- ing of integral membrane proteins directly to actin filaments. More common are complex linkages that connect actin fila- ments to integral membrane proteins through peripheral mem- brane proteins that function as adapter proteins. Such linkages between the cytoskeleton and certain plasma-membrane pro- teins are considered in Chapter 6. 176 CHAPTER 5 • Biomembranes and Cell Architecture a b Band 4.1 Spectrin tetramer Plasma membrane Glycophorin Band 3 dimer Ankyrin Tropomodulin Tropomyosin Adducin Actin Band 4.1 0.1 µ m ▲ FIGURE 5-31 Cortical cytoskeleton supporting the plasma membrane in human erythrocytes. a Electron micrograph of the erythrocyte membrane showing the spoke- and-hub organization of the cytoskeleton. The long spokes are composed mainly of spectrin and can be seen to intersect at the hubs or membrane-attachment sites. The darker spots along the spokes are ankyrin molecules which cross-link spectrin to integral membrane proteins. b Diagram of the erythrocyte cytoskeleton showing the various components. See text for discussion. Part a from T. J. Byers and D. Branton 1985 Proc. Nat’l. Acad. Sci. USA 82:6153. Courtesy of D. Branton. Part b adapted from S. E. Lux 1979 Nature 281:426 and E. J. Luna and A. L. Hitt 1992 Science 258:955.

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The richest area of actin filaments in many cells lies in the cortex a narrow zone just beneath the plasma membrane. In this region most actin filaments are arranged in a network that excludes most organelles from the cortical cytoplasm. Perhaps the simplest cytoskeleton is the two-dimensional network of actin filaments adjacent to the erythrocyte plasma membrane. In more complicated cortical cytoskele- tons such as those in platelets epithelial cells and muscle actin filaments are part of a three-dimensional network that fills the cytosol and anchors the cell to the substratum. A red blood cell must squeeze through narrow blood cap- illaries without rupturing its membrane. The strength and flexibility of the erythrocyte plasma membrane depend on a dense cytoskeletal network that underlies the entire mem- brane and is attached to it at many points. The primary com- ponent of the erythrocyte cytoskeleton is spectrin a 200-nm-long fibrous protein. The entire cytoskeleton is arranged in a spoke-and-hub network Figure 5-31a. Each spoke is composed of a single spectrin molecule which ex- tends from two hubs and cross-links them. Each hub com- prises a short 14-subunit actin filament plus adducin tropomyosin and tropomodulin Figure 5-31b inset. The last two proteins strengthen the network by preventing the actin filament from depolymerizing. Six or seven spokes ra- diate from each hub suggesting that six or seven spectrin molecules are bound to the same actin filament. To ensure that the erythrocyte retains its characteristic shape the spectrin-actin cytoskeleton is firmly attached to the overlying erythrocyte plasma membrane by two periph- eral membrane proteins each of which binds to a specific integral membrane protein and to membrane phospholipids. Ankyrin connects the center of spectrin to band 3 protein an anion-transport protein in the membrane. Band 4.1 protein a component of the hub binds to the integral membrane pro- tein glycophorin whose structure was discussed previously see Figure 5-12. Both ankyrin and band 4.1 protein also contain lipid-binding motifs which help bind them to the membrane see Table 5-3. The dual binding by ankyrin and band 4.1 ensures that the membrane is connected to both the spokes and the hubs of the spectrin-actin cytoskeleton see Figure 5-31b. Intermediate Filaments Support the Nuclear Membrane and Help Connect Cells into Tissues Intermediate filaments typically crisscross the cytosol form- ing an internal framework that stretches from the nuclear en- velope to the plasma membrane Figure 5-32. A network of intermediate filaments is located adjacent to some cellular membranes where it provides mechanical support. For ex- ample lamin A and lamin C filaments form an orthogonal lattice that is associated with lamin B. The entire supporting structure called the nuclear lamina is anchored to the inner nuclear membrane by prenyl anchors on lamin B. At the plasma membrane intermediate filaments are at- tached by adapter proteins to specialized cell junctions called desmosomes and hemidesmosomes which mediate cell–cell adhesion and cell–matrix adhesion respectively particularly in epithelial tissues. In this way intermediate filaments in one cell are indirectly connected to intermediate filaments in a neighboring cell or to the extracellular matrix. Because of the important role of cell junctions in cell adhesion and the sta- bility of tissues we consider their structure and relation to cytoskeletal filaments in detail in Chapter 6. Microtubules Radiate from Centrosomes and Organize Certain Subcellular Structures Like microfilaments and intermediate filaments micro- tubules are not randomly distributed in cells. Rather micro- tubules radiate from the centrosome which is the primary microtubule-organizing center MTOC in animal cells Fig- ure 5-33. As detailed in Chapter 20 the two ends of a mi- crotubule differ in their dynamic properties and are commonly designated as the and ends. For this rea- son microtubles can have two distinct orientations relative to one another and to other cell structures. In many nondi- viding animal cells the MTOC is located at the center of the cell near the nucleus and the radiating microtubules are all oriented with their ends directed toward the cell periph- ery. Although most interphase animal cells contain a single perinuclear MTOC epithelial cells and plant cells contain hundreds of MTOCs. Both of these cell types exhibit distinct 5.4 • The Cytoskeleton: Components and Structural Functions 177 ▲ FIGURE 5-32 Fluorescence micrograph of a PtK2 fibroblast cell stained to reveal keratin intermediate filaments. A network of filaments crisscrosses the cell from the nucleus to the plasma membrane. At the plasma membrane the filaments are linked by adapter proteins to two types of anchoring junctions: desmosomes between adjacent cells and hemidesmosomes between the cell and the matrix. Courtesy of R. D. Goldman.

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functional or structural properties or both in different re- gions of the cell. The functional and structural polarity of these cells is linked to the orientation of microtubules within them. Findings from studies discussed in Chapter 20 show that the association of microtubules with the endoplasmic retic- ulum and other membrane-bounded organelles may be criti- cal to the location and organization of these organelles within the cell. For instance if microtubules are destroyed by drugs such as nocodazole or colcemid the ER loses its net- worklike organization. Microtubules are also critical to the formation of the mitotic apparatus—the elaborate transient structure that captures and subsequently separates replicated chromosomes in cell division. KEY CONCEPTS OF SECTION 5.4 The Cytoskeleton: Components and Structural Functions ■ The cytosol is the internal aqueous medium of a cell ex- clusive of all organelles and the cytoskeleton. It contains numerous soluble enzymes responsible for much of the cell’s metabolic activity. ■ Three major types of protein filaments—actin filaments microtubules and intermediate filaments—make up the cy- toskeleton see Figure 5-29. ■ Microfilaments are assembled from monomeric actin subunits microtubules from -tubulin subunits and in- termediate filaments from lamin subunits and other tissue- specific proteins. ■ In all animal and plant cells the cytoskeleton provides structural stability for the cell and contributes to cell move- ment. Some bacteria have a primitive cytoskeleton. ■ Actin bundles form the core of microvilli and other fin- gerlike projections of the plasma membrane. ■ Cortical spectrin-actin networks are attached to the cell membrane by bivalent membrane–microfilament binding proteins such as ankyrin and band 4.1 see Figure 5-31. ■ Intermediate filaments are assembled into networks and bundles by various intermediate filament–binding proteins which also cross-link intermediate filaments to the plasma and nuclear membranes microtubules and microfilaments. ■ In some animal cells microtubules radiate out from a single microtubule-organizing center lying at the cell cen- ter see Figure 5-33. Intact microtubules appear to be nec- essary for endoplasmic reticulum and Golgi membranes to form into organized structures. Purification of Cells and Their Parts Many studies on cell structure and function require samples of a particular type of cell or subcellular organelle. Most an- imal and plant tissues however contain a mixture of cell types likewise most cells are filled with a variety of or- ganelles. In this section we describe several commonly used techniques for separating different cell types and organelles. The purification of membrane proteins presents some unique problems also considered here. Flow Cytometry Separates Different Cell Types Some cell types differ sufficiently in density that they can be separated on the basis of this physical property. White blood cells leukocytes and red blood cells erythrocytes for in- stance have very different densities because erythrocytes have no nucleus thus these cells can be separated by equi- librium density centrifugation described shortly. Because most cell types cannot be differentiated so easily other tech- niques such as flow cytometry must be used to separate them. A flow cytometer identifies different cells by measuring the light that they scatter and the fluorescence that they emit as they flow through a laser beam thus it can sort out cells of a particular type from a mixture. Indeed a fluorescence- activated cell sorter FACS an instrument based on flow cy- tometry can select one cell from thousands of other cells Figure 5-34. For example if an antibody specific to a cer- tain cell-surface molecule is linked to a fluorescent dye any 5.5 178 CHAPTER 5 • Biomembranes and Cell Architecture ▲ FIGURE 5-33 Fluorescence micrograph of a Chinese hamster ovary cell stained to reveal microtubles and the MTOC. The microtubules green detected with an antibody to tubulin are seen to radiate from a central point the microtubule- organizing center MTOC near the nucleus. The MTOC yellow is detected with an antibody to a protein localized to the centrosome. Courtesy of R. Kuriyame.

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cell bearing this molecule will bind the antibody and will then be separated from other cells when it fluoresces in the FACS. Having been sorted from other cells the selected cells can be grown in culture. The FACS procedure is commonly used to purify the dif- ferent types of white blood cells each of which bears on its surface one or more distinctive proteins and will thus bind monoclonal antibodies specific for that protein. Only the T cells of the immune system for instance have both CD3 and Thy1.2 proteins on their surfaces. The presence of these sur- face proteins allows T cells to be separated easily from other types of blood cells or spleen cells Figure 5-35. In a varia- tion of the use of monoclonal antibodies for separating cells small magnetic beads are coated with a monoclonal antibody specific for a surface protein such as CD3 or Thy1.2. Only cells with these proteins will stick to the beads and can be 5.5 • Purification of Cells and Their Parts 179 Drops with lesser charge Sorted charged droplets containing fluorescent cells Drops with no charge Drops with greater charge − − − − + Laser beam Filter Fluorescent light detector Cell suspension Sheath fluid Scattered light detector Fluorescent cells Nonfluorescent cell Fluorescent cell droplets Nonfluorescent cell droplet Condenser 1 2 3 2 4 1 EXPERIMENTAL FIGURE 5-34 Fluorescence-activated cell sorter FACS separates cells that are labeled differentially with a fluorescent reagent. Step : A concentrated suspension of labeled cells is mixed with a buffer the sheath fluid so that the cells pass single-file through a laser light beam. Step : Both the fluorescent light emitted and the light scattered by each cell are measured from measurements of the scattered light the size and shape of the cell can be determined. Step : The suspension is then forced through a nozzle which forms tiny droplets containing at most a single cell. At the time of formation each droplet is given a negative electric charge proportional to the amount of fluorescence of its cell. Step : Droplets with no charge and those with different electric charges are separated by an electric field and collected. It takes only milliseconds to sort each droplet and so as many as 10 million cells per hour can pass through the machine. In this way cells that have desired properties can be separated and then grown. Adapted from D. R. Parks and L. A. Herzenberg 1982 Meth. Cell Biol. 26:283. 4 3 2 1 Red fluorescence Non-T cells T cells CD3 10 4 10 3 10 2 10 1 10 0 Green fluorescence Thy1.2 10 4 10 3 10 2 10 1 10 0 EXPERIMENTAL FIGURE 5-35 T cells bound to fluorescence-tagged antibodies to two cell-surface proteins are separated from other white blood cells by FACS. Spleen cells from a mouse reacted with a fluorescence-tagged monoclonal antibody green specific for the CD3 cell-surface protein and with a fluorescence-tagged monoclonal antibody red specific for a second cell-surface protein Thy1.2. As cells were passed through a FACS machine the intensity of the green and red fluorescence emitted by each cell was recorded. This plot of the red fluorescence vertical axis versus green fluorescence horizontal axis for thousands of cells shows that about half of the cells—the T cells—express both CD3 and Thy1.2 proteins on their surfaces upper-right quadrant. The remaining cells which exhibit low fluorescence lower-left quadrant express only background levels of these proteins and are other types of white blood cells. Note the logarithmic scale on both axes. Courtesy of Chengcheng Zhang.

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recovered from the preparation by adhesion to a small mag- net on the side of the test tube. Other uses of flow cytometry include the measurement of a cell’s DNA and RNA content and the determination of its general shape and size. The FACS can make simultaneous measurements of the size of a cell from the amount of scat- tered light and the amount of DNA that it contains from the amount of fluorescence emitted from a DNA-binding dye. Disruption of Cells Releases Their Organelles and Other Contents The initial step in purifying subcellular structures is to rup- ture the plasma membrane and the cell wall if present. First the cells are suspended in a solution of appropriate pH and salt content usually isotonic sucrose 0.25 M or a combi- nation of salts similar in composition to those in the cell’s interior. Many cells can then be broken by stirring the cell suspension in a high-speed blender or by exposing it to ultrahigh-frequency sound sonication. Plasma membranes can also be sheared by special pressurized tissue homogeniz- ers in which the cells are forced through a very narrow space between the plunger and the vessel wall. As noted earlier water flows into cells when they are placed in a hypotonic solution see Figure 5-18. This osmotic flow causes cells to swell weakening the plasma membrane and facilitating its rupture. Generally the cell solution is kept at 0 C to best preserve enzymes and other constituents after their release from the stabilizing forces of the cell. Disrupting the cell produces a mix of suspended cellular components the homogenate from which the desired or- ganelles can be retrieved. Homogenization of the cell and di- lution of the cytosol cause the depolymerization of actin microfilaments and microtubules releasing their monomeric subunits and shear intermediate filaments into short frag- ments. Thus other procedures described in Chapters 19 and 20 are used to study these important constituents. Because rat liver contains an abundance of a single cell type this tissue has been used in many classic studies of cell organelles. However the same isolation principles apply to virtually all cells and tis- sues and modifications of these cell-fractionation techniques can be used to separate and purify any desired components. 180 CHAPTER 5 • Biomembranes and Cell Architecture Filter homogenate to remove clumps of unbroken cells connective tissue etc. Filtered homogenate Nuclei Mitochondria chloroplasts lysosomes and peroxisomes Plasma membrane microsomal fraction fragments of endoplasmic reticulum and large polyribosomes Ribosomal subunits small polyribo- somes Soluble part of cytoplasm cytosol 600g × 10 min Pour out: 15000g × 5 min Pour out: 100000g × 60 min Centrifuge Pour out: 300000g × 2 h Pour out ▲ EXPERIMENTAL FIGURE 5-36 Differential centrifugation is a common first step in fractionating a cell homogenate. The homogenate resulting from disrupting cells is usually filtered to remove unbroken cells and then centrifuged at a fairly low speed to selectively pellet the nucleus—the largest organelle. The undeposited material the supernatant is next centrifuged at a higher speed to sediment the mitochondria chloroplasts lysosomes and peroxisomes. Subsequent centrifugation in the ultracentrifuge at 100000g for 60 minutes results in deposition of the plasma membrane fragments of the endoplasmic reticulum and large polyribosomes. The recovery of ribosomal subunits small polyribosomes and particles such as complexes of enzymes requires additional centrifugation at still higher speeds. Only the cytosol—the soluble aqueous part of the cytoplasm—remains in the supernatant after centrifugation at 300000g for 2 hours.

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Centrifugation Can Separate Many Types of Organelles In Chapter 3 we considered the principles of centrifugation and the uses of centrifugation techniques for separating pro- teins and nucleic acids. Similar approaches are used for sep- arating and purifying the various organelles which differ in both size and density and thus undergo sedimentation at dif- ferent rates. Most cell-fractionation procedures begin with differen- tial centrifugation of a filtered cell homogenate at increas- ingly higher speeds Figure 5-36. After centrifugation at each speed for an appropriate time the supernatant is poured off and centrifuged at higher speed. The pelleted fractions obtained by differential centrifugation generally contain a mixture of organelles although nuclei and viral particles can sometimes be purified completely by this pro- cedure. An impure organelle fraction obtained by differen- tial centrifugation can be further purified by equilibrium density-gradient centrifugation which separates cellular components according to their density. After the fraction is resuspended it is layered on top of a solution that con- tains a gradient of a dense nonionic substance e.g. sucrose or glycerol. The tube is centrifuged at a high speed about 40000 rpm for several hours allowing each particle to mi- grate to an equilibrium position where the density of the surrounding liquid is equal to the density of the particle Figure 5-37. Because each organelle has unique morphological fea- tures the purity of organelle preparations can be assessed by examination in an electron microscope. Alternatively organelle-specific marker molecules can be quantified. For example the protein cytochrome c is present only in mito- chondria so the presence of this protein in a fraction of lysosomes would indicate its contamination by mitochon- dria. Similarly catalase is present only in peroxisomes acid phosphatase only in lysosomes and ribosomes only in the rough endoplasmic reticulum or the cytosol. Organelle-Specific Antibodies Are Useful in Preparing Highly Purified Organelles Cell fractions remaining after differential and equilibrium density-gradient centrifugation may still contain more than one type of organelle. Monoclonal antibodies for various organelle-specific membrane proteins are a powerful tool for further purifying such fractions. One example is the pu- rification of coated vesicles whose outer surface is covered with clathrin Figure 5-38. An antibody to clathrin bound to a bacterial carrier can selectively bind these vesicles in a crude preparation of membranes and the whole antibody complex can then be isolated by low-speed centrifugation. A related technique uses tiny metallic beads coated with spe- cific antibodies. Organelles that bind to the antibodies and are thus linked to the metallic beads are recovered from the preparation by adhesion to a small magnet on the side of the test tube. All cells contain a dozen or more different types of small membrane-limited vesicles of about the same size 50–100 nm in diameter and density. Because of their sim- ilar size and density these vesicles are difficult to separate from one another by centrifugation techniques. Immuno- logical techniques are particularly useful for purifying spe- cific classes of such vesicles. Fat and muscle cells for instance contain a particular glucose transporter GLUT4 that is localized to the membrane of a specific kind of vesi- cle. When insulin is added to the cells these vesicles fuse with the cell-surface membrane and increase the number of glucose transporters able to take up glucose from the blood. As will be seen in Chapter 15 this process is criti- cal to maintaining the appropriate concentration of sugar in the blood. The GLUT4-containing vesicles can be puri- fied by using an antibody that binds to a segment of the GLUT4 protein that faces the cytosol. Likewise the various transport vesicles discussed in Chapter 17 are characterized by unique surface proteins that permit their separation with the aid of specific antibodies. 5.5 • Purification of Cells and Their Parts 181 Organelle fraction Lysosomes 1.12 g/cm 3 Mitochondria 1.18 g/cm 3 Peroxisomes 1.23 g/cm 3 Before centrifugation 1.09 1.11 1.15 1.19 1.22 1.25 After centrifugation Increasing density of sucrose g/cm 3 ▲ EXPERIMENTAL FIGURE 5-37 A mixed organelle fraction can be further separated by equilibrium density- gradient centrifugation. In this example material in the pellet from centrifugation at 15000g see Figure 5-36 is resuspended and layered on a gradient of increasingly more dense sucrose solutions in a centrifuge tube. During centrifugation for several hours each organelle migrates to its appropriate equilibrium density and remains there. To obtain a good separation of lysosomes from mitochondria the liver is perfused with a solution containing a small amount of detergent before the tissue is disrupted. During this perfusion period detergent is taken into the cells by endocytosis and transferred to the lysosomes making them less dense than they would normally be and permitting a “clean” separation of lysosomes from mitochondria.

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Proteins Can Be Removed from Membranes by Detergents or High-Salt Solutions Detergents are amphipathic molecules that disrupt mem- branes by intercalating into phospholipid bilayers and solubi- lizing lipids and proteins. The hydrophobic part of a detergent molecule is attracted to hydrocarbons and mingles with them readily the hydrophilic part is strongly attracted to water. Some detergents are natural products but most are synthetic molecules developed for cleaning and for dispers- ing mixtures of oil and water Figure 5-39. Ionic detergents such as sodium deoxycholate and sodium dodecylsulfate SDS contain a charged group nonionic detergents such as Triton X-100 and octylglucoside lack a charged group. At very low concentrations detergents dissolve in pure water as isolated molecules. As the concentration increases the mole- cules begin to form micelles—small spherical aggregates in which hydrophilic parts of the molecules face outward and the hydrophobic parts cluster in the center see Figure 2-20. The critical micelle concentration CMC at which micelles form is characteristic of each detergent and is a function of the structures of its hydrophobic and hydrophilic parts. Ionic detergents bind to the exposed hydrophobic regions of membrane proteins as well as to the hydrophobic cores of water-soluble proteins. Because of their charge these de- tergents also disrupt ionic and hydrogen bonds. At high con- centrations for example sodium dodecylsulfate completely denatures proteins by binding to every side chain a prop- erty that is exploited in SDS gel electrophoresis see Figure 3-32. Nonionic detergents do not denature proteins and are thus useful in extracting proteins from membranes before purifying them. These detergents act in different ways at dif- ferent concentrations. At high concentrations above the CMC they solubilize biological membranes by forming mixed micelles of detergent phospholipid and integral membrane proteins Figure 5-40. At low concentrations below the CMC these detergents bind to the hydrophobic regions of most integral membrane proteins making them soluble in aqueous solution. Treatment of cultured cells with a buffered salt solution containing a nonionic detergent such as Triton X-100 extracts water-soluble proteins as well as integral membrane proteins. As noted earlier the exoplasmic and cytosolic domains of in- tegral membrane proteins are generally hydrophilic and sol- 182 CHAPTER 5 • Biomembranes and Cell Architecture Clathrin a Antibody to clathrin Protein A Coated vesicle Bacterial cell Coated vesicles b 0.1 m ▲ EXPERIMENTAL FIGURE 5-38 Small vesicles can be purified by binding of antibody specific for a vesicle surface protein and linkage to bacterial cells. In this example a suspension of membranes from rat liver is incubated with an antibody specific for clathrin a protein that coats the outer surface of certain cytosolic vesicles. To this mixture is added a suspension of Staphylococcus aureus bacteria whose surface membrane contains protein A which binds to the Fc constant region of antibodies. a Interaction of protein A with antibodies bound to clathrin-coated vesicles links the vesicles to the bacterial cells. The vesicle–bacteria complexes can then be recovered by low-speed centrifugation. b A thin-section electron micrograph reveals clathrin-coated vesicles bound to an S. aureus cell. See E. Merisko et al. 1982 J. Cell Biol. 93:846. Micrograph courtesy of G. Palade.

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uble in water. The membrane-spanning domains however are rich in hydrophobic and uncharged residues see Figure 5-12. When separated from membranes these exposed hy- drophobic segments tend to interact with one another caus- ing the protein molecules to aggregate and precipitate from aqueous solutions. The hydrophobic parts of nonionic deter- gent molecules preferentially bind to the hydrophobic seg- ments of transmembrane proteins preventing protein aggre- gation and allowing the proteins to remain in the aqueous so- lution. Detergent-solubilized transmembrane proteins can then be purified by affinity chromatography and other tech- niques used in purifying water-soluble proteins Chapter 3. As discussed previously most peripheral proteins are bound to specific transmembrane proteins or membrane 5.5 • Purification of Cells and Their Parts 183 Sodium deoxycholate Sodium dodecylsulfate SDS CH 2 11 H 3 C O O O SO Na IONIC DETERGENTS CH 3 CH 2 H 3 C OH HC HO CH 2 COO Na CH 3 CH 2 CH 2 H 3 C H 3 CCH 3 CH 3 H 3 C CC O CH 2 H O 9.5 Average Triton X-100 polyoxyethylene9.5p-t-octylphenol CH 2 7 CH 3 HOCH 2 OH OH HO O O Octylglucoside octyl- -D-glucopyranoside NONIONIC DETERGENTS ▲ FIGURE 5-39 Structures of four common detergents. The hydrophobic part of each molecule is shown in yellow the hydrophilic part in blue. The bile salt sodium deoxycholate is a natural product the others are synthetic. Although ionic detergents commonly cause denaturation of proteins nonionic detergents do not and are thus useful in solubilizing integral membrane proteins. Concentration above CMC Detergent Micelles Dissolved but not forming micelles Concentration below CMC ▲ FIGURE 5-40 Solubilization of integral membrane proteins by nonionic detergents. At a concentration higher than its critical micelle concentration CMC a detergent solubilizes lipids and integral membrane proteins forming mixed micelles containing detergent protein and lipid molecules. At concentrations below the CMC nonionic detergents e.g. octylglucoside Triton X-100 can dissolve membrane proteins without forming micelles by coating the membrane-spanning regions.

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phospholipids by ionic or other weak interactions. Generally peripheral proteins can be removed from the membrane by solutions of high ionic strength high salt concentrations which disrupt ionic bonds or by chemicals that bind divalent cations such as Mg 2 . Unlike integral proteins most periph- eral proteins are soluble in aqueous solution and need not be solubilized by nonionic detergents. KEY CONCEPTS OF SECTION 5.5 Purification of Cells and Their Parts ■ Flow cytometry can identify different cells on the basis of the light that they scatter and the fluorescence that they emit. The fluorescence-activated cell sorter FACS is useful in sep- arating different types of cells see Figures 5-34 and 5-35. ■ Disruption of cells by vigorous homogenization sonica- tion or other techniques releases their organelles. Swelling of cells in a hypotonic solution weakens the plasma mem- brane making it easier to rupture. ■ Sequential differential centrifugation of a cell ho- mogenate yields fractions of partly purified organelles that differ in mass and density see Figure 5-36. ■ Equilibrium density-gradient centrifugation which sep- arates cellular components according to their densities can further purify cell fractions obtained by differential centrifugation. ■ Immunological techniques using antibodies against organelle-specific membrane proteins are particularly use- ful in purifying organelles and vesicles of similar sizes and densities. ■ Transmembrane proteins are selectively solubilized and purified with the use of nonionic detergents. Visualizing Cell Architecture In the 1830s Matthias Schleiden and Theodore Schwann proposed that individual cells constitute the fundamental unit of life. This first formulation of the cell theory was based on observations made with rather primitive light mi- croscopes. Modern cell biologists have many more-powerful tools for revealing cell architecture. For example variations of standard light microscopy permit scientists to view ob- jects that were undetectable several decades ago. Electron microscopy which can reveal extremely small objects has yielded much information about subcellular particles and the organization of plant and animal tissues. Each technique is most suitable for detecting and imaging particular struc- tural features of the cell Figure 5-41. Digital recording sys- tems and appropriate computer algorithms represent another advance in visualizing cell architecture that has spread widely in the past decade. Digital systems not only 5.6 can provide microscopic images of improved quality but also permit three-dimensional reconstructions of cell com- ponents from two-dimensional images. A Microscope Detects Magnifies and Resolves Small Objects All microscopes produce a magnified image of a small object but the nature of the images depends on the type of micro- scope employed and on the way in which the specimen is pre- pared. The compound microscope used in conventional bright-field light microscopy contains several lenses that magnify the image of a specimen under study Figure 5-42a b. The total magnification is a product of the magni- fication of the individual lenses: if the objective lens magni- fies 100-fold a 100 lens the maximum usually employed and the projection lens or eyepiece magnifies 10-fold the final magnification recorded by the human eye or on film will be 1000-fold. However the most important property of any micro- scope is not its magnification but its resolving power or resolution—the ability to distinguish between two very closely positioned objects. Merely enlarging the image of a specimen accomplishes nothing if the image is blurred. The resolution of a microscope lens is numerically equivalent to D the minimum distance between two distinguishable ob- jects. The smaller the value of D the better the resolution. The value of D is given by the equation 5-1 where is the angular aperture or half-angle of the cone of light entering the objective lens from the specimen N is the refractive index of the medium between the specimen and the objective lens i.e. the relative velocity of light in the medium compared with the velocity in air and is the wavelength of the incident light. Resolution is improved by using shorter wavelengths of light decreasing the value of D 0.61 N sin 184 CHAPTER 5 • Biomembranes and Cell Architecture Atoms Proteins Organelles Cells 1 nm 1 µ m 1 mm Fluorescence microscopy Conventional light microscopy Transmission electron microscopy Scanning electron microscopy ▲ FIGURE 5-41 The range in sizes of objects imaged by different microscopy techniques. The smallest object that can be imaged by a particular technique is limited by the resolving power of the equipment and other factors.

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or gathering more light increasing either N or . Note that the magnification is not part of this equation. Owing to limitations on the values of and N the limit of resolution of a light microscope using visible light is about 0.2 m 200 nm. No matter how many times the image is magnified the microscope can never resolve ob- jects that are less than ≈0.2 m apart or reveal details smaller than ≈0.2 m in size. Despite this limit on resolu- tion the light microscope can be used to track the location of a small bead of known size to a precision of only a few nanometers. If we know the precise size and shape of an object—say a 5-nm sphere of gold—and if we use a video camera to record the microscopic image as a digital image then a computer can calculate the position of the center of the object to within a few nanometers. This technique has been used to measure nanometer-size steps as molecules and vesicles move along cytoskeletal filaments see Figures 19-17 19-18 and 20-18. Samples for Microscopy Must Be Fixed Sectioned and Stained to Image Subcellular Details Live cells and tissues lack compounds that absorb light and are thus nearly invisible in a light microscope. Although such specimens can be visualized by special techniques to be dis- cussed shortly these methods do not reveal the fine details of structure and require cells to be housed in special glass-faced chambers called culture chambers that can be mounted on a microscope stage. For these reasons cells are often fixed sectioned and stained to reveal subcellular structures. Specimens for light and electron microscopy are com- monly fixed with a solution containing chemicals that cross- link most proteins and nucleic acids. Formaldehyde a common fixative cross-links amino groups on adjacent mol- ecules these covalent bonds stabilize protein–protein and protein–nucleic acid interactions and render the molecules 5.6 • Visualizing Cell Architecture 185 Detector Dichroic mirror Projection lens Lamp Lamp Objective Condenser Mirror Specimen stage a Optical microscope b Bright-field light path c Epifluorescence light path Excitation filter Collector lens ▲ EXPERIMENTAL FIGURE 5-42 Optical microscopes are commonly configured for both bright-field transmitted and epifluorescence microscopy. a In a typical light microscope the specimen is usually mounted on a transparent glass slide and positioned on the movable specimen stage. The two imaging methods require separate illumination systems but use the same light gathering and detection systems. b In bright-field light microscopy light from a tungsten lamp is focused on the specimen by a condenser lens below the stage the light travels the pathway shown. c In epifluorescence microscopy ultraviolet light from a mercury lamp positioned above the stage is focused on the specimen by the objective lens. Filters in the light path select a particular wavelength of ultraviolet light for illumination and are matched to capture the wavelength of the emitted light by the specimen.

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insoluble and stable for subsequent procedures. After fixa- tion a sample is usually embedded in paraffin or plastic and cut into sections 0.5–50 m thick Figure 5-43. Alterna- tively the sample can be frozen without prior fixation and then sectioned such treatment preserves the activity of en- zymes for later detection by cytochemical reagents. A final step in preparing a specimen for light microscopy is to stain it so as to visualize the main structural features of the cell or tissue. Many chemical stains bind to molecules that have specific features. For example hematoxylin binds to basic amino acids lysine and arginine on many different kinds of proteins whereas eosin binds to acidic molecules such as DNA and side chains of aspartate and glutamate. Because of their different binding properties these dyes stain various cell types sufficiently differently that they are distin- guishable visually. If an enzyme catalyzes a reaction that pro- duces a colored or otherwise visible precipitate from a colorless precursor the enzyme may be detected in cell sec- tions by their colored reaction products. Such staining tech- niques although once quite common have been largely replaced by other techniques for visualizing particular pro- teins or structures as discussed next. Phase-Contrast and Differential Interference Contrast Microscopy Visualize Unstained Living Cells Two common methods for imaging live cells and unstained tissues generate contrast by taking advantage of differences in the refractive index and thickness of cellular materials. These methods called phase-contrast microscopy and dif- ferential interference contrast DIC microscopy or Nomarski interference microscopy produce images that differ in appearance and reveal different features of cell ar- chitecture. Figure 5-44 compares images of live cultured cells obtained with these two methods and standard bright- field microscopy. In phase-contrast images the entire object and subcellular structures are highlighted by interference rings—concentric halos of dark and light bands. This artifact is inherent in the method which generates contrast by interference between diffracted and undiffracted light by the specimen. Because the interference rings around an object obscure many details this technique is suitable for observing only single cells or thin cell layers but not thick tissues. It is particularly useful for exam- ining the location and movement of larger organelles in live cells. DIC microscopy is based on interference between polar- ized light and is the method of choice for visualizing extremely small details and thick objects. Contrast is generated by dif- ferences in the index of refraction of the object and its sur- rounding medium. In DIC images objects appear to cast a shadow to one side. The “shadow” primarily represents a dif- ference in the refractive index of a specimen rather than its to- pography. DIC microscopy easily defines the outlines of large organelles such as the nucleus and vacuole. In addition to hav- ing a “relief”-like appearance a DIC image is a thin optical section or slice through the object. Thus details of the nucleus 186 CHAPTER 5 • Biomembranes and Cell Architecture Block Specimen holder Specimen block Knife Specimen Knife Cut section Sections Microscope slide Copper mesh grid ▲ EXPERIMENTAL FIGURE 5-43 Tissues for microscopy are commonly fixed embedded in a solid medium and cut into thin sections. A fixed tissue is dehydrated by soaking in a series of alcohol-water solutions ending with an organic solvent compatible with the embedding medium. To embed the tissue for sectioning the tissue is placed in liquid paraffin for light microscopy or in liquid plastic for electron microscopy after the block containing the specimen has hardened it is mounted on the arm of a microtome and slices are cut with a knive. Typical sections cut for electron microscopy 50–100 nm thick sections cut for light microscopy are 0.5–50 m thick. The sections are collected either on microscope slides light microscopy or copper mesh grids electron microscopy and stained with an appropriate agent.

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in thick specimens e.g. an intact Caenorhabditis elegans roundworm can be observed in a series of such optical sec- tions and the three-dimensional structure of the object can be reconstructed by combining the individual DIC images. Fluorescence Microscopy Can Localize and Quantify Specific Molecules in Fixed and Live Cells Perhaps the most versatile and powerful technique for local- izing proteins within a cell by light microscopy is fluorescent staining of cells and observation by fluorescence microscopy. A chemical is said to be fluorescent if it absorbs light at one wavelength the excitation wavelength and emits light fluo- resces at a specific and longer wavelength. Most fluorescent dyes or flurochromes emit visible light but some such as Cy5 and Cy7 emit infrared light. In modern fluorescence mi- croscopes only fluorescent light emitted by the sample is used to form an image light of the exciting wavelength in- duces the fluorescence but is then not allowed to pass the fil- ters placed between the objective lens and the eye or camera see Figure 5-42a c. Immunological Detection of Specific Proteins in Fixed Cells The common chemical dyes just mentioned stain nucleic acids or broad classes of proteins. However investigators often want to detect the presence and location of specific proteins. A widely used method for this purpose employs specific antibodies covalently linked to flurochromes. Com- monly used flurochromes include rhodamine and Texas red which emit red light Cy3 which emits orange light and fluo- rescein which emits green light. These flurochromes can be chemically coupled to purified antibodies specific for almost any desired macromolecule. When a flurochrome–antibody complex is added to a permeabilized cell or tissue section the complex will bind to the corresponding antigens which then light up when illuminated by the exciting wavelength a tech- nique called immunfluorescence microscopy Figure 5-45. Staining a specimen with two or three dyes that fluoresce at different wavelengths allows multiple proteins to be localized within a cell see Figure 5-33. 5.6 • Visualizing Cell Architecture 187 ▲ EXPERIMENTAL FIGURE 5-44 Live cells can be visualized by microscopy techniques that generate contrast by interference. These micrographs show live cultured macrophage cells viewed by bright-field microscopy left phase- contrast microscopy middle and differential interference contrast DIC microscopy right. In a phase-contrast image cells are surrounded by alternating dark and light bands in-focus and out-of-focus details are simultaneously imaged in a phase- contrast microscope. In a DIC image cells appear in pseudorelief. Because only a narrow in-focus region is imaged a DIC image is an optical slice through the object. Courtesy of N. Watson and J. Evans. Brush border Lateral membrane Lamina propia 20 m ▲ EXPERIMENTAL FIGURE 5-45 One or more specific proteins can be localized in fixed tissue sections by immunofluorescence microscopy. A section of the rat intestinal wall was stained with Evans blue which generates a nonspecific red fluorescence and with a yellow green–fluorescing antibody specific for GLUT2 a glucose transport protein. As evident from this fluorescence micrograph GLUT2 is present in the basal and lateral sides of the intestinal cells but is absent from the brush border composed of closely packed microvilli on the apical surface facing the intestinal lumen. Capillaries run through the lamina propria a loose connective tissue beneath the epithelial layer. See B. Thorens et al. 1990 Am. J. Physio. 259:C279 courtesy of B. Thorens.

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Expression of Fluorescent Proteins in Live Cells A natu- rally fluorescent protein found in the jellyfish Aequorea vic- toria can be exploited to visualize live cells and specific proteins within them. This 238-residue protein called green fluorescent protein GFP contains a serine tyrosine and glycine sequence whose side chains have spontaneously cyclized to form a green-fluorescing chromophore. With the use of recombinant DNA techniques discussed in Chapter 9 the GFP gene can be introduced into living cultured cells or into specific cells of an entire animal. Cells containing the in- troduced gene will express GFP and thus emit a green fluo- rescence when irradiated this GFP fluorescence can be used to localize the cells within a tissue. Figure 5-46 illustrates the results of this approach in which a variant of GFP that emits blue fluorescence was used. In a particularly useful application of GFP a cellular pro- tein of interest is “tagged” with GFP to localize it. In this tech- nique the gene for GFP is fused to the gene for a particular cellular protein producing a recombinant DNA encoding one long chimeric protein that contains the entirety of both pro- teins. Cells in which this recombinant DNA has been intro- duced will synthesize the chimeric protein whose green fluorescence reveals the subcellular location of the protein of interest. This GFP-tagging technique for example has been used to visualize the expression and distribution of specific proteins that mediate cell–cell adhesion see Figure 6-8. In some cases a purified protein chemically linked to a fluorescent dye can be microinjected into cells and followed by fluorescence microscopy. For example findings from careful biochemical studies have established that purified actin “tagged” with a flurochrome is indistinguishable in function from its normal counterpart. When the tagged pro- tein is microinjected into a cultured cell the endogenous cel- lular and injected tagged actin monomers copolymerize into normal long actin fibers. This technique can also be used to study individual microtubules within a cell. Determination of Intracellular Ca 2 and H Levels with Ion-Sensitive Fluoresent Dyes Flurochromes whose fluo- rescence depends on the concentration of Ca 2 or H have proved useful in measuring the concentration of these ions within live cells. As discussed in later chapters intracellular Ca 2 and H concentrations have pronounced effects on many cellular processes. For instance many hormones or other stimuli cause a rise in cytosolic Ca 2 from the resting level of about 10 7 M to 10 6 M which induces various cel- lular responses including the contraction of muscle. The fluorescent dye fura-2 which is sensitive to Ca 2 contains five carboxylate groups that form ester linkages with ethanol. The resulting fura-2 ester is lipophilic and can 188 CHAPTER 5 • Biomembranes and Cell Architecture ▲ EXPERIMENTAL FIGURE 5-46 Expression of fluorescent proteins in early and late mouse embryos is detected by emitted blue and yellow light. The genes encoding blue fluorescent protein ECFP and yellow fluorescent protein EYFP were introduced into mouse embryonic stem cells which then were grown into early-stage embryos top and late-stage embryos bottom. These bright-field left and fluorescence right micrographs reveal that all but four of the early-stage embryos display a blue or yellow fluorescence indicating expression of the introduced ECFP and EYFP genes. Of the two late-stage embryos shown one expressed the ECFP gene left and one expressed the EYFP gene right. From A.-K. Hadjantonakis et al. 2002 BMC Biotechnol. 2:11 . ▲ EXPERIMENTAL FIGURE 5-47 Fura-2 a Ca 2+ -sensitive flurochrome can be used to monitor the relative cytosolic Ca 2+ concentrations in different regions of live cells. Left In a moving leukocyte a Ca 2+ gradient is established. The highest levels green are at the rear of the cell where cortical contractions take place and the lowest levels blue are at the cell front where actin undergoes polymerization. Right When a pipette filled with chemotactic molecules placed to the side of the cell induces the cell to turn the Ca 2+ concentration momentarily increases throughout the cytoplasm and a new gradient is established. The gradient is oriented such that the region of lowest Ca 2+ blue lies in the direction that the cell will turn whereas a region of high Ca 2+ yellow always forms at the site that will become the rear of the cell. From R. A. Brundage et al. 1991 Science 254:703 courtesy of F . Fay.

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diffuse from the medium across the plasma membrane into cells. Within the cytosol esterases hydrolyze fura-2 ester yielding fura-2 whose free carboxylate groups render the molecule nonlipophilic and so it cannot cross cellular mem- branes and remains in the cytosol. Inside cells each fura-2 molecule can bind a single Ca 2 ion but no other cellular cation. This binding which is proportional to the cytosolic Ca 2 concentration over a certain range increases the fluo- rescence of fura-2 at one particular wavelength. At a second wavelength the fluorescence of fura-2 is the same whether or not Ca 2 is bound and provides a measure of the total amount of fura-2 in a region of the cell. By examining cells continuously in the fluorescence microscope and measuring rapid changes in the ratio of fura-2 fluorescence at these two wavelengths one can quantify rapid changes in the fraction of fura-2 that has a bound Ca 2 ion and thus in the concen- tration of cytosolic Ca 2 Figure 5-47. Similarly to fura-2 fluorescent dyes e.g. SNARF-1 that are sensitive to the H concentration can be used to moni- tor the cytosolic pH of living cells. Confocal Scanning and Deconvolution Microscopy Provide Sharp Images of Three-Dimensional Objects Conventional fluorescence microscopy has two major limi- tations. First the physical process of cutting a section de- stroys material and so in consecutive serial sectioning a small part of a cell’s structure is lost. Second the fluorescent light emitted by a sample comes from molecules above and below the plane of focus thus the observer sees a blurred image caused by the superposition of fluorescent images from molecules at many depths in the cell. The blurring effect makes it difficult to determine the actual three-dimensional molecular arrangement Figure 5-48a. Two powerful refine- ments of fluorescence microscopy produce much sharper im- ages by reducing the image-degrading effects of out-of-focus light. In confocal scanning microscopy exciting light from a fo- cused laser beam illuminates only a single small part of a sample for an instant and then rapidly moves to different spots in the sample focal plane. The emitted fluorescent light passes through a pinhole that rejects out-of-focus light thereby producing a sharp image. Because light in focus with the image is collected by the pinhole the scanned area is an optical section through the specimen. The intensity of light from these in-focus areas is recorded by a photomultiplier tube and the image is stored in a computer Figure 5-48b. Deconvolution microscopy achieves the same image- sharpening effect as confocal scanning microscopy but through a different process. In this method images from con- secutive focal planes of the specimen are collected. A sepa- rate focal series of images from a test slide of subresolution size i.e. 0.2 m diameter bead are also collected. Each bead represents a pinpoint of light that becomes an object blurred by the imperfect optics of the microscope. Deconvolution 5.6 • Visualizing Cell Architecture 189 a Conventional fluorescence microscopy b Confocal fluorescence microscopy 40 m Focal plane Imaged volume Focal plane Imaged volume ▲ EXPERIMENTAL FIGURE 5-48 Confocal microscopy produces an in-focus optical section through thick cells. A mitotic fertilized egg from a sea urchin Psammechinus was lysed with a detergent exposed to an anti-tubulin antibody and then exposed to a fluorescein-tagged antibody that binds to the first antibody. a When viewed by conventional fluorescence microscopy the mitotic spindle is blurred. This blurring occurs because background fluorescence is detected from tubulin above and below the focal plane as depicted in the sketch. b The confocal microscopic image is sharp particularly in the center of the mitotic spindle. In this case fluorescence is detected only from molecules in the focal plane generating a very thin optical section. Micrographs from J. G. White et al. 1987 J. Cell Biol. 104:41 .

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reverses the degradation of the image by using the blurred beads as a reference object. The out-of-focus light is mathe- matically reassigned with the aid of deconvolution algo- rithms. Images restored by deconvolution display impressive detail without any blurring Figure 5-49. Astronomers use deconvolution algorithms to sharpen images of distant stars. Resolution of Transmission Electron Microscopy Is Vastly Greater Than That of Light Microscopy The fundamental principles of electron microscopy are simi- lar to those of light microscopy the major difference is that electromagnetic lenses rather than optical lenses focus a high-velocity electron beam instead of visible light. In the transmission electron microscope TEM electrons are emit- ted from a filament and accelerated in an electric field. A condenser lens focuses the electron beam onto the sample objective and projector lenses focus the electrons that pass through the specimen and project them onto a viewing screen or other detector Figure 5-50 left. Because electrons are absorbed by atoms in air the entire tube between the electron source and the detector is maintained under an ultrahigh vacuum. The short wavelength of electrons means that the limit of resolution for the transmission electron microscope is the- oretically 0.005 nm less than the diameter of a single atom or 40000 times better than the resolution of the light microscope and 2 million times better than that of the un- aided human eye. However the effective resolution of the transmission electron microscope in the study of biological systems is considerably less than this ideal. Under optimal conditions a resolution of 0.10 nm can be obtained with transmission electron microscopes about 2000 times better than the best resolution of light microscopes. Several exam- ples of cells and subcellular structures imaged by TEM are included in Section 5.3. Because TEM requires very thin fixed sections about 50 nm only a small part of a cell can be observed in any one section. Sectioned specimens are prepared in a manner simi- lar to that for light microscopy by using a knife capable of producing sections 50–100 nm in thickness see Figure 5-43. The generation of the image depends on differential scattering of the incident electrons by molecules in the prepa- ration. Without staining the beam of electrons passes through a specimen uniformly and so the entire sample ap- pears uniformly bright with little differentiation of compo- nents. To obtain useful images by TEM sections are commonly stained with heavy metals such as gold or os- mium. Metal-stained areas appear dark on a micrograph be- cause the metals scatter diffract most of the incident 190 CHAPTER 5 • Biomembranes and Cell Architecture ▲ EXPERIMENTAL FIGURE 5-49 Deconvolution fluorescence microscopy yields high-resolution optical sections that can be reconstructed into one three- dimensional image. A macrophage cell was stained with fluorochrome-labeled reagents specific for DNA blue microtubules green and actin microfilaments red. The series of fluorescent images obtained at consecutive focal planes optical sections through the cell were recombined in three dimensions. a In this three-dimensional reconstruction of the raw images the DNA microtubules and actin appear as diffuse zones in the cell. b After application of the deconvolution algorithm to the images the fibrillar organization of microtubules and the localization of actin to adhesions become readily visible in the reconstruction. Courtesy of J. Evans.

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electrons scattered electrons are not focused by the electro- magnetic lenses and do not contribute to the image. Areas that take up less stain appear lighter. Osmium tetroxide pref- erentially stains certain cellular components such as mem- branes see Figure 5-2a. Specific proteins can be detected in thin sections by the use of electron-dense gold particles coated with protein A a bacterial protein that binds anti- body molecules nonspecifically Figure 5-51. Standard electron microscopy cannot be used to study live cells because they are generally too vulnerable to the re- quired conditions and preparatory techniques. In particular the absence of water causes macromolecules to become de- natured and nonfunctional. However the technique of cryo- electron microscopy allows examination of hydrated un- fixed and unstained biological specimens directly in a transmission electron microscope. In this technique an aqueous suspension of a sample is applied in an extremely thin film to a grid. After it has been frozen in liquid nitro- gen and maintained in this state by means of a special mount the sample is observed in the electron microscope. The very low temperature 196 C keeps water from evaporating even in a vacuum and the sample can be observed in detail in its native hydrated state without fixing or heavy metal 5.6 • Visualizing Cell Architecture 191 Tungsten filament cathode Anode SEM TEM Beam of electrons Scanning coils Condenser lens Specimen Specimen Electromagnetic objective lens Projector lens Detector ▲ EXPERIMENTAL FIGURE 5-50 In electron microscopy images are formed from electrons that pass through a specimen or are released from a metal-coated specimen. In a transmission electron microscope TEM electrons are extracted from a heated filament accelerated by an electric field and focused on the specimen by a magnetic condenser lens. Electrons that pass through the specimen are focused by a series of magnetic objective and projector lenses to form a magnified image of the specimen on a detector which may be a fluorescent viewing screen photographic film or a charged- couple-device CCD camera. In a scanning electron microscope SEM electrons are focused by condensor and objective lenses on a metal-coated specimen. Scanning coils move the beam across the specimen and electrons from the metal are collected by a photomultiplier tube detector. In both types of microscopes because electrons are easily scattered by air molecules the entire column is maintained at a very high vacuum. Gold Fc domain Protein A Antigen catalase Antibody a b 0.5 µ m Peroxisomes ▲ EXPERIMENTAL FIGURE 5-51 Gold particles coated with protein A are used to detect an antibody-bound protein by transmission electron microscopy. a First antibodies are allowed to interact with their specific antigen e.g. catalase in a section of fixed tissue. Then the section is treated with a complex of protein A from the bacterium S. aureus and electron- dense gold particles. Binding of this complex to the Fc domains of the antibody molecules makes the location of the target protein catalase in this case visible in the electron microscope. b A slice of liver tissue was fixed with glutaraldehyde sectioned and then treated as described in part a to localize catalase. The gold particles black dots indicating the presence of catalase are located exclusively in peroxisomes. From H. J. Geuze et al. 1981 J. Cell Biol. 89:653. Reproduced from the Journal of Cell Biology by copyright permission of The Rockefeller University Press.

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staining. By computer-based averaging of hundreds of im- ages a three-dimensional model almost to atomic resolution can be generated. For example this method has been used to generate models of ribosomes see Figure 4-27 the mus- cle calcium pump discussed in Chapter 7 and other large proteins that are difficult to crystallize. Electron Microscopy of Metal-Coated Specimens Can Reveal Surface Features of Cells and Their Components Transmission electron microscopy is also used to obtain in- formation about the shapes of purified viruses fibers en- zymes and other subcellular particles by using a technique called metal shadowing in which a thin layer of metal such as platinum is evaporated on a fixed and sectioned or rap- idly frozen biological sample Figure 5-52. Acid treatment dissolves away the cell leaving a metal replica that is viewed in a transmission electron microscope. Alternatively the scanning electron microscope allows in- vestigators to view the surfaces of unsectioned metal-coated specimens. An intense electron beam inside the microscope scans rapidly over the sample. Molecules in the coating are excited and release secondary electrons that are focused onto a scintillation detector the resulting signal is displayed on a cathode-ray tube see Figure 5-50 right. Because the num- ber of secondary electrons produced by any one point on the sample depends on the angle of the electron beam in rela- tion to the surface the scanning electron micrograph has a three-dimensional appearance Figure 5-53. The resolving power of scanning electron microscopes which is limited by the thickness of the metal coating is only about 10 nm much less than that of transmission instruments. Three-Dimensional Models Can Be Constructed from Microscopy Images In the past decade digital cameras have largely replaced op- tical cameras to record microscopy images. Digital images can be stored in a computer and manipulated by conven- tional photographic software as well as specialized algo- rithms. As mentioned earlier the deconvolution algorithm 192 CHAPTER 5 • Biomembranes and Cell Architecture Sample Mica surface Metal replica Evaporated carbon Evaporated platinum Carbon film Acid Metal replica ready for visualization 1 2 3 4 5 ▲ EXPERIMENTAL FIGURE 5-52 Metal shadowing makes surface details on very small particles visible by transmission electron microscopy. The sample is spread on a mica surface and then dried in a vacuum evaporator . A filament of a heavy metal such as platinum or gold is heated electrically so that the metal evaporates and some of it falls over the sample grid in a very thin film . To stabilize the replica the specimen is then coated with a carbon film evaporated from an overhead electrode . The biological material is then dissolved by acid leaving a metal replica of the sample which is viewed in a TEM. In electron micrographs of such preparations the carbon-coated areas appear light—the reverse of micrographs of simple metal- stained preparations in which the areas of heaviest metal staining appear the darkest. 5 4 3 2 1 Absorptive epithelial cells Basal lamina Microvilli 5 m ▲ EXPERIMENTAL FIGURE 5-53 Scanning electron microscopy SEM produces a three-dimensional image of the surface of an unsectioned specimen. Shown here is an SEM image of the epithelium lining the lumen of the intestine. Abundant fingerlike microvilli extend from the lumen-facing surface of each cell. The basal lamina beneath the epithelium helps support and anchor it to the underlying connective tissue Chapter 6. Compare this image of intestinal cells with those in Figure 5-28 a transmission electron micrograph and in Figure 5-45 a fluorescence micrograph. From R. Kessel and R. Kardon 1979 Tissues and Organs A Text-Atlas of Scanning Electron Microscopy W. H. Freeman and Company p. 176.

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can sharpen an image by restoring out-of-focus photons to their origin—an example of a computational method that improves the quality of the image. The details in stored dig- ital images also can be quantified and objects in images can be reconstructed in three dimensions. For example the three- dimensional model of an object can be calculated by tomo- graphic methods from a collection of images that cover different views of the object. In light microscopy a stack of optical sections collected with either a confocal or a decon- volution microscope can be recombined into one three- dimensional image see Figure 5-49. If a TEM specimen is tilted through various degrees the resulting images also can be recombined to generate a three-dimensional view of the object see Figure 5-23. KEY CONCEPTS OF SECTION 5.6 Visualizing Cell Architecture ■ The limit of resolution of a light microscope is about 200 nm of a scanning electron microscope about 10 nm and of a transmission electron microscope about 0.1 nm. ■ Because cells and tissues are almost transparent various types of stains and optical techniques are used to generate sufficient contrast for imaging. ■ Phase-contrast and differential interference contrast DIC microscopy are used to view the details of live un- stained cells and to monitor cell movement. ■ In immunofluorescence microscopy specific proteins and organelles in fixed cells are stained with fluorescence- labeled monoclonal antibodies. Multiple proteins can be localized in the same sample by staining with antibodies labeled with different fluorochromes. ■ When proteins tagged with naturally occurring green flu- orescent protein GFP or its variants are expressed in live cells they can be visualized in a fluorescence microscope. ■ With the use of dyes whose fluorescence is proportional to the concentration of Ca 2 or H ions fluorescence mi- croscopy can measure the local concentration of Ca 2 ions and intracellular pH in living cells. ■ Confocal microscopy and deconvolution microscopy use different methods to optically section a specimen thereby reducing the blurring due to out-of-focus fluorescence light. Both methods provide much sharper images partic- ularly of thick specimens than does standard fluorescence microscopy. ■ Specimens for electron microscopy generally must be fixed sectioned dehydrated and then stained with electron- dense heavy metals. ■ Surface details of objects can be revealed by transmission electron microscopy of metal-coated specimens. Scanning electron microscopy of metal-coated unsectioned cells or tis- sues produces images that appear to be three-dimensional. PERSPECTIVES FOR THE FUTURE Advances in bioengineering will make major contributions not only to our understanding of cell and tissue function but also to the quality of human health. In a glass slide consisting of microfabricated wells and channels for example reagents can be introduced and exposed to selected parts of individual cells the responses of the cells can then be detected by light microscopy and analyzed by powerful image-processing soft- ware. These types of studies will lead to discovery of new drugs detection of subtle phenotypes of mutant cells e.g. tumor cells and development of comprehensive models of cellular processes. Bioengineers also are fabricating artificial tissues based on a synthetic three-dimensional architecture incorporating layers of different cells. Eventually such artifi- cial tissues will provide replacements for defective tissues in sick injured or aging individuals. Microscopy will continue to be a major tool in cell biol- ogy providing images that relate to both the chemistryi.e. interactions among proteins and the mechanics i.e. move- ments involved in various cell processes. The forces causing molecular and cellular movements will be directly detected by fluorescent sensors in cells and the extracellular matrix. Improvements to high-resolution imaging methods will per- mit studies of single molecules in live cells something that is currently possible only in vitro. Finally cells will be stud- ied in more natural contexts not on glass coverslips but in 3D gels of extracellular matrix molecules. To aid in the im- aging the use of more fluorescent labels and tags will allow visualization of five or six different types of molecules si- multaneously. With more labeled proteins the complex in- teractions among proteins and organelles will become better understood. Finally the electron microscope will become the domi- nant instrument for studying protein machines in vitro and in situ. Tomographic methods applied to single cells and mole- cules combined with automated reconstruction methods will generate models of protein-based structures that cannot be determined by x-ray crystallography. High resolution three- dimensional models of molecules in cells will help explain the intricate biochemical interactions among proteins. KEY TERMS actin filaments 174 apical 153 basolateral 153 chloroplast 172 cytoskeleton 147 cytosol 147 cytosolic face 150 endoplasmic reticulum ER 168 Key Terms 193 endosome 165 exoplasmic face 150 fluorescent staining 187 glycolipid 151 Golgi complex 169 GPI anchor 161 immunofluorescence microscopy 187

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REVIEW THE CONCEPTS 1. When viewed by electron microscopy the lipid bilayer is often described as looking like a railroad track. Explain how the structure of the bilayer creates this image. 2. Biomembranes contain many different types of lipid molecules. What are the three main types of lipid molecules found in biomembranes How are the three types similar and how are they different 3. Lipid bilayers are considered to be two-dimensional flu- ids what does this mean What drives the movement of lipid molecules and proteins within the bilayer How can such movement be measured What factors affect the degree of membrane fluidity 4. Explain the following statement: The structure of all biomembranes depends on the chemical properties of phos- pholipids whereas the function of each specific biomem- brane depends on the specific proteins associated with that membrane. 5. Name the three groups into which membrane-associated proteins may be classified. Explain the mechanism by which each group associates with a biomembrane. 6. Although both faces of a biomembrane are composed of the same general types of macromolecules principally lipids and proteins the two faces of the bilayer are not identical. What accounts for the asymmetry between the two faces 7. One of the defining features of eukaryotic cells is the presence of organelles. What are the major organelles of eu- karyotic cells and what is the function of each What is the cytosol What cellular processes occur within the cytosol 8. Cell organelles such as mitochondria chloroplasts and the Golgi apparatus each have unique structures. How is the structure of each organelle related to its function 9. Much of what we know about cellular function depends on experiments utilizing specific cells and specific parts e.g. organelles of cells. What techniques do scientists commonly use to isolate cells and organelles from complex mixtures and how do these techniques work 10. Isolation of some membrane proteins requires the use of detergents isolation of others can be accomplished with the use of high-salt solutions. What types of membrane pro- teins require detergents as part of the isolation procedure What types of membrane proteins may be isolated with high- salt solutions Describe how the chemical properties of detergents and high salt facilitate the isolation process of each type of membrane protein. 11. Three systems of cytoskeletal filaments exist in most eukaryotic cells. Compare them in terms of composition function and structure. 12. Individual cytoskeletal filaments are typically organized into more complex structures within the cytosol. What two general types of structures do individual filaments combine to form in the cytosol How are these structures created and maintained 13. Both light and electron microscopy are commonly used to visualize cells cell structures and the location of specific molecules. Explain why a scientist may choose one or the other microscopy technique for use in research. 14. Why are chemical stains required for visualizing cells and tissues with the basic light microscope What advantage does fluorescent microscopy provide in comparison to the chemical dyes used to stain specimens for light microscopy What advantages do confocal scanning microscopy and deconvolution microscopy provide in comparison to con- ventional fluorescence microscopy 15. In certain electron microscopy methods the specimen is not directly imaged. How do these methods provide infor- mation about cellular structure and what types of structures do they visualize ANALYZE THE DATA Mouse liver cells were homogenized and the homogenate subjected to equilibrium density-gradient centrifugation with sucrose gradients. Fractions obtained from these gradients were assayed for marker molecules i.e. molecules that are limited to specific organelles. The results of these assays are shown in the figure. The marker molecules have the follow- ing functions: Cytochrome oxidase is an enzyme involved in the process by which ATP is formed in the complete aerobic degradation of glucose or fatty acids ribosomal RNA forms part of the protein-synthesizing ribosomes catalase catalyzes decomposition of hydrogen peroxide acid phosphatase hydrolyzes monophosphoric esters at acid pH cytidylyl transferase is involved in phospholipid biosynthesis and amino acid permease aids in transport of amino acids across membranes. 194 CHAPTER 5 • Biomembranes and Cell Architecture integral membrane protein 157 intermediate filament 174 lipid raft 156 lumen 147 lysosome 165 microfilament 174 microtubule 174 mitochondrion 171 nuclear lamina 177 nucleolus 171 peripheral membrane protein 157 peroxisome 168 phosphoglyceride 150 phospholipid bilayer 149 pleckstrin homology PH domain 163 porin 160 prenyl anchor 160 resolution 184 sphingolipid 151

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a. Name the marker molecule and give the number of the fraction that is most enriched for each of the follow- ing: lysosomes peroxisomes mitochondria plasma mem- brane rough endoplasmic reticulum smooth endoplasmic reticulum. b. Is the rough endoplasmic reticulum more or less dense than the smooth endoplasmic reticulum Why c. Describe an alternative approach by which you could identify which fraction was enriched for which organelle. d. How would addition of a detergent to the homogenate affect the equilibrium density-gradient results REFERENCES General Histology Texts and Atlases Cross P. A. and K. L. Mercer. 1993. Cell and Tissue Ultra- structure: A Functional Perspective. W. H. Freeman and Company. Fawcett D. W. 1993. Bloom and Fawcett: A Textbook of His- tology 12th ed. Chapman Hall. Kessel R. and R. Kardon. 1979. Tissues and Organs: A Text- Atlas of Scanning Electron Microscopy. W. H. Freeeman and Company. Biomembranes: Lipid Composition and Structural Organization Simons K. and D. Toomre. 2000. Lipid rafts and signal trans- duction. Nature Rev. Mol. Cell Biol. 1:31–41. Sprong H. P. van der Sluijs and G. van Meer. 2001. How pro- teins move lipids and lipids move proteins. Nature Rev. Mol. Cell Biol. 2:504–513. Tamm L. K. V. K. Kiessling and M. L. Wagner. 2001. Mem- brane dynamics. Encyclopedia of Life Sciences. Nature Publishing Group. Vance D. E. and J. E. Vance. 2002. Biochemistry of Lipids Lipoproteins and Membranes 4th ed. Elsevier. Yeager P. L. 2001. Lipids. Encyclopedia of Life Sciences. Na- ture Publishing Group. Biomembranes: Protein Components and Basic Functions Cullen P. J. G. E. Cozier G. Banting and H. Mellor. 2001. Modular phosphoinositide-binding domains: their role in signalling and membrane trafficking. Curr. Biol. 11:R882–R893. Lanyi J. K. and H. Luecke. 2001. Bacteriorhodopsin. Curr. Opin. Struc. Biol. 11:415–519. MacKenzie K. R. J. H. Prestegard and D. M. Engelman. 1997. A transmembrane helix dimer: structure and implications. Science 276:131–133. Minor D. L. 2001. Potassium channels: life in the post-structural world. Curr. Opin. Struc. Biol. 11:408–414. Schulz G. E. 2000. -Barrel membrane proteins. Curr. Opin. Struc. Biol. 10:443–447. Organelles of the Eukaryotic Cell Bainton D. 1981. The discovery of lysosomes. J. Cell Biol. 91:66s–76s. Cuervo A. M. and J. F. Dice. 1998. Lysosomes: a meeting point of proteins chaperones and proteases. J. Mol. Med. 76:6–12. de Duve C. 1996. The peroxisome in retrospect. Ann. NY Acad. Sci. 804:1–10. Holtzman E. 1989. Lysosomes. Plenum Press. Lamond A. and W. Earnshaw. 1998. Structure and function in the nucleus. Science 280:547–553. Masters C. and D. Crane. 1996. Recent developments in per- oxisome biology. Endeavour 20:68–73. Palade G. 1975. Intracellular aspects of the process of protein synthesis. Science 189:347–358. The Nobel Prize lecture of a pioneer in the study of cellular organelles. See also de Duve 1996. Subramani S. 1998. Components involved in peroxisome im- port biogenesis proliferation turnover and movement. Physiol. Rev. 78:171–188. The Cytoskeleton: Components and Structural Functions Bray D. 2001. Cell Movements: From Molecules to Motility. Garland. Excellent overview of the cytoskeleton and motility. Various authors. Curr. Topics Cell Biol. February issue is always devoted to the cytoskeleton. Purification of Cells and Their Parts Battye F. L. and K. Shortman. 1991. Flow cytometry and cell- separation procedures. Curr. Opin. Immunol. 3:238–241. de Duve C. 1975. Exploring cells with a centrifuge. Science 189:186–194. The Nobel Prize lecture of a pioneer in the study of cellular organelles. de Duve C. and H. Beaufay. 1981. A short history of tissue fractionation. J. Cell Biol. 91:293s–299s. Howell K. E. E. Devaney and J. Gruenberg. 1989. Subcellular fractionation of tissue culture cells. Trends Biochem. Sci. 14:44–48. References 195 100 60 80 40 20 0 05 10 20 15 AC DE F B Fraction number 50 Sucrose 0 of maximum Curve A cytochrome oxidase Curve B ribosomal RNA Curve C catalase Curve D acid phosphatase Curve E cytidylyl transferase Curve F amino acid permease

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Ormerod M. G. ed. 1990. Flow Cytometry: A Practical Ap- proach. IRL Press. Rickwood D. 1992. Preparative Centrifugation: A Practical Ap- proach. IRL Press. Visualizing Cell Architecture Bastiaens P. I. H. and R. Pepperkok. 2000. Observing proteins in their natural habitat: the living cell. Trends Biochem. Sci. 25:631–637. Baumeister W. and A. C. Steven. 2000. Macromolecular elec- tron microscopy in the era of structural genomics. Trends Biochem. Sci. 25:624–630. Bozzola J. J. and L. D. Russell. 1992. Electron Microscopy. Jones and Bartlett. Dykstra M. J. 1992. Biological Electron Microscopy: Theory Techniques and Troubleshooting. Plenum Press. Gilroy S. 1997. Fluorescence microscopy of living plant cells. Ann. Rev. Plant Physiol. Plant Mol. Biol. 48:165–190. Inoué S. and K. Spring. 1997. Video Microscopy 2d ed. Plenum Press. Lippincott-Schwartz J. and C. L Smith. 1997. Insights into se- cretory and endocytic membrane traffic using green fluorescent pro- tein chimeras. Curr. Opin. Neurobiol. 7:631–639. Mason W. T. 1999. Fluorescent and Luminescent Probes for Biological Activity 2d ed. Academic Press. Matsumoto B. ed. 2002. Methods in Cell Biology Vol. 70: Cell Biological Applications of Confocal Microscopy. Academic Press. Misteli T. and D. L. Spector. 1997. Applications of the green fluorescent protein in cell biology and biotechnology. Nature Biotech. 15:961–964. Sluder G. and D. Wolf eds. 1998. Methods in Cell Biology Vol. 56: Video Microscopy. Academic Press. 196 CHAPTER 5 • Biomembranes and Cell Architecture

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5.1 • Last A Head 197

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material energy and time during the development of an in- dividual organism. Although the physiological costs of com- plex tissues and organs are high they provide organisms with the ability to thrive in varied and variable environ- ments a major evolutionary advantage. The complex and diverse morphologies of plants and animals are examples of the whole being greater than the sum of the individual parts more technically described as the emergent properties of a complex system. For example the root-stem-leaf organization of plants permits them to simul- taneously obtain energy sunlight and carbon CO 2 from 6 Model of inflammatory bowel disease in which cultured flat colonic smooth muscle cells were induced to secrete cables of hyaluronan green that bind to spheroidal mononuclear leukocytes via their CD44 receptors red. Nuclei are stained blue. Courtesy of C. de la Motte et al. Lerner Research Institute. INTEGRATING CELLS INTO TISSUES I n the development of complex multicellular organisms such as plants and animals progenitor cells differentiate into distinct “types” that have characteristic composi- tions structures and functions. Cells of a given type often aggregate into a tissue to cooperatively perform a common function: muscle contracts nervous tissues conduct electrical impulses xylem tissue in plants transports water. Different tissues can be organized into an organ again to perform one or more specific functions. For instance the muscles valves and blood vessels of a heart work together to pump blood through the body. The coordinated functioning of many types of cells within tissues as well as of multiple special- ized tissues permits the organism as a whole to move me- tabolize reproduce and carry out other essential activities. The adult form of the roundworm Caenorhabditis elegans contains a mere 959 cells yet these cells fall into 12 different general cell types and many distinct subtypes. Vertebrates have hundreds of different cell types including leukocytes white blood cells erythrocytes and macrophages in the blood photoreceptors in the retina adipocytes that store fat secretory and cells in the pancreas fibroblasts in connec- tive tissue and hundreds of different subtypes of neurons in the human brain. Despite their diverse forms and functions all animal cells can be classified as being components of just five main classes of tissue: epithelial tissue connective tissue muscular tissue nervous tissue and blood. Various cell types are arranged in precise patterns of staggering complexity to generate the different tissues and organs. The costs of such complexity include increased requirements for information 197 OUTLINE 6.1 Cell–Cell and Cell–Matrix Adhesion: An Overview 6.2 Sheetlike Epithelial Tissues: Junctions and Adhesion Molecules 6.3 The Extracellular Matrix of Epithelial Sheets 6.4 The Extracellular Matrix of Nonepithelial Tissues 6.5 Adhesive Interactions and Nonepithelial Cells 6.6 Plant Tissues 6.7 Growth and Use of Cultured Cells

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the atmosphere and water and nutrients e.g. minerals from the soil. The distinct mechanical properties of rigid bones flexible joints and contracting muscles permit vertebrates to move efficiently and achieve substantial size. Sheets of tightly attached epithelial cells can act as regulatable selec- tive permeability barriers which permit the generation of chemically and functionally distinct compartments in an or- ganism e.g. stomach bloodstream. As a result distinct and sometimes opposite functions e.g. digestion and synthesis can efficiently proceed simultaneously within an organism. Such compartmentalization also permits more sophisticated regulation of diverse biological functions. In many ways the roles of complex tissues and organs in an organism are anal- ogous to those of organelles and membranes in individual cells. The assembly of distinct tissues and their organization into organs are determined by molecular interactions at the cellular level and would not be possible without the tempo- rally and spatially regulated expression of a wide array of ad- hesive molecules. Cells in tissues can adhere directly to one another cell–cell adhesion through specialized integral membrane proteins called cell-adhesion molecules CAMs that often cluster into specialized cell junctions Figure 6-1. Cells in animal tissues also adhere indirectly cell–matrix 198 CHAPTER 6 • Integrating Cells into Tissues Actin Intermediate filament Adhesion receptors Connexon Tight junction Desmosome Hemi- desmosome Gap junction Basal lamina Apical surface Basal surface Extracellular matrix ECM Adherens junction 1 2 3 6 7 10 9 8 Cell adhesion molecules CAMs Adapters CELL CELL CELL ECM Adapter CELL-CELL ADHESIONS CELL-MATRIX ADHESIONS 5 4 ▲ FIGURE 6-1 Schematic overview of major adhesive interactions that bind cells to each other and to the extracellular matrix. Schematic cutaway drawing of a typical epithelial tissue such as the intestines. The apical upper surface of these cells is packed with fingerlike microvilli that project into the intestinal lumen and the basal bottom surface rests on extracellular matrix ECM. The ECM associated with epithelial cells is usually organized into various interconnected layers e.g. the basal lamina connecting fibers connective tissue in which large interdigitating ECM macromolecules bind to one another and to the cells . Cell-adhesion molecules CAMs bind to CAMs on other cells mediating cell–cell adhesions and adhesion receptors bind to various components of the ECM mediating cell–matrix adhesions . Both types of cell-surface adhesion molecules are usually integral membrane proteins whose cytosolic domains often bind to multiple intracellular adapter proteins. These adapters directly or indirectly link the CAM to the cytoskeleton actin or intermediate filaments and to 5 4 3 2 1 intracellular signaling pathways. As a consequence information can be transferred by CAMs and the macromolecules to which they bind from the cell exterior into the intracellular environment and vice versa. In some cases a complex aggregate of CAMs adapters and associated proteins is assembled. Specific localized aggregates of CAMs or adhesion receptors form various types of cell junctions that play important roles in holding tissues together and facilitating communication between cells and their environment. Tight junctions lying just under the microvilli prevent the diffusion of many substances through the extracellular spaces between the cells. Gap junctions allow the movement through connexon channels of small molecules and ions between the cytosols of adjacent cells. The remaining three types of junctions adherens junctions spot desmosomes and hemidesmosomes link the cytoskeleton of a cell to other cells or the ECM. See V. Vasioukhin and E. Fuchs 2001 Curr. Opin. Cell Biol. 13:76. 10 9 8 7 6

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adhesion through the binding of adhesion receptors in the plasma membrane to components of the surrounding extra- cellular matrix ECM a complex interdigitating meshwork of proteins and polysaccharides secreted by cells into the spaces between them. These two basic types of interactions not only allow cells to aggregate into distinct tissues but also provide a means for the bidirectional transfer of informa- tion between the exterior and the interior of cells. In this chapter we examine the various types of adhesive molecules and how they interact. The evolution of plants and animals is thought to have diverged before multicellular organisms arose. Thus multicellularity and the molecular means for assembling tissues and organs must have arisen in- dependently in animal and plant lineages. Not surprisingly then animals and plants exhibit many differences in the or- ganization and development of tissues. For this reason we first consider the organization of epithelial and nonepithe- lial tissues in animals and then deal separately with plant tis- sues. Although most cells in living organisms exist within tissues our understanding about cells depends greatly on the study of isolated cells. Hence we present some general fea- tures of working with populations of cells removed from tis- sues and organisms in the last section of this chapter. Cell–Cell and Cell–Matrix Adhesion: An Overview We begin with a brief orientation to the various types of ad- hesive molecules their major functions in organisms and their evolutionary origin. In subsequent sections we examine in detail the unique structures and properties of the vari- ous participants in cell–cell and cell–matrix interactions in animals. Cell-Adhesion Molecules Bind to One Another and to Intracellular Proteins A large number of CAMs fall into four major families: the cadherins immunoglobulin Ig superfamily integrins and selectins. As the schematic structures in Figure 6-2 illustrate many CAMs are mosaics of multiple distinct domains many 6.1 6.1 • Cell–Cell and Cell–Matrix Adhesion: An Overview 199 Ig domain Type III fibronectin repeat Lectin domain Cadherins E-cadherin lg-superfamily CAMs NCAM Homophilic interactions Heterophilic interactions Selectins P-selectin Glycoprotein Sugars Integrins αvβ3 Fibronectin Cadherin domain Calcium- binding sites ▲ FIGURE 6-2 Major families of cell-adhesion molecules CAMs and adhesion receptors. Dimeric E-cadherins most commonly form homophilic self cross-bridges with E-cadherins on adjacent cells. Members of the immunoglobulin Ig superfamily of CAMs can form both homophilic linkages shown here and heterophilic nonself linkages. Selectins shown as dimers contain a carbohydrate-binding lectin domain that recognizes specialized sugar structures on glycoproteins shown here and glycolipids on adjacent cells. Heterodimeric integrins for example v and 3 chains function as CAMs or as adhesion receptors shown here that bind to very large multiadhesive matrix proteins such as fibronectin only a small part of which is shown here see also Figure 6-25. Note that CAMs often form higher-order oligomers within the plane of the plasma membrane. Many adhesive molecules contain multiple distinct domains some of which are found in more than one kind of CAM. The cytoplasmic domains of these proteins are often associated with adapter proteins that link them to the cytoskeleton or to signaling pathways. See R. O. Hynes 1999 Trends Cell Biol. 912:M33 and R. O. Hynes 2002 Cell 110:673–687 .

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of which can be found in more than one kind of CAM. They are called “repeats” when they exist multiple times in the same molecule. Some of these domains confer the binding specificity that characterizes a particular protein. Some other membrane proteins whose structures do not belong to any of the major classes of CAMs also participate in cell–cell ad- hesion in various tissues. CAMs mediate through their extracellular domains ad- hesive interactions between cells of the same type homotypic adhesion or between cells of different types heterotypic adhesion. A CAM on one cell can directly bind to the same kind of CAM on an adjacent cell homophilic binding or to a different class of CAM heterophilic binding. CAMs can be broadly distributed along the regions of plasma mem- branes that contact other cells or clustered in discrete patches or spots called cell junctions. Cell–cell adhesions can be tight and long lasting or relatively weak and transient. The asso- ciations between nerve cells in the spinal cord or the meta- bolic cells in the liver exhibit tight adhesion. In contrast immune-system cells in the blood can exhibit only weak short-lasting interactions allowing them to roll along and pass through a blood vessel wall on their way to fight an in- fection within a tissue. The cytosol-facing domains of CAMs recruit sets of mul- tifunctional adapter proteins see Figure 6-1. These adapters act as linkers that directly or indirectly connect CAMs to el- ements of the cytoskeleton Chapter 5 they can also recruit intracellular molecules that function in signaling pathways to control protein activity and gene expression Chapters 13 and 14. In some cases a complex aggregate of CAMs adapter proteins and other associated proteins is assembled at the inner surface of the plasma membrane. Because cell–cell adhesions are intrinsically associated with the cy- toskeleton and signaling pathways a cell’s surroundings influence its shape and functional properties “outside-in” effects likewise cellular shape and function influence a cell’s surroundings “inside-out” effects. Thus connectivity and communication are intimately related properties of cells in tissues. The formation of many cell–cell adhesions entails two types of molecular interactions Figure 6-3. First CAMs on one cell associate laterally through their extracellular domains or cytosolic domains or both into homodimers or higher-order oligomers in the plane of the cell’s plasma membrane these interactions are called intracellular lat- eral or cis interactions. Second CAM oligomers on one cell bind to the same or different CAMs on an adjacent cell these interactions are called intercellular or trans in- teractions. Trans interactions sometimes induce additional cis interactions and as a consequence yet even more trans interactions. Adhesive interactions between cells vary considerably depending on the particular CAMs participating and the tis- sue. Just like Velcro very tight adhesion can be generated when many weak interactions are combined together in a small well-defined area. Furthermore the association of in- tracellular molecules with the cytosolic domains of CAMs can dramatically influence the intermolecular interactions of CAMs by promoting their cis association clustering or by altering their conformation. Among the many variables that determine the nature of adhesion between two cells are the binding affinity of the interacting molecules thermodynamic properties the overall “on” and “off” rates of association and dissociation for each interacting molecule kinetic prop- erties the spatial distribution clustering high or low den- sity of adhesion molecules geometric properties the active versus inactive states of CAMs with respect to adhesion bio- chemical properties and external forces such as the lami- nar and turbulent flow of cells in the circulatory system mechanical properties. 200 CHAPTER 6 • Integrating Cells into Tissues Trans + + Trans Cis + trans Cis + trans Cis lateral CELL 1 CELL 2 Cis lateral ▲ FIGURE 6-3 Schematic model for the generation of cell–cell adhesions. Lateral interactions between cell-adhesion molecules CAMs within the plasma membrane of a cell form dimers and larger oligomers. The parts of the molecules that participate in these cis interactions vary among the different CAMs. Subsequent trans interactions between distal domains of CAMs on adjacent cells generate a zipperlike strong adhesion between the cells. Adapted from M. S. Steinberg and P . M. McNutt 1999 Curr. Opin. Cell Biol. 11:554.

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The Extracellular Matrix Participates in Adhesion and Other Functions Certain cell-surface receptors including some integrins can bind components of the extracellular matrix ECM thereby indirectly adhering cells to each other through their interac- tions with the matrix. Three abundant ECM components are proteoglycans a unique type of glycoprotein collagens pro- teins that often form fibers and soluble multiadhesive matrix proteins e.g. fibronectin. The relative volumes of cells ver- sus matrix vary greatly among different animal tissues and organs. Some connective tissue for instance is mostly ma- trix whereas many organs are composed of very densely packed cells with relatively little matrix. Although the extracellular matrix generally provides me- chanical support to tissues it serves several other functions as well. Different combinations of ECM components tailor the extracellular matrix for specific purposes: strength in a tendon tooth or bone cushioning in cartilage and adhesion in most tissues. In addition the composition of the matrix which can vary depending on the anatomical site and phys- iological status of a tissue can let a cell know where it is and what it should do environmental cues. Changes in ECM components which are constantly being remodeled de- graded and resynthesized locally can modulate the interac- tions of a cell with its environment. The matrix also serves as a reservoir for many extracellular signaling molecules that control cell growth and differentiation. In addition the ma- trix provides a lattice through or on which cells can move particularly in the early stages of tissue assembly. Morpho- genesis—the later stage of embryonic development in which tissues organs and body parts are formed by cell movements and rearrangements—also is critically dependent on cell– matrix adhesion as well as cell–cell adhesion. Diversity of Animal Tissues Depends on Evolution of Adhesion Molecules with Various Properties Cell–cell adhesions and cell–matrix adhesions are responsible for the formation composition architecture and function of animal tissues. Not surprisingly adhesion molecules of ani- mals are evolutionarily ancient and are some of the most highly conserved proteins among multicellular metazoan organisms. Sponges the most primitive metazoans express certain CAMs and multiadhesive ECM molecules whose structures are strikingly similar to those of the corresponding human proteins. The evolution of organisms with complex tissues and organs has depended on the evolution of diverse CAMs adhesion receptors and ECM molecules with novel properties and functions whose levels of expression differ in different types of cells. The diversity of adhesive molecules arises in large part from two phenomena that can generate numerous closely re- lated proteins called isoforms that constitute a protein fam- ily. In some cases the different members of a protein family are encoded by multiple genes that arose from a common an- cestor by gene duplication and divergent evolution Chapter 9. Analyses of gene and cDNA sequences can provide evi- dence for the existence of such a set of related genes or gene family. In other cases a single gene produces an RNA tran- script that can undergo alternative splicing to yield multiple mRNAs each encoding a distinct isoform Chapter 4. Al- ternative splicing thus increases the number of proteins that can be expressed from one gene. Both of these phenomena contribute to the diversity of some protein families such as the cadherins. Particular isoforms of an adhesive protein are often expressed in some cell types but not others account- ing for their differential distribution in various tissues. KEY CONCEPTS OF SECTION 6.1 Cell–Cell and Cell–Matrix Adhesion: An Overview ■ Cell-adhesion molecules CAMs mediate direct cell–cell adhesions homotypic and heterotypic and cell-surface ad- hesion receptors mediate cell–matrix adhesions see Figure 6-1. These interactions bind cells into tissues and facilitate communication between cells and their environments. ■ The cytosolic domains of CAMs and adhesion receptors bind multifunctional adapter proteins that mediate inter- action with cytoskeletal fibers and intracellular signaling proteins. ■ The major families of cell-surface adhesion molecules are the cadherins selectins Ig-superfamily CAMs and inte- grins see Figure 6-2. ■ Tight cell–cell adhesions entail both cis lateral or in- tracellular oligomerization of CAMs and trans intercel- lular interaction of like homophilic or different hetero- philic CAMs see Figure 6-3. ■ The extracellular matrix ECM is a complex meshwork of proteins and polysaccharides that contributes to the structure and function of a tissue. ■ The evolution of CAMs adhesion receptors and ECM molecules with specialized structures and functions permits cells to assemble into diverse classes of tissues with vary- ing functions. Sheetlike Epithelial Tissues: Junctions and Adhesion Molecules In general the external and internal surfaces of organs are covered by a sheetlike layer of epithelial tissue called an ep- ithelium. Cells that form epithelial tissues are said to be po- larized because their plasma membranes are organized into at least two discrete regions. Typically the distinct surfaces of a polarized epithelial cell are called the apical top basal 6.2 6.2 • Sheetlike Epithelial Tissues: Junctions and Adhesion Molecules 201

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base or bottom and lateral side surfaces Figure 6-4. The basal surface usually contacts an underlying extracellular matrix called the basal lamina whose composition and func- tion are discussed in Section 6.3. Often the basal and lateral surfaces are similar in composition and together are called the basolateral surface. The basolateral surfaces of most ep- ithelia are usually on the side of the cell closest to the blood vessels. In animals with closed circulatory systems blood flows through vessels whose inner lining is composed of flat- tened epithelial cells called endothelial cells. The apical side of endothelial cells which faces the blood is usually called the luminal surface and the opposite basal side the ablumi- nal surface. Epithelia in different body locations have characteristic morphologies and functions see Figure 6-4. Stratified mul- tilayered epithelia commonly serve as barriers and protec- tive surfaces e.g. the skin whereas simple single-layer epithelia often selectively move ions and small molecules from one side of the layer to the other. For instance the sim- ple columnar epithelium lining the stomach secretes hy- drochloric acid into the stomach lumen a similar epithelium lining the small intestine transports products of digestion e.g. glucose and amino acids from the lumen of the intes- tine across the basolateral surface into the blood Chapter 7. The simple columnar epithelium lining the small intestine has numerous fingerlike projections 100 nm in diameter called microvilli singular microvillus that extend from the luminal apical surface see Figure 5-45. The upright orientation of a microvillus is maintained by numerous connections be- tween the surrounding plasma membrane and a central bun- dle of actin microfilaments which extend into the cell and interact with keratin intermediate filaments see Figure 5-28. Microvilli greatly increase the area of the apical sur- face and thus the number of proteins that it can contain en- hancing the absorptive capacity of the intestinal epithelium. Here we describe the various cell junctions and CAMs that play key roles in the assembly and functioning of epithe- lial sheets. In Section 6.3 we consider the components of the extracellular matrix intimately associated with epithelia. Specialized Junctions Help Define the Structure and Function of Epithelial Cells All epithelial cells in a sheet are connected to one another and the extracellular matrix by specialized cell junctions consist- ing of dense clusters of CAMs. Although hundreds of indi- vidual CAM-mediated interactions are sufficient to cause cells to adhere junctions play special roles in imparting strength and rigidity to a tissue transmitting information between the extracellular and the intracellular space controlling the pas- sage of ions and molecules across cell layers and serving as conduits for the movement of ions and molecules from the cy- toplasm of one cell to that of its immediate neighbor. Three major classes of animal cell junctions are promi- nent features of the intestinal epithelium Figure 6-5 see also Figure 6-1. Anchoring junctions and tight junctions perform the key task of holding cells together into tissues. These junc- tions are organized into three parts: adhesive proteins in the plasma membrane that connect one cell to another cell CAMs or to the extracellular matrix adhesion receptors adapter proteins which connect the CAMs or adhesion re- 202 CHAPTER 6 • Integrating Cells into Tissues c Transitional d Stratified squamous nonkeratinized b Simple squamous a Simple columnar Basal surface Basal lamina Apical surface Lateral surface Connective tissue ▲ FIGURE 6-4 Principal types of epithelium. The apical and basolateral surfaces of epithelial cells exhibit distinctive characteristics. a Simple columnar epithelia consist of elongated cells including mucus-secreting cells in the lining of the stomach and cervical tract and absorptive cells in the lining of the small intestine. b Simple squamous epithelia composed of thin cells line the blood vessels endothelial cells/endothelium and many body cavities. c Transitional epithelia composed of several layers of cells with different shapes line certain cavities subject to expansion and contraction e.g. the urinary bladder. d Stratified squamous nonkeratinized epithelia line surfaces such as the mouth and vagina these linings resist abrasion and generally do not participate in the absorption or secretion of materials into or out of the cavity. The basal lamina a thin fibrous network of collagen and other ECM components supports all epithelia and connects them to the underlying connective tissue.

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ceptors to cytoskeletal filaments and signaling molecules and the cytoskeletal filaments themselves. Tight junctions also control the flow of solutes between the cells forming an epithelial sheet. Gap junctions permit the rapid diffusion of small water-soluble molecules between the cytoplasm of ad- jacent cells. Although present in epithelia gap junctions are also abundant in nonepithelial tissues and structurally are very different from anchoring junctions and tight junctions they also bear some resemblance to an important cell–cell junction in plants. For these reasons we wait to consider gap junctions at the end of Section 6.5. Of the three types of anchoring junctions present in ep- ithelial cells two participate in cell–cell adhesion whereas the third participates in cell–matrix adhesion. Adherens junc- tions which connect the lateral membranes of adjacent ep- ithelial cells are usually located near the apical surface just below the tight junctions see Figures 6-1 and 6-5. A cir- cumferential belt of actin and myosin filaments in a complex with the adherens junction functions as a tension cable that can internally brace the cell and thereby control its shape. Epithelial and some other types of cells such as smooth mus- cle are also bound tightly together by desmosomes button- like points of contact sometimes called spot desmosomes. Hemidesmosomes found mainly on the basal surface of ep- ithelial cells anchor an epithelium to components of the un- derlying extracellular matrix much like nails holding down a carpet. Bundles of intermediate filaments running parallel to the cell surface or through the cell rather than actin fila- ments interconnect spot desmosomes and hemidesmosomes imparting shape and rigidity to the cell. Desmosomes and hemidesmosomes also transmit shear forces from one region of a cell layer to the epithelium as a whole providing strength and rigidity to the entire epithe- lial cell layer. These junctions are especially important in maintaining the integrity of skin epithelia. For instance mu- tations that interfere with hemidesmosomal anchoring in the skin can lead to blistering in which the epithelium becomes detached from its matrix foundation and extracellular fluid accumulates at the basolateral surface forcing the skin to balloon outward. 6.2 • Sheetlike Epithelial Tissues: Junctions and Adhesion Molecules 203 Basal surface Apical surface Tight junction Adherens junction Microvillus Desmosome Gap junction Lateral surface a Microvillus Tight junction Adherens junction Desmosome Gap junction Intermediate filaments Hemidesmosome Basal lamina Actin and myosin filaments Connective tissue b ▲ FIGURE 6-5 The principal types of cell junctions that connect the columnar epithelial cells lining the small intestine. a Schematic cutaway drawing of intestinal epithelial cells. The basal surface of the cells rests on a basal lamina and the apical surface is packed with fingerlike microvilli that project into the intestinal lumen. Tight junctions lying just under the microvilli prevent the diffusion of many substances between the intestinal lumen and the blood through the extracellular space between cells. Gap junctions allow the movement of small molecules and ions between the cytosols of adjacent cells. The remaining three types of junctions—adherens junctions spot desmosomes and hemidesmosomes—are critical to cell–cell and cell–matrix adhesion and signaling. b Electron micrograph of a thin section of intestinal epithelial cells showing relative locations of the different junctions. Part b C. Jacobson et al. 2001 Journal Cell Biol. 152:435–450.

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Ca 2 -Dependent Homophilic Cell–Cell Adhesion in Adherens Junctions and Desmosomes Is Mediated by Cadherins The primary CAMs in adherens junctions and desmosomes belong to the cadherin family. In vertebrates and inverte- brates this protein family of more than 100 members can be grouped into at least six subfamilies. The diversity of cad- herins arises from the presence of multiple cadherin genes and alternative RNA splicing which generates multiple mRNAs from one gene. Cadherins are key molecules in cell–cell adhesion and cell signaling and they play a critical role during tissue differen- tiation. The “classical” E- P- and N-cadherins are the most widely expressed particularly during early differentiation. Sheets of polarized epithelial cells such as those that line the small intestine or kidney tubules contain abundant E-cadherin along their lateral surfaces. Although E-cadherin is concentrated in adherens junctions it is present through- out the lateral surfaces where it is thought to link adjacent cell membranes. The brain expresses the largest number of different cadherins presumably owing to the necessity of forming many very specific cell–cell contacts to help establish its complex wiring diagram. Classical Cadherins The results of experiments with L cells a line of cultured mouse fibroblasts grown in the laboratory demonstrated that E-cadherin and P-cadherin preferentially mediate homophilic interactions. L cells express no cadherins and adhere poorly to themselves or to other types of cultured cells. When genes encoding either E-cadherin or P-cadherin were introduced into L cells with the use of techniques de- scribed in Chapter 9 the resulting engineered L cells expressed the encoded cadherin. These cadherin-expressing L cells were found to adhere preferentially to cells expressing the same type of cadherin molecules that is they mediate homophilic inter- actions. The L cells expressing E-cadherin also exhibited the polarized distribution of a membrane protein similar to that in epithelial cells and they formed epithelial-like aggregates with one another and with epithelial cells isolated from lungs. The adhesiveness of cadherins depends on the presence of extracellular Ca 2 the property that gave rise to their name calcium adhering. For example the adhesion of engineered L cells expressing E-cadherin is prevented when the cells are bathed in a solution growth medium that is low in Ca 2 . The role of E-cadherin in adhesion can also be demonstrated 204 CHAPTER 6 • Integrating Cells into Tissues Culture dish Basal lamina Monolayer of MDCK cells Porous filter Apical medium Basal medium Apical surface ▲ EXPERIMENTAL FIGURE 6-6 Madin-Darby canine kidney MDCK cells grown in specialized containers provide a useful experimental system for studying epithelial cells. MDCK cells form a polarized epithelium when grown on a porous membrane filter coated on one side with collagen and other components of the basal lamina. With the use of the special culture dish shown here the medium on each side of the filter apical and basal sides of the monolayer can be experimentally manipulated and the movement of molecules across the layer monitored. Anchoring junctions and tight junctions form only if the growth medium contains sufficient Ca 2 . Extracellular space E-cadherin F-actin -Catenin Plasma membrane Cell 1 Cell 2 -Actinin p120-catenin Vinculin ZO1 -Catenin Cytosol Cytosol VASP Plasma membrane ▲ FIGURE 6-7 Protein constitutents of typical adherens junctions. The exoplasmic domains of E-cadherin dimers clustered at adherens junctions on adjacent cells 1 and 2 form Ca 2 -dependent homophilic interactions. The cytosolic domains of the E-cadherins bind directly or indirectly to multiple adapter proteins that connect the junctions to actin filaments F-actin of the cytoskeleton and participate in intracellular signaling pathways e.g. -catenin. Somewhat different sets of adapter proteins are illustrated in the two cells shown to emphasize that a variety of adapters can interact with adherens junctions which can thereby participate in diverse activities. Adapted from V. Vasioukhin and E. Fuchs 2001 Curr. Opin. Cell Biol.13:76.

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in experiments with cultured cells called Madin-Darby ca- nine kidney MDCK cells. When grown in specialized con- tainers these cells form a continuous one-cell-thick sheet monolayer of polarized kidneylike epithelial cells Figure 6-6. In this experimental system the addition of an antibody that binds to E-cadherin preventing its homophilic interac- tions blocks the Ca 2 -dependent attachment of suspended MDCK cells to a substrate and the subsequent formation of intercellular adherens junctions. Each classical cadherin contains a single transmembrane domain a relatively short C-terminal cytosolic domain and five extracellular “cadherin” domains see Figure 6-2. The extracellular domains are necessary for Ca 2 binding and cadherin-mediated cell–cell adhesion. Cadherin-mediated ad- hesion entails both lateral intracellular and trans intercel- lular molecular interactions as described previously see Figure 6-3. The Ca 2 -binding sites located between the cadherin repeats serve to rigidify the cadherin oligomers. The cadherin oligomers subsequently form intercellular com- plexes to generate cell–cell adhesion and then additional lat- eral contacts resulting in a “zippering up” of cadherins into clusters. In this way multiple low-affinity interactions sum to produce a very tight intercellular adhesion. The results of domain swap experiments in which an ex- tracellular domain of one kind of cadherin is replaced with the corresponding domain of a different cadherin have in- dicated that the specificity of binding resides at least in part in the most distal extracellular domain the N-terminal do- main. In the past cadherin-mediated adhesion was com- monly thought to require only head-to-head interactions between the N-terminal domains of cadherin oligomers on adjacent cells as depicted in Figure 6-3. However the results of some experiments suggest that under some experimental conditions at least three cadherin domains from each mole- cule not just the N-terminal domains participate by inter- digitation in trans associations. The C-terminal cytosolic domain of classical cadherins is linked to the actin cytoskeleton by a number of cytosolic adapter proteins Figure 6-7. These linkages are essential for strong adhesion apparently owing primarily to their con- tributing to increased lateral associations. For example disrup- tion of the interactions between classical cadherins and - or -catenin—two common adapter proteins that link these cad- herins to actin filaments—dramatically reduces cadherin- mediated cell–cell adhesion. This disruption occurs sponta- neously in tumor cells which sometimes fail to express - catenin and can be induced experimentally by depleting the cytosolic pool of accessible -catenin. The cytosolic domains of cadherins also interact with intracellular signaling molecules such as -catenin and p120-catenin. Interestingly -catenin not only mediates cytoskeletal attachment but can also translocate to the nucleus and alter gene transcription see Figure 15-32. Although E-cadherins exhibit primarily homophilic bind- ing some cadherins mediate heterophilic interactions. Im- portantly each classical cadherin has a characteristic tissue distribution. In the course of differentiation the amount or nature of the cell-surface cadherins changes affecting many aspects of cell–cell adhesion and cell migration. For instance the reorganization of tissues during morphogenesis is often accompanied by the conversion of nonmotile epithelial cells into motile precursor cells for other tissues mesenchymal cells. Such epithelial-to-mesenchymal transitions are asso- ciated with a reduction in the expression of E-cadherin. The conversion of epithelial cells into cancerous melanoma cells also is marked by a loss of E-cadherin activity. The resulting decrease in cell–cell adhesion permits melanoma cells to in- vade the underlying tissue and spread throughout the body. Desmosomal Cadherins Desmosomes Figure 6-8 con- tain two specialized cadherin proteins desmoglein and 6.2 • Sheetlike Epithelial Tissues: Junctions and Adhesion Molecules 205 a Plasma membrane Intercellular space Cytoplasmic plaque plakoglobin‚ desmoplakins Desmoglein and desmocollin cadherins Intermediate filaments b Intermediate filaments Cytoplasmic plaques Plasma membranes 0.2 µ m ▲ FIGURE 6-8 Desmosomes. a Schematic model showing components of a desmosome between epithelial cells and attachments to the sides of keratin intermediate filaments which crisscross the interior of cells. The transmembrane CAMs desmoglein and desmocollin belong to the cadherin family. b Electron micrograph of a thin section of a desmosome connecting two cultured differentiated human keratinocytes. Bundles of intermediate filaments radiate from the two darkly staining cytoplasmic plaques that line the inner surface of the adjacent plasma membranes. Part a see B. M. Gumbiner 1993 Neuron 11:551 and D. R. Garrod 1993 Curr. Opin. Cell Biol. 5:30. Part b courtesy of R. van Buskirk.

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desmocollin whose cytosolic domains are distinct from those in the classical cadherins. The cytosolic domains of desmosomal cadherins interact with plakoglobin similar in structure to -catenin and the plakophilins. These adapter proteins which form the thick cytoplasmic plaques charac- teristic of desmosomes in turn interact with intermediate fil- aments. Thus desmosomes and adherens junctions are linked to different cytoskeletal fibers. The cadherin desmoglein was first identified by an unusual but revealing skin disease called pemphi- gus vulgaris an autoimmune disease. Patients with autoimmune disorders synthesize antibodies that bind to a normal body protein. In this case the autoantibodies disrupt adhesion between epithelial cells causing blisters of the skin and mucous membranes. The predominant autoantibody was shown to be specific for desmoglein indeed the addition of such antibodies to normal skin induces the formation of blisters and disruption of cell adhesion.❚ Tight Junctions Seal Off Body Cavities and Restrict Diffusion of Membrane Components For polarized epithelial cells to carry out their functions as barriers and mediators of selective transport extracellular fluids surrounding their apical and basolateral membranes must be kept separate. The tight junctions between adjacent epithelial cells are usually located just below the apical sur- face and help establish and maintain cell polarity see Figures 6-1 and 6-5. These specialized regions of the plasma mem- brane form a barrier that seals off body cavities such as the intestine the stomach lumen the blood e.g. the blood–brain barrier and the bile duct in the liver. 206 CHAPTER 6 • Integrating Cells into Tissues a Tight junction Microvilli Microvilli Tight junction Linkage of protein particles in adjacent cells Intercellular space Rows of protein particles b 50 nm c N C Occludin N CC N JAM Claudin-1 FIGURE 6-9 Tight junctions. a Freeze-fracture preparation of tight junction zone between two intestinal epithelial cells. The fracture plane passes through the plasma membrane of one of the two adjacent cells. A honeycomb-like network of ridges and grooves below the microvilli constitutes the tight junction zone. b Schematic drawing shows how a tight junction might be formed by the linkage of rows of protein particles in adjacent cells. In the inset micrograph of an ultrathin sectional view of a tight junction the adjacent cells can be seen in close contact where the rows of proteins interact. c As shown in these schematic drawings of the major proteins in tight junctions both occludin and claudin-1 contain four transmembrane helices whereas the junction adhesion molecule JAM has a single transmembrane domain and a large extracellular region. See text for discussion. Part a courtesy of L. A. Staehelin. Drawing in part b adapted from L. A. Staehelin and B. E. Hull 1978 Sci. Am. 2385:140 and D. Goodenough 1999 Proc. Nat’l. Acad. Sci. USA 96:319. Photograph in part b courtesy of S. Tsukita et al. 2001 Nature Rev. Mol. Cell Biol. 2:285. Drawing in part c adapted from S. Tsukita et al. 2001 Nature Rev. Mol. Cell Biol. 2:285.

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Tight junctions prevent the diffusion of macromolecules and to varying degrees impede the diffusion of small water- soluble molecules and ions across an epithelial sheet in the spaces between cells. They also maintain the polarity of ep- ithelial cells by preventing the diffusion of membrane pro- teins and glycolipids lipids with covalently attached sugars between the apical and the basolateral regions of the plasma membrane ensuring that these regions contain different membrane components. As a consequence movement of many nutrients across the intestinal epithelium is in large part through the transcellular pathway. In this pathway spe- cific transport proteins in the apical membrane import small molecules from the intestinal lumen into cells other trans- port proteins located in the basolateral membrane then ex- port these molecules into the extracellular space. Such transcellular transport is covered in detail in Chapter 7. Tight junctions are composed of thin bands of plasma- membrane proteins that completely encircle a polarized cell and are in contact with similar thin bands on adjacent cells. When thin sections of cells are viewed in an electron micro- scope the lateral surfaces of adjacent cells appear to touch each other at intervals and even to fuse in the zone just below the apical surface see Figure 6-5b. In freeze-fracture prepa- rations tight junctions appear as an interlocking network of ridges in the plasma membrane Figure 6-9a. More specifi- cally there appear to be ridges on the cytosolic face of the plasma membrane of each of the two contacting cells. Cor- responding grooves are found on the exoplasmic face. Very high magnification reveals that rows of protein par- ticles 3–4 nm in diameter form the ridges seen in freeze- fracture micrographs of tight junctions. In the model shown in Figure 6-9b the tight junction is formed by a double row of these particles one row donated by each cell. The two principal integral-membrane proteins found in tight junc- tions are occludin and claudin. Initially investigators thought that occludin was the only essential protein compo- nent of tight junctions. However when investigators engi- neered mice with mutations inactivating the occludin gene the mice still had morphologically distinct tight junctions. This technique called gene knockout is described in Chap- ter 9. Further analysis led to the discovery of claudin. Each of these proteins has four membrane-spanning helices Fig- ure 6-9c. The claudin multigene family encodes numerous homologous proteins isoforms that exhibit distinct tissue- specific patterns of expression. Recently a group of junction adhesion molecules JAMs have been found to contribute to homophilic adhesion and other functions of tight junc- tions. These molecules which contain a single transmem- brane helix belong to the Ig superfamily of CAMs. The extracellular domains of rows of occludin claudin and JAM proteins in the plasma membrane of one cell apparently form extremely tight links with similar rows of the same proteins in an adjacent cell creating a tight seal. Treatment of an ep- ithelium with the protease trypsin destroys the tight junc- tions supporting the proposal that proteins are essential structural components of these junctions. The long C-terminal cytosolic segment of occludin binds to PDZ domains in certain large cytosolic adapter proteins. These domains are found in various cytosolic proteins and mediate binding to the C-termini of particular plasma- membrane proteins. PDZ-containing adapter proteins asso- ciated with occludin are bound in turn to other cytoskele- tal and signaling proteins and to actin fibers. These interactions appear to stabilize the linkage between occludin and claudin molecules that is essential for maintaining the in- tegrity of tight junctions. A simple experiment demonstrates the impermeability of certain tight junctions to many water-soluble substances. In this experiment lanthanum hydroxide an electron-dense colloid of high molecular weight is injected into the pancre- atic blood vessel of an experimental animal a few minutes later the pancreatic acinar cells which are specialized ep- ithelial cells are fixed and prepared for microscopy. As shown in Figure 6-10 the lanthanum hydroxide diffuses from the blood into the space that separates the lateral sur- faces of adjacent acinar cells but cannot penetrate past the tight junction. The importance of Ca 2 to the formation and integrity of tight junctions has been demonstrated in studies with MDCK cells in the experimental system described previously see Figure 6-7. If the growth medium in the chamber contains very low concentrations of Ca 2 MDCK cells form a mono- layer in which the cells are not connected by tight junctions. As a result fluids and salts flow freely across the cell layer. When sufficient Ca 2 is added to the medium tight junctions form within an hour and the cell layer becomes impermeable 6.2 • Sheetlike Epithelial Tissues: Junctions and Adhesion Molecules 207 Apical surface of left cell Lanthanum hydroxide between cells Apical surface of right cell Tight junction Lateral surface of right cell Lateral surface of left cell ▲ EXPERIMENTAL FIGURE 6-10 Tight junctions prevent passage of large molecules through extracellular space between epithelial cells. This experiment described in the text demonstrates the impermeability of tight junctions in the pancreas to the large water-soluble colloid lanthanum hydroxide. Courtesy of D. Friend.

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to fluids and salts. Thus Ca 2 is required for the formation of tight junctions as well as for cell–cell adhesion mediated by cadherins. Plasma-membrane proteins cannot diffuse in the plane of the membrane past tight junctions. These junctions also re- strict the lateral movement of lipids in the exoplasmic leaflet of the plasma membrane in the apical and basolateral regions of epithelial cells. Indeed the lipid compositions of the exo- plasmic leaflet in these two regions are distinct. Essentially all glycolipids are present in the exoplasmic face of the apical membrane as are all proteins linked to the membrane by a glycosylphosphatidylinositol GPI anchor see Figure 5-15. In contrast lipids in the cytosolic leaflet in the apical and ba- solateral regions of epithelial cells have the same composi- tion and can apparently diffuse laterally from one region of the membrane to the other. Differences in Permeability of Tight Junctions Can Control Passage of Small Molecules Across Epithelia The barrier to diffusion provided by tight junctions is not ab- solute. Owing at least in part to the varying properties of the different isoforms of claudin located in different tight junc- tions their permeability to ions small molecules and water varies enormously among different epithelial tissues. In ep- ithelia with “leaky” tight junctions small molecules can move from one side of the cell layer to the other through the paracellular pathway in addition to the transcellular path- way Figure 6-11. The leakiness of tight junctions can be altered by intra- cellular signaling pathways especially G protein–coupled pathways entailing cyclic AMP and protein kinase C Chap- ter 13. The regulation of tight junction permeability is often studied by measuring ion flux electrical resistance or the movement of radioactive or fluorescent molecules across monolayers of MDCK cells. The importance of paracellular transport is illus- trated in several human diseases. In hereditary hy- pomagnesemia defects in the claudin16 gene prevent the normal paracellular flow of magnesium through tight junctions in the kidney. This results in an abnormally low blood level of magnesium which can lead to convul- sions. Furthermore a mutation in the claudin14 gene causes hereditary deafness apparently by altering transport around hair cells in the cochlea of the inner ear. Toxins produced by Vibrio cholerae which causes cholera and several other enteric gastrointestinal tract bacteria alter the permeability barrier of the intestinal ep- ithelium by altering the composition or activity of tight junc- tions. Other bacterial toxins can affect the ion-pumping activity of membrane transport proteins in intestinal epithe- lial cells. Toxin-induced changes in tight junction permeabil- ity increased paracellular transport and in protein-mediated ion-pumping proteins increased transcellular transport can result in massive loss of internal body ions and water into the gastrointestinal tract which in turn leads to diarrhea and po- tentially lethal dehydration.❚ Many Cell–Matrix and Some Cell–Cell Interactions Are Mediated by Integrins The integrin family comprises heterodimeric integral mem- brane proteins that function as adhesion receptors mediat- ing many cell–matrix interactions see Figure 6-2. In vertebrates at least 24 integrin heterodimers composed of 18 types of subunits and 8 types of subunits in various combinations are known. A single chain can interact with any one of multiple chains forming integrins that bind dif- ferent ligands. This phenomenon of combinatorial diversity which is found throughout the biological world allows a rel- atively small number of components to serve a large num- ber of distinct functions. In epithelial cells integrin 6 4 is concentrated in hemidesmosomes and plays a major role in adhering cells to matrix in the underlying basal lamina as discussed in detail in Section 6.3. Some integrins particularly those expressed by certain blood cells participate in heterophilic cell–cell interactions. The members of this large family play impor- tant roles in adhesion and signaling in both epithelial and nonepithelial tissues. Integrins typically exhibit low affinities for their ligands with dissociation constants K D between 10 6 and 10 8 mol/L. However the multiple weak interactions generated by the binding of hundreds or thousands of integrin molecules to their ligands on cells or in the extracellular matrix allow a cell to remain firmly anchored to its ligand-expressing target. Moreover the weakness of individual integrin-mediated in- teractions facilitates cell migration. 208 CHAPTER 6 • Integrating Cells into Tissues Basolateral membrane Transcellular pathway Tight junction Paracellular pathway Apical membrane ▲ FIGURE 6-11 Transcellular and paracellular pathways of transepithelial transport. Transcellular transport requires the cellular uptake of molecules on one side and subsequent release on the opposite side by mechanisms discussed in Chapters 7 and 17 . In paracellular transport molecules move extracellularly through parts of tight junctions whose permeability to small molecules and ions depends on the composition of the junctional components and the physiologic state of the epithelial cells. Adapted from S. Tsukita et al. 2001 Nature Rev. Mol. Cell Biol. 2:285.

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Parts of both the and the subunits of an integrin molecule contribute to the primary extracellular ligand- binding site see Figure 6-2. Ligand binding to integrins also requires the simultaneous binding of divalent cations positively charged ions. Like other cell-surface adhesive molecules the cytosolic region of integrins interacts with adapter proteins that in turn bind to the cytoskeleton and intracellular signaling molecules. Although most integrins are linked to the actin cytoskeleton the cytosolic domain of the 4 chain in the 6 4 integrin in hemidesmosomes which is much longer than those of other integrins binds to specialized adapter proteins e.g. plectin that in turn interact with keratin-based intermediate filaments. In addition to their adhesion function integrins can me- diate outside-in and inside-out transfer of information sig- naling. In outside-in signaling the engagement of integrins with their extracellular ligands can through adapter proteins bound to the integrin cytosolic region influence the cy- toskeleton and intracellular signaling pathways. Conversely in inside-out signaling intracellular signaling pathways can alter from the cytoplasm the structure of integrins and con- sequently their abilities to adhere to their extracellular lig- ands and mediate cell–cell and cell–matrix interactions. Integrin-mediated signaling pathways influence processes as diverse as cell survival cell proliferation and programmed cell death Chapter 22. Many cells express several different integrins that bind the same ligand. By selectively regulating the activity of each type of integrin these cells can fine-tune their cell–cell and cell–matrix interactions and the associated signaling processes. We will consider various integrins and the regulation of their activity in detail in Section 6.5. KEY CONCEPTS OF SECTION 6.2 Sheetlike Epithelial Tissues: Junctions and Adhesion Molecules ■ Polarized epithelial cells have distinct apical basal and lateral surfaces. Microvilli projecting from the apical sur- faces of many epithelial cells considerably expand their sur- face areas. ■ Three major classes of cell junctions—anchoring junc- tions tight junctions and gap junctions—assemble ep- ithelial cells into sheets and mediate communication be- tween them see Figures 6-1 and 6-5. ■ Adherens junctions and desmosomes are cadherin- containing anchoring junctions that bind the membranes of adjacent cells giving strength and rigidity to the entire tissue. Hemidesmosomes are integrin-containing anchoring junctions that attach cells to elements of the underlying ex- tracellular matrix. ■ Cadherins are cell-adhesion molecules CAMs respon- sible for Ca 2 -dependent interactions between cells in epithelial and other tissues. They promote strong cell– cell adhesion by mediating both lateral and intercellular interactions. ■ Adapter proteins that bind to the cytosolic domain of cadherins and other CAMs mediate the association of cytoskeletal and signaling molecules with the plasma membrane see Figure 6-9. Strong cell–cell adhesion depends on the linkage of the interacting CAMs to the cytoskeleton. ■ Tight junctions block the diffusion of proteins and some lipids in the plane of the plasma membrane contributing to the polarity of epithelial cells. They also limit and reg- ulate the extracellular paracellular flow of water and solutes from one side of the epithelium to the other see Figure 6-11. ■ Integrins are a large family of heterodimeric cell- surface proteins that mediate both cell–cell and cell– matrix adhesions and inside-out and outside-in signaling in numerous tissues. The Extracellular Matrix of Epithelial Sheets In animals the extracellular matrix helps organize cells into tissues and coordinates their cellular functions by ac- tivating intracellular signaling pathways that control cell growth proliferation and gene expression. Many func- tions of the matrix require transmembrane adhesion re- ceptors that bind directly to ECM components and that also interact through adapter proteins with the cy- toskeleton. The principal class of adhesion receptors that mediate cell–matrix adhesion are integrins which were in- troduced in Section 6.2. However other types of molecules also function as important adhesion receptors in some nonepithelial tissues. Three types of molecules are abundant in the extracellu- lar matrix of all tissues. ■ Highly viscous proteoglycans a group of glycoproteins that cushion cells and bind a wide variety of extracellular molecules ■ Collagen fibers which provide mechanical strength and resilience ■ Soluble multiadhesive matrix proteins which bind to and cross-link cell-surface adhesion receptors and other ECM components We begin our description of the structures and functions of these major ECM components in this section focusing on the molecular components and organization of the basal lamina—the specialized extracellular matrix that helps de- termine the overall architecture of an epithelial tissue. In Sec- tion 6.4 we extend our discussion to specific ECM molecules that are commonly present in nonepithelial tissues. 6.3 6.3 • The Extracellular Matrix of Epithelial Sheets 209

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The Basal Lamina Provides a Foundation for Epithelial Sheets In animals epithelia and most organized groups of cells are underlain or surrounded by the basal lamina a sheetlike meshwork of ECM components usually no more than 60–120 nm thick Figure 6-12 see also Figures 6-1 and 6-4. The basal lamina is structured differently in different tissues. In columnar and other epithelia e.g. intestinal lining skin it is a foundation on which only one surface of the cells rests. In other tissues such as muscle or fat the basal lamina surrounds each cell. Basal laminae play im- portant roles in regeneration after tissue damage and in em- bryonic development. For instance the basal lamina helps 210 CHAPTER 6 • Integrating Cells into Tissues Cell-surface receptor proteins Collagen fibers Basal lamina Connective tissue Basal surface Cytosol a b Plasma membrane Basal lamina ▲ EXPERIMENTAL FIGURE 6-12 The basal lamina separates epithelial cells and some other cells from connective tissue. a Transmission electron micrograph of a thin section of cells top and underlying connective tissue bottom. The electron-dense layer of the basal lamina can be seen to follow the undulation of the basal surface of the cells. b Electron micrograph of a quick-freeze deep-etch preparation of skeletal muscle showing the relation of the plasma membrane basal lamina and surrounding connective tissue. In this preparation the basal lamina is revealed as a meshwork of filamentous proteins that associate with the plasma membrane and the thicker collagen fibers of the connective tissue. Part a courtesy of P . FitzGerald. Part b from D. W. Fawcett 1981 The Cell 2d ed. Saunders/Photo Researchers courtesy of John Heuser. Type IV collagen Perlecan Laminin Entactin FIGURE 6-13 Major components of the basal lamina. Schematic model of basal lamina showing the organization of the major protein components. Type IV collagen and laminin each form two-dimensional networks which are cross-linked by entactin and perlecan molecules. Adapted from B. Alberts et al. 1994 Molecular Biology of the Cell 3d ed. Garland p. 991.

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four- and eight-celled embryos adhere together in a ball. In the development of the nervous system neurons migrate along ECM pathways that contain basal lamina components. Thus the basal lamina is important not only for organizing cells into tissues but also for tissue repair and for guiding mi- grating cells during tissue formation. Most of the ECM components in the basal lamina are synthesized by the cells that rest on it. Four ubiquitous pro- tein components are found in basal laminae Figure 6-13: ■ Type IV collagen trimeric molecules with both rodlike and globular domains that form a two-dimensional network ■ Laminins a family of multiadhesive proteins that form a fibrous two-dimensional network with type IV collagen and that also bind to integrins ■ Entactin also called nidogen a rodlike molecule that cross-links type IV collagen and laminin and helps incorporate other components into the ECM ■ Perlecan a large multidomain proteoglycan that binds to and cross-links many ECM components and cell-surface molecules As depicted in Figure 6-1 one side of the basal lamina is linked to cells by adhesion receptors including 6 4 integrin that binds to laminin in the basal lamina. The other side of the basal lamina is anchored to the adjacent connective tissue by a layer of fibers of collagen embedded in a proteoglycan- rich matrix. In stratified squamous epithelia e.g. skin this linkage is mediated by anchoring fibrils of type VII collagen. Together the basal lamina and this collagen-containing layer see the micrograph on page 197 form the structure called the basement membrane. Sheet-Forming Type IV Collagen Is a Major Structural Component in Basal Laminae Type IV collagen the principal component of all basal lam- ina is one of more than 20 types of collagen that participate in the formation of the extracellular matrix in various tis- sues. Although they differ in certain structural features and tissue distribution all collagens are trimeric proteins made from three polypeptides called collagen chains. All three chains can be identical homotrimeric or different het- erotrimeric. A trimeric collagen molecule contains one or more three-stranded segments each with a similar triple- helical structure Figure 6-14a. Each strand contributed by one of the chains is twisted into a left-handed helix and three such strands from the three chains wrap around each other to form a right-handed triple helix. The collagen triple helix can form because of an un- usual abundance of three amino acids: glycine proline and a modified form of proline called hydroxyproline see Figure 3-12. They make up the characteristic re- peating motif Gly-X-Y where X and Y can be any amino acid but are often proline and hydroxyproline and less often lysine and hydroxylysine. Glycine is essential be- cause its small side chain a hydrogen atom is the only one that can fit into the crowded center of the three- stranded helix Figure 6-14b. Hydrogen bonds help hold the three chains together. Although the rigid peptidyl- proline and peptidyl-hydroxyproline linkages are not compatible with formation of a classic single-stranded helix they stabilize the distinctive three-stranded colla- gen helix. The hydroxyl group in hydroxyproline helps hold its ring in a conformation that stabilizes the three- stranded helix. The unique properties of each type of collagen are due mainly to differences in 1 the number and lengths of the collagenous triple-helical segments 2 the segments that flank or interrupt the triple-helical segments and that fold into other kinds of three-dimensional structures and 3 the covalent modification of the chains e.g. hydroxylation glycosylation oxidation cross-linking. For example the chains in type IV collagen which is unique to basal laminae are designated IV chains. Mammals express six homolo- gous IV chains which assemble into a series of type IV 6.3 • The Extracellular Matrix of Epithelial Sheets 211 a b ▲ FIGURE 6-14 The collagen triple helix. a Left Side view of the crystal structure of a polypeptide fragment whose sequence is based on repeating sets of three amino acids Gly-X- Y characteristic of collagen chains. Center Each chain is twisted into a left-handed helix and three chains wrap around each other to form a right-handed triple helix. The schematic model right clearly illustrates the triple helical nature of the structure. b View down the axis of the triple helix. The proton side chains of the glycine residues orange point into the very narrow space between the polypeptide chains in the center of the triple helix. In mutations in collagen in which other amino acids replace glycine the proton in glycine is replaced by larger groups that disrupt the packing of the chains and destablize the triple-helical structure. Adapted from R. Z. Kramer et al. 2001 J. Mol. Biol. 3111:131 .

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collagens with distinct properties. All subtypes of type IV collagen however form a 400-nm-long triple helix that is in- terrupted about 24 times with nonhelical segments and flanked by large globular domains at the C-termini of the chains and smaller globular domains at the N-termini. The nonhelical regions introduce flexibility into the molecule. Through both lateral associations and interactions entailing the globular N- and C-termini type IV collagen molecules assemble into a branching irregular two-dimensional fibrous network that forms the lattice on which the basal lamina is built Figure 6-15. In the kidney a double basal lamina the glomeru- lar basement membrane separates the epithelium that lines the urinary space from the endothelium that lines the surrounding blood-filled capillaries. Defects in this structure which is responsible for ultrafiltration of the blood and initial urine formation can lead to renal failure. For instance mutations that alter the C-terminal globular domain of certain IV chains are associated with progres- sive renal failure as well as sensorineural hearing loss and ocular abnormalities a condition known as Alport’s syndrome. In Goodpasture’s syndrome a relatively rare autoimmune disease self-attacking or “auto” antibodies bind to the 3 chains of type IV collagen found in the glomerular basement membrane and lungs. This binding sets off an immune response that causes cellular damage resulting in progressive renal failure and pulmonary hemorrhage.❚ Laminin a Multiadhesive Matrix Protein Helps Cross-link Components of the Basal Lamina Multiadhesive matrix proteins are long flexible molecules that contain multiple domains responsible for binding vari- ous types of collagen other matrix proteins polysaccharides cell-surface adhesion receptors and extracellular signaling molecules e.g. growth factors and hormones. These pro- teins are important for organizing the other components of the extracellular matrix and for regulating cell–matrix ad- hesion cell migration and cell shape in both epithelial and nonepithelial tissues. Laminin the principal multiadhesive matrix protein in basal laminae is a heterotrimeric cross-shaped protein with a total molecular weight of 820000 Figure 6-16. Many laminin isoforms containing slightly different polypeptide chains have been identified. Globular LG domains at the C- terminus of the laminin subunit mediate Ca 2 -dependent binding to specific carbohydrates on certain cell-surface molecules such as syndecan and dystroglycan. LG domains are found in a wide variety of proteins and can mediate binding to steroids and proteins as well as carbohydrates. For example LG domains in the chain of laminin can me- diate binding to certain integrins including 6 4 integrin on epithelial cells. 212 CHAPTER 6 • Integrating Cells into Tissues Triple helical C-terminal globular domain N-terminal globular domain Collagen IV monomer Association Dimer Tetramer a 250 nm b Type IV network Nonhelical ▲ FIGURE 6-15 Structure and assembly of type IV collagen. a Schematic representation of type IV collagen. This 400-nm- long molecule has a small noncollagenous globular domain at the N-terminus and a large globular domain at the C-terminus. The triple helix is interrupted by nonhelical segments that introduce flexible kinks in the molecule. Lateral interactions between triple helical segments as well as head-to-head and tail-to-tail interactions between the globular domains form dimers tetramers and higher-order complexes yielding a sheetlike network. b Electron micrograph of type IV collagen network formed in vitro. The lacy appearance results from the flexibility of the molecule the side-to-side binding between triple-helical segments thin arrows and the interactions between C-terminal globular domains thick arrows. Part a adapted from A. Boutaud 2000 J. Biol. Chem. 275:30716. Part b courtesy of P . Yurchenco see P . Yurchenco and G. C. Ruben 1987 J. Cell Biol. 105:2559.

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Secreted and Cell-Surface Proteoglycans Are Expressed by Many Cell Types Proteoglycans are a subset of glycoproteins containing co- valently linked specialized polysaccharide chains called glycosaminoglycans GAGs which are long linear polymers of specific repeating disaccharides. Usually one sugar is ei- ther a uronic acid D-glucuronic acid or L-iduronic acid or D-galactose the other sugar is N-acetylglucosamine or N-acetylgalactosamine Figure 6-17. One or both of the sug- ars contain at least one anionic group carboxylate or sul- fate. Thus each GAG chain bears many negative charges. 6.3 • The Extracellular Matrix of Epithelial Sheets 213 Binds sulfated lipids Binds type IV collagen β Chain 215000 MW α Chain 400000 MW γ Chain 205000 MW α-Helical coiled coil Binds neurites LG domains bind carbohydrates and integrins Binds collagen sulfated lipids 25 nm a b 10 nm 50 nm ▲ FIGURE 6-16 Laminin a heterotrimeric multiadhesive matrix protein found in all basal laminae. a Schematic model showing the general shape location of globular domains and coiled-coil region in which laminin’s three chains are covalently linked by several disulfide bonds. Different regions of laminin bind to cell-surface receptors and various matrix components. b Electron micrographs of intact laminin molecule showing its characteristic cross appearance left and the carbohydrate- binding LG domains near the C-terminus right. Part a adapted from G. R. Martin and R. Timpl 1987 Ann. Rev. Cell Biol. 3:57 and K. Y amada 1991 J. Biol. Chem. 266:12809. Part b from R. Timpl et al. 2000 Matrix Biol. 19:309 photograph at right courtesy of Jürgen Engel. D-Glucuronic acid or L-iduronic acid COO − CH 2 OH O OH OH HO O NHCOCH 3 O O n 4 1 5 32 β1→4 D-Glucuronic acid N-Acetyl- D-glucosamine a Hyaluronan n 25000 ∼ COO − CH 2 OH O OH OH HO O NHCOCH 3 O O n SO 3 − SO 3 − N-Acetyl- D-galactosamine COO − CH 2 OH O OH O NHSO 3 − O SO 3 − COCH 3 SO 3 − α1→4 c Heparin/Heparan sulfate n 200 D-Glucuronic or L-iduronic acid N-Acetyl- or N-sulfo- D-glucosamine OH OH O O O D-Galactose N-Acetyl- D-glucosamine CH 2 OH OH O NHCOCH 3 SO 3 − β1→3 d Keratan sulfate n 20–40 HO CH 2 OH O O O OH b Chondroitin or dermatan sulfate n 250 ∼ 6 α/β1→4 α/β1→3 β1→3 β1→4 β1→4 SO 3 − O O n n ▲ FIGURE 6-17 The repeating disaccharides of glycosaminoglycans GAGs the polysaccharide components of proteoglycans. Each of the four classes of GAGs is formed by polymerization of monomer units into repeats of a particular disaccharide and subsequent modifications including addition of sulfate groups and inversion epimerization of the carboxyl group on carbon 5 of D-glucuronic acid to yield L-iduronic acid. Heparin is generated by hypersulfation of heparan sulfate whereas hyaluronan is unsulfated. The number n of disaccharides typically found in each glycosaminoglycan chain is given. The squiggly lines represent covalent bonds that are oriented either above D-glucuronic acid or below L-iduronic acid the ring.

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GAGs are classified into several major types based on the na- ture of the repeating disaccharide unit: heparan sulfate chondroitin sulfate dermatan sulfate keratan sulfate and hyaluronan. A hypersulfated form of heparan sulfate called heparin produced mostly by mast cells plays a key role in allergic reactions. It is also used medically as an anticlotting drug because of its ability to activate a natural clotting in- hibitor called antithrombin III. As we will see in later chapters complex signaling path- ways direct the emergence of various cell types in the proper position and at the proper time in normal embryonic develop- ment. Laboratory generation and analysis of mutants with de- fects in proteoglycan production in Drosophila melanogaster fruit fly C. elegans roundworm and mice have clearly shown that proteoglycans play critical roles in development most likely as modulators of various signaling pathways. Biosynthesis of Proteoglycans With the exception of hyaluronan which is discussed in the next section all the major GAGs occur naturally as components of proteogly- cans. Like other secreted and transmembrane glycoproteins proteoglycan core proteins are synthesized on the endoplas- mic reticulum Chapter 16. The GAG chains are assembled on these cores in the Golgi complex. To generate heparan or chondroitin sulfate chains a three-sugar “linker” is first at- tached to the hydroxyl side chains of certain serine residues in a core protein Figure 6-18. In contrast the linkers for the addition of keratan sulfate chains are oligosaccharide chains attached to asparagine residues such N-linked oligosaccha- rides are present in most glycoproteins although only a sub- set carry GAG chains. All GAG chains are elongated by the alternating addition of sugar monomers to form the disac- charide repeats characteristic of a particular GAG the chains are often modified subsequently by the covalent linkage of small molecules such as sulfate. The mechanisms responsi- ble for determining which proteins are modified with GAGs the sequence of disaccharides to be added the sites to be sul- fated and the lengths of the GAG chains are unknown. The ratio of polysaccharide to protein in all proteoglycans is much higher than that in most other glycoproteins. Diversity of Proteoglycans The proteoglycans constitute a remarkably diverse group of molecules that are abundant in the extracellular matrix of all animal tissues and are also ex- pressed on the cell surface. For example of the five major classes of heparan sulfate proteoglycans three are located in the extracellular matrix perlecan agrin and type XVIII collagen and two are cell-surface proteins. The latter include integral membrane proteins syndecans and GPI-anchored proteins glypicans the GAG chains in both types of cell- surface proteoglycans extend into the extracellular space. The sequences and lengths of proteoglycan core proteins vary considerably and the number of attached GAG chains ranges from just a few to more than 100. Moreover a core protein is often linked to two different types of GAG chains e.g. heparan sulfate and chondroitin sulfate generating a “hybrid” proteoglycan. Thus the molecular weight and charge density of a population of proteoglycans can be ex- pressed only as an average the composition and sequence of individual molecules can differ considerably. Perlecan the major secreted proteoglycan in basal lami- nae consists of a large multidomain core protein ≈400 kDa with three or four specialized GAG chains. Both the protein and the GAG components of perlecan contribute to its abil- ity to incorporate into and define the structure and function of basal laminae. Because of its multiple domains with dis- tinctive binding properties perlecan can cross-link not only ECM components to one another but also certain cell- surface molecules to ECM components. Syndecans are expressed by epithelial cells and many other cell types. These cell-surface proteoglycans bind to collagens and multiadhesive matrix proteins such as the fibronectins which are discussed in Section 6.4. In this way cell-surface proteoglycans can anchor cells to the extracellular matrix. Like that of many integral membrane proteins the cytosolic domain of syndecan interacts with the actin cytoskeleton and in some cases with intracellular regulatory molecules. In ad- dition cell-surface proteoglycans bind many protein growth factors and other external signaling molecules thereby helping to regulate cellular metabolism and function. For instance syndecans in the hypothalamic region of the brain modulate feeding behavior in response to food deprivation fasted state. They do so by participating in the binding of antisatiety pep- tides to cell-surface receptors that help control feeding behav- ior. In the fed state the syndecan extracellular domain decorated with heparan sulfate chains is released from the sur- face by proteolysis thus suppressing the activity of the anti- satiety peptides and feeding behavior. In mice engineered to overexpress the syndecan-1 gene in the hypothalamic region of the brain and other tissues normal control of feeding by anti- satiety peptides is disrupted and the animals overeat and be- come obese. Other examples of proteoglycans interacting with external signaling molecules are described in Chapter 14. 214 CHAPTER 6 • Integrating Cells into Tissues GlcUA Gal Gal Ser Xyl GlcUA glucuronic acid Xyl xylose Core protein SO 4 GlcUA GalNAc n Chondroitin sulfate repeats Gal galactose GalNAc N-acetylgalactosamine Linking sugars ▲ FIGURE 6-18 Biosynthesis of heparan and chondroitin sulfate chains in proteoglycans. Synthesis of a chondroitin sulfate chain shown here is initiated by transfer of a xylose residue to a serine residue in the core protein most likely in the Golgi complex followed by sequential addition of two galactose residues. Glucuronic acid and N-acetylgalactosamine residues are then added sequentially to these linking sugars forming the chondroitin sulfate chain. Heparan sulfate chains are connected to core proteins by the same three-sugar linker.

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Modifications in Glycosaminoglycan GAG Chains Can Determine Proteoglycan Functions As is the case with the sequence of amino acids in proteins the arrangement of the sugar residues in GAG chains and the modification of specific sugars e.g. addition of sulfate in the chains can determine their function and that of the pro- teoglycans containing them. For example groupings of cer- tain modified sugars in the GAG chains of heparin sulfate proteoglycans can control the binding of growth factors to certain receptors the activities of proteins in the blood- clotting cascade and the activity of lipoprotein lipase a membrane-associated enzyme that hydrolyzes triglycerides to fatty acids Chapter 18. For years the chemical and structural complexity of proteoglycans posed a daunting barrier to an analysis of their structures and an understanding of their many diverse functions. In recent years investigators employing classi- cal and new state-of-the-art biochemical techniques e.g. capillary high-pressure liquid chromatography mass spec- trometry and genetics have begun to elucidate the detailed structures and functions of these ubiquitous ECM mole- cules. The results of ongoing studies suggest that sets of sugar-residue sequences containing some modifications in common rather than single unique sequences are respon- sible for specifying distinct GAG functions. A case in point is a set of five-residue pentasaccharide sequences found in a subset of heparin GAGs that control the activity of antithrombin III ATIII an inhibitor of the key blood- clotting protease thrombin. When these pentasaccharide se- quences in heparin are sulfated at two specific positions heparin can activate ATIII thereby inhibiting clot forma- tion Figure 6-19. Several other sulfates can be present in the active pentasaccharide in various combinations but they are not essential for the anticlotting activity of he- parin. The rationale for generating sets of similar active sequences rather than a single unique sequence and the mechanisms that control GAG biosynthetic pathways per- mitting the generation of such active sequences are not well understood. KEY CONCEPTS OF SECTION 6.3 The Extracellular Matrix of Epithelial Sheets ■ The basal lamina a thin meshwork of extracellular ma- trix ECM molecules separates most epithelia and other organized groups of cells from adjacent connective tissue. Together the basal lamina and collagenous reticular lam- ina form a structure called the basement membrane. ■ Four ECM proteins are found in all basal laminae see Fig- ure 6-13: type IV collagen laminin a multiadhesive matrix protein entactin nidogen and perlecan a proteoglycan. ■ Cell-surface adhesion receptors e.g. 6 4 integrin in hemidesmosomes anchor cells to the basal lamina which in turn is connected to other ECM components see Figure 6-1. ■ Repeating sequences of Gly-X-Y give rise to the colla- gen triple-helical structure see Figure 6-14. Different col- lagens are distinguished by the length and chemical mod- ifications of their chains and by the segments that interrupt or flank their triple-helical regions. ■ The large flexible molecules of type IV collagen inter- act end to end and laterally to form a meshlike scaffold to which other ECM components and adhesion receptors can bind see Figure 6-15. ■ Laminin and other multiadhesive matrix proteins are multidomain molecules that bind multiple adhesion recep- tors and ECM components. ■ Proteoglycans consist of membrane-associated or se- creted core proteins covalently linked to one or more gly- cosaminoglycan GAG chains which are linear polymers of sulfated disaccharides. ■ Perlecan a large secreted proteoglycan present primarily in the basal lamina binds many ECM components and ad- hesion receptors. ■ Cell-surface proteoglycans such as the syndecans facili- tate cell–matrix interactions and help present certain ex- ternal signaling molecules to their cell-surface receptors. 6.3 • The Extracellular Matrix of Epithelial Sheets 215 O HO HO O RHN R Ac or SO 3 OSO 3 HO OH O O O O 3 SO OOC O O O OH OSO 3 O 3 SHN O 3 SO OOC O HO O OH OSO 3 O 3 SHN FIGURE 6-19 Pentasaccharide GAG sequence that regulates the activity of antithrombin III ATIII. Sets of modified five-residue sequences in heparin with the composition shown here bind to ATIII and activate it thereby inhibiting blood clotting. The sulfate groups in red type are essential for this heparin function the modifications in blue type may be present but are not essential. Other sets of modified GAG sequences are thought to regulate the activity of other target proteins. Courtesy of Robert Rosenberg and Balagurunathan Kuberan.

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The Extracellular Matrix of Nonepithelial Tissues We have seen how diverse CAMs and adhesion receptors participate in the assembly of animal cells into epithelial sheets that rest on and adhere to a well-defined ECM struc- ture the basal lamina. The same or similar molecules me- diate and control cell–cell and cell–matrix interactions in 6.4 connective muscle and neural tissues and between blood cells and the surrounding vessels. In this section we con- sider some of the ECM molecules characteristic of these nonepithelial tissues. We also describe the synthesis of fib- rillar collagens which are the most abundant proteins in animals. The interactions entailing CAMs and adhesion receptors expressed by various nonepithelial cells which serve a wide variety of distinctive functions are covered in Section 6.5. 216 CHAPTER 6 • Integrating Cells into Tissues TABLE 6-1 Selected Collagens Molecule Type Composition Structural Features Representative Tissues FIBRILLAR COLLAGENS I 1I 2 2I 300-nm-long fibrils Skin tendon bone liga- ments dentin interstitial tissues II 1II 3 300-nm-long fibrils Cartilage vitreous humor III 1III 3 300-nm-long fibrils often with type I Skin muscle blood vessels V 1V 2 2V 390-nm-long fibrils with globular Cornea teeth bone 1V 3 N-terminal extension often with type I placenta skin smooth muscle FIBRIL-ASSOCIATED COLLAGENS VI 1VI 2VI Lateral association with type I periodic Most interstitial tissues globular domains IX 1IX 2IX 3IX Lateral association with type II Cartilage vitreous humor N-terminal globular domain bound GAG SHEET-FORMING AND ANCHORING COLLAGENS IV 1IV 2 2IV Two-dimensional network All basal laminae VII 1VII 3 Long fibrils Below basal lamina of the skin XV 1XV 3 Core protein of chondroitin sulfate Widespread near basal proteoglycan lamina in muscle TRANSMEMBRANE COLLAGENS XIII 1XIII 3 Integral membrane protein Hemidesmosomes in skin XVII 1XVII 3 Integral membrane protein Hemidesmosomes in skin HOST DEFENSE COLLAGENS Collectins Oligomers of triple helix lectin domains Blood alveolar space C1q Oligomers of triple helix Blood complement Class A scavenger Homotrimeric membrane proteins Macrophages receptors SOURCES: K. Kuhn 1987 in R. Mayne and R. Burgeson eds. Structure and Function of Collagen Types Academic Press p. 2 and M. van der Rest and R. Garrone 1991 FASEB J. 5:2814.

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Fibrillar Collagens Are the Major Fibrous Proteins in the Extracellular Matrix of Connective Tissues Connective tissue such as tendon and cartilage differs from other solid tissues in that most of its volume is made up of extracellular matrix rather than cells. This matrix is packed with insoluble protein fibers and contains proteoglycans various multiadhesive proteins and hyaluronan a very large nonsulfated GAG. The most abundant fibrous protein in connective tissue is collagen. Rubberlike elastin fibers which can be stretched and relaxed also are present in deformable sites e.g. skin tendons heart. As discussed later the fibronectins a family of multiadhesive matrix proteins form their own distinct fibrils in the matrix of some connective tis- sues. Although several types of cells are found in connective tissues the various ECM components are produced largely by cells called fibroblasts. About 80–90 percent of the collagen in the body consists of types I II and III collagens located primarily in connec- tive tissues. Because of its abundance in tendon-rich tissue such as rat tail type I collagen is easy to isolate and was the first collagen to be characterized. Its fundamental structural unit is a long 300-nm thin 1.5-nm-diameter triple helix consisting of two 1I chains and one 2I chain each pre- cisely 1050 amino acids in length see Figure 6-14. The triple-stranded molecules associate into higher-order poly- mers called collagen fibrils which in turn often aggregate into larger bundles called collagen fibers. The minor classes of collagen include fibril-associated col- lagens which link the fibrillar collagens to one another or to other ECM components sheet-forming and anchoring colla- gens which form two-dimensional networks in basal laminae type IV and connect the basal lamina in skin to the underly- ing connective tissue type VII transmembrane collagens which function as adhesion receptors and host defense collagens which help the body recognize and eliminate pathogens. Table 6-1 lists specific examples in the various classes of collagens. Interestingly several collagens e.g. types XVIII and XV function as core proteins in proteoglycans. Formation of Collagen Fibrils Begins in the Endoplasmic Reticulum and Is Completed Outside the Cell Collagen biosynthesis and secretion follow the normal pathway for a secreted protein which is described in detail in Chapters 16 and 17. The collagen chains are synthesized as longer precursors called pro- chains by ribosomes attached to the endoplasmic reticulum ER. The pro- chains undergo a series of covalent modifications and fold into triple-helical procolla- gen molecules before their release from cells Figure 6-20. 6.4 • The Extracellular Matrix of Nonepithelial Tissues 217 Cytosol Golgi complex Rough ER Collagen fibril 1 2 3 4 5 7 6 8 Fibril assembly and crosslinking Propeptide cleavage Lateral association Procollagen Hsp47 N OH OH OH N N N N N OH OH Propeptide α1 α1 α2 OH O O Collagen molecule Extracellular space Cross-striations 67 nm 250 nm 67 nm S−S FIGURE 6-20 Major events in biosynthesis of fibrillar collagens.Step : Procollagen chains are synthesized on ribosomes associated with the endoplasmic reticulum ER membrane and asparagine-linked oligosaccharides are added to the C-terminal propeptide. Step : Propeptides associate to form trimers and are covalently linked by disulfide bonds and selected residues in the Gly-X-Y triplet repeats are covalently modified certain prolines and lysines are hydroxylated galactose Gal or galactose-glucose hexagons is attached to some hydroxylysines prolines are cis → trans isomerized. Step : The modifications facilitate zipperlike formation stabilization of triple helices and binding by the chaperone protein Hsp47 Chapter 16 which may stabilize the helices or prevent premature aggregation of the trimers or both. Steps and : The folded procollagens are transported to and through the Golgi apparatus where some lateral association into small bundles takes place. The chains are then secreted step the N- and C-terminal propeptides are removed step and the trimers assemble into fibrils and are covalently cross-linked step . The 67-nm staggering of the trimers gives the fibrils a striated appearance in electron micrographs inset. Adapted from A. V. Persikov and B. Brodsky 2002 Proc. Nat’l. Acad. Sci. USA 993:1101–1103. 8 7 6 5 4 3 2 1

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After the secretion of procollagen from the cell extracel- lular peptidases e.g. bone morphogenetic protein-1 remove the N-terminal and C-terminal propeptides. In regard to fib- rillar collagens the resulting molecules which consist almost entirely of a triple-stranded helix associate laterally to gen- erate fibrils with a diameter of 50–200 nm. In fibrils adja- cent collagen molecules are displaced from one another by 67 nm about one-quarter of their length. This staggered array produces a striated effect that can be seen in electron micrographs of collagen fibrils see Figure 6-20 inset. The unique properties of the fibrous collagens e.g. types I II III are mainly due to the formation of fibrils. Short non-triple-helical segments at either end of the colla- gen chains are of particular importance in the formation of collagen fibrils. Lysine and hydroxylysine side chains in these segments are covalently modified by extracellular lysyl oxidases to form aldehydes in place of the amine group at the end of the side chain. These reactive aldehyde groups form covalent cross- links with lysine hydroxylysine and histidine residues in adja- cent molecules. These cross-links stabilize the side-by-side packing of collagen molecules and generate a strong fibril. The removal of the propeptides and covalent cross-linking take place in the extracellular space to prevent the potentially cata- strophic assembly of fibrils within the cell. The post-translational modifications of pro- chains are crucial for the formation of mature col- lagen molecules and their assembly into fibrils. De- fects in these modifications have serious consequences as ancient mariners frequently experienced. For example ascor- bic acid vitamin C is an essential cofactor for the hydroxy- lases responsible for adding hydroxyl groups to proline and lysine residues in pro- chains. In cells deprived of ascorbate as in the disease scurvy the pro- chains are not hydroxy- lated sufficiently to form stable triple-helical procollagen at normal body temperature and the procollagen that forms cannot assemble into normal fibrils. Without the structural support of collagen blood vessels tendons and skin become fragile. Because fresh fruit in the diet can supply sufficient vi- tamin C to support the formation of normal collagen early British sailors were provided with limes to prevent scurvy leading to their being called “limeys.” Rare mutations in lysyl hydroxylase genes cause Bruck syndrome and one form of Ehlers-Danlos syndrome. Both disorders are marked by connective-tissue defects although their clinical symptoms differ. ❚ Type I and II Collagens Form Diverse Structures and Associate with Different Nonfibrillar Collagens Collagens differ in their ability to form fibers and to organ- ize the fibers into networks. In tendons for instance long type I collagen fibrils are packed side by side in parallel bun- dles forming thick collagen fibers. Tendons connect muscles to bones and must withstand enormous forces. Because type I collagen fibers have great tensile strength tendons can be stretched without being broken. Indeed gram for gram type I collagen is stronger than steel. Two quantitatively minor fibrillar collagens type V and type XI coassemble into fibers with type I collagen thereby regulating the structures and properties of the fibers. Incorporation of type V collagen for example results in smaller-diameter fibers. Type I collagen fibrils are also used as the reinforcing rods in the construction of bone. Bones and teeth are hard and strong because they contain large amounts of dahllite a crystalline calcium- and phosphate-containing mineral. Most bones are about 70 percent mineral and 30 percent protein the vast majority of which is type I collagen. Bones form when certain cells chondrocytes and osteoblasts secrete col- lagen fibrils that are then mineralized by deposition of small dahllite crystals. In many connective tissues type VI collagen and proteo- glycans are noncovalently bound to the sides of type I fibrils and may bind the fibrils together to form thicker collagen fibers Figure 6-21a. Type VI collagen is unusual in that the molecule consists of a relatively short triple helix with glob- 218 CHAPTER 6 • Integrating Cells into Tissues Type-I collagen fibrils Type-VI collagen Type-IX collagen Kink Proteoglycan a Type-II collagen fibril Chondroitin sulfate b ▲ FIGURE 6-21 Interactions of fibrous collagens with nonfibrous fibril-associated collagens. a In tendons type I fibrils are all oriented in the direction of the stress applied to the tendon. Proteoglycans and type VI collagen bind noncovalently to fibrils coating the surface. The microfibrils of type VI collagen which contain globular and triple-helical segments bind to type I fibrils and link them together into thicker fibers. b In cartilage type IX collagen molecules are covalently bound at regular intervals along type II fibrils. A chondroitin sulfate chain covalently linked to the 2IX chain at the flexible kink projects outward from the fibril as does the globular N-terminal region. Part a see R. R. Bruns et al. 1986 J. Cell Biol. 103:393. Part b see L. M. Shaw and B. Olson 1991 Trends Biochem. Sci. 18:191 .

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ular domains at both ends. The lateral association of two type VI monomers generates an “antiparallel” dimer. The end-to-end association of these dimers through their globu- lar domains forms type VI “microfibrils.” These microfi- brils have a beads-on-a-string appearance with about 60- nm-long triple-helical regions separated by 40-nm-long glob- ular domains. The fibrils of type II collagen the major collagen in car- tilage are smaller in diameter than type I fibrils and are ori- ented randomly in a viscous proteoglycan matrix. The rigid collagen fibrils impart a strength and compressibility to the matrix and allow it to resist large deformations in shape. This property allows joints to absorb shocks. Type II fibrils are cross-linked to matrix proteoglycans by type IX collagen another fibril-associated collagen. Type IX collagen and sev- eral related types have two or three triple-helical segments connected by flexible kinks and an N-terminal globular seg- ment Figure 6-22b. The globular N-terminal segment of type IX collagen extends from the fibrils at the end of one of its helical segments as does a GAG chain that is some- times linked to one of the type IX chains. These protruding nonhelical structures are thought to anchor the type II fibril to proteoglycans and other components of the matrix. The interrupted triple-helical structure of type IX and related col- lagens prevents them from assembling into fibrils although they can associate with fibrils formed from other collagen types and form covalent cross-links to them. Certain mutations in the genes encoding collagen 1I or 2I chains which form type I collagen lead to osteogenesis imperfecta or brittle-bone dis- ease. Because every third position in a collagen chain must be a glycine for the triple helix to form see Figure 6-14 mu- tations of glycine to almost any other amino acid are delete- rious resulting in poorly formed and unstable helices. Only one defective chain of the three in a collagen molecule can disrupt the whole molecule’s triple-helical structure and func- tion. A mutation in a single copy allele of either the 1I gene or the 2I gene which are located on nonsex chro- mosomes autosomes can cause this disorder. Thus it nor- mally shows autosomal dominant inheritance Chapter 9. ❚ Hyaluronan Resists Compression and Facilitates Cell Migration Hyaluronan also called hyaluronic acid HA or hy- aluronate is a nonsulfated GAG formed as a disaccharide re- peat composed of glucuronic acid and N-acetylglucosamine see Figure 6-17a by a plasma-membrane-bound enzyme HA synthase and is directly secreted into the extracellular space. It is a major component of the extracellular matrix that surrounds migrating and proliferating cells particularly in embryonic tissues. In addition as will be described shortly hyaluronan forms the backbone of complex proteoglycan ag- gregates found in many extracellular matrices particularly cartilage. Because of its remarkable physical properties hyaluronan imparts stiffness and resilience as well as a lu- bricating quality to many types of connective tissue such as joints. Hyaluronan molecules range in length from a few disac- charide repeats to ≈25000. The typical hyaluronan in joints such as the elbow has 10000 repeats for a total mass of 4 10 6 Da and length of 10 µm about the diameter of a small cell. Individual segments of a hyaluronan molecule fold into a rodlike conformation because of the glycosidic linkages between the sugars and extensive intrachain hydro- gen bonding. Mutual repulsion between negatively charged carboxylate groups that protrude outward at regular inter- vals also contributes to these local rigid structures. Overall however hyaluronan is not a long rigid rod as is fibrillar col- lagen rather in solution it is very flexible bending and twist- ing into many conformations forming a random coil. Because of the large number of anionic residues on its surface the typical hyaluronan molecule binds a large amount of water and behaves as if it were a large hydrated sphere with a diameter of ≈500 nm. As the concentration of hyaluronan increases the long chains begin to entangle forming a viscous gel. Even at low concentrations hyaluro- nan forms a hydrated gel when placed in a confining space such as in a matrix between two cells the long hyaluronan molecules will tend to push outward. This outward pushing creates a swelling or turgor pressure within the extracellu- lar space. In addition the binding of cations by COO groups on the surface of hyaluronan increases the concen- tration of ions and thus the osmotic pressure in the gel. As a result large amounts of water are taken up into the matrix contributing to the turgor pressure. These swelling forces give connective tissues their ability to resist compression forces in contrast with collagen fibers which are able to re- sist stretching forces. Hyaluronan is bound to the surface of many migrating cells by a number of adhesion receptors e.g. one called CD44 containing HA-binding domains each with a similar three-dimensional conformation. Because of its loose hy- drated porous nature the hyaluronan “coat” bound to cells appears to keep cells apart from one another giving them the freedom to move about and proliferate. The cessation of cell movement and the initiation of cell–cell attachments are frequently correlated with a decrease in hyaluronan a de- crease in HA-binding cell-surface molecules and an increase in the extracellular enzyme hyaluronidase which degrades hyaluronan in the matrix. These functions of hyaluronan are particularly important during the many cell migrations that facilitate differentiation and in the release of a mammalian egg cell oocyte from its surrounding cells after ovulation. Association of Hyaluronan and Proteoglycans Forms Large Complex Aggregates The predominant proteoglycan in cartilage called aggrecan assembles with hyaluronan into very large aggregates illus- trative of the complex structures that proteoglycans some- times form. The backbone of the cartilage proteoglycan 6.4 • The Extracellular Matrix of Nonepithelial Tissues 219

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aggregate is a long molecule of hyaluronan to which multiple aggrecan molecules are bound tightly but noncovalently Fig- ure 6-22a. A single aggrecan aggregate one of the largest macromolecular complexes known can be more than 4 mm long and have a volume larger than that of a bacterial cell. These aggregates give cartilage its unique gel-like properties and its resistance to deformation essential for distributing the load in weight-bearing joints. The aggrecan core protein ≈250000 MW has one N- terminal globular domain that binds with high affinity to a specific decasaccharide sequence within hyaluronan. This specific sequence is generated by covalent modification of some of the repeating disaccharides in the hyaluronan chain. The interaction between aggrecan and hyaluronan is facili- tated by a link protein that binds to both the aggrecan core protein and hyaluronan Figure 6-22b. Aggrecan and the link protein have in common a “link” domain ≈100 amino acids long that is found in numerous matrix and cell- surface hyaluronan-binding proteins in both cartilaginous and noncartilaginous tissues. Almost certainly these proteins arose in the course of evolution from a single ancestral gene that encoded just this domain. The importance of the GAG chains that are part of various matrix proteoglycans is illustrated by the rare humans who have a genetic defect in one of the enzymes required for synthesis of the GAG dermatan sul- fate. These persons have many defects in their bones joints and muscles do not grow to normal height and have wrin- kled skin giving them a prematurely aged appearance. ❚ Fibronectins Connect Many Cells to Fibrous Collagens and Other Matrix Components Many different cell types synthesize fibronectin an abundant multiadhesive matrix protein found in all vertebrates. The discovery that fibronectin functions as an adhesive molecule stemmed from observations that it is present on the surfaces of normal fibroblastic cells which adhere tightly to petri dishes in laboratory experiments but is absent from the sur- faces of tumorigenic cells which adhere weakly. The 20 or so isoforms of fibronectin are generated by alternative splicing of the RNA transcript produced from a single gene see Fig- ure 4-15. Fibronectins are essential for the migration and differentiation of many cell types in embryogenesis. These proteins are also important for wound healing because they promote blood clotting and facilitate the migration of macrophages and other immune cells into the affected area. Fibronectins help attach cells to the extracellular matrix by binding to other ECM components particularly fibrous collagens and heparan sulfate proteoglycans and to cell- surface adhesion receptors such as integrins see Figure 6-2. Through their interactions with adhesion receptors e.g. 5 1 integrin fibronectins influence the shape and move- ment of cells and the organization of the cytoskeleton. Con- versely by regulating their receptor-mediated attachments to fibronectin and other ECM components cells can sculpt the immediate ECM environment to suit their needs. Fibronectins are dimers of two similar polypeptides linked at their C-termini by two disulfide bonds each chain is about 60–70 nm long and 2–3 nm thick. Partial digestion 220 CHAPTER 6 • Integrating Cells into Tissues b Hyaluronan molecule Link protein Linking sugars N-terminal Hyaluronan-binding domain Aggrecan core protein Keratan sulfate Chondroitin sulfate 300 nm a Aggrecan Hyaluronan molecule ▲ FIGURE 6-22 Structure of proteoglycan aggregate from cartilage. a Electron micrograph of an aggrecan aggregate from fetal bovine epiphyseal cartilage. Aggrecan core proteins are bound at ≈40-nm intervals to a molecule of hyaluronan. b Schematic representation of an aggrecan monomer bound to hyaluronan. In aggrecan both keratan sulfate and chondroitin sulfate chains are attached to the core protein. The N-terminal domain of the core protein binds noncovalently to a hyaluronan molecule. Binding is facilitated by a link protein which binds to both the hyaluronan molecule and the aggrecan core protein. Each aggrecan core protein has 127 Ser-Gly sequences at which GAG chains can be added. The molecular weight of an aggrecan monomer averages 2 10 6 . The entire aggregate which may contain upward of 100 aggrecan monomers has a molecular weight in excess of 2 10 8 . Part a from J. A. Buckwalter and L. Rosenberg 1983 Coll. Rel. Res. 3:489 courtesy of L. Rosenberg.

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of fibronectin with low amounts of proteases and analysis of the fragments showed that each chain comprises six func- tional regions with different ligand-binding specificities Fig- ure 6-23. Each region in turn contains multiple copies of certain sequences that can be classified into one of three types. These classifications are designated fibronectin type I II and III repeats on the basis of similarities in amino acid sequence although the sequences of any two repeats of a given type are not always identical. These linked repeats give the molecule the appearance of beads on a string. The com- bination of different repeats composing the regions another example of combinatorial diversity confers on fibronectin its ability to bind multiple ligands. One of the type III repeats in the cell-binding region of fi- bronectin mediates binding to certain integrins. The results of studies with synthetic peptides corresponding to parts of this repeat identified the tripeptide sequence Arg-Gly-Asp usually called the RGD sequence as the minimal sequence within this repeat required for recognition by those integrins. In one study heptapeptides containing the RGD sequence or a variation of this sequence were tested for their ability to mediate the adhesion of rat kidney cells to a culture dish. The results showed that heptapeptides containing the RGD sequence mimicked intact fibronectin’s ability to stimulate integrin-mediated adhesion whereas variant heptapeptides lacking this sequence were ineffective Figure 6-24. 6.4 • The Extracellular Matrix of Nonepithelial Tissues 221 S S NH 2 COOH COOH Type I Type II Type III Repeating amino acid sequences: IIICS Fibrin heparan sulfate– binding repeats Collagen- binding repeats Integrin- binding repeats Fibrin- binding repeats Heparan sulfate– binding repeat SS EIIIB EIIIA ▲ FIGURE 6-23 Organization of fibronectin chains. Only one of the two chains present in the dimeric fibronectin molecule is shown both chains have very similar sequences. Each chain contains about 2446 amino acids and is composed of three types of repeating amino acid sequences. Circulating fibronectin lacks one or both of the type III repeats designated EIIIA and EIIIB owing to alternative mRNA splicing see Figure 4-15. At least five different sequences may be present in the IIICS region as a result of alternative splicing. Each chain contains six domains tan boxes some of which contain specific binding sites for heparan sulfate fibrin a major constituent of blood clots collagen and cell-surface integrins. The integrin-binding domain is also known as the cell-binding domain. Adapted from G. Paolella M. Barone and F . Baralle 1993 in M. Zern and L. Reid eds. Extracellular Matrix Marcel Dekker pp. 3–24. Relative amounts of bound cells stain intensity 0.2 0.4 0.6 0.8 1.0 1.2 1.4 GRADSPC GRGESPC GKGDSPC DREDSRC GRGDSPC YKPGEGKRGDACEGDSG GRGDAPC PRGDVDC Peptide concentration nmol/ml 110 100 1000 EXPERIMENTAL FIGURE 6-24 A specific tripeptide sequence RGD in the cell-binding region of fibronectin is required for adhesion of cells. The cell-binding region of fibronectin contains an integrin-binding heptapeptide sequence GRDSPC in the single-letter amino acid code see Figure 2-13. This heptapeptide and several variants were synthesized chemically. Different concentrations of each synthetic peptide were added to polystyrene dishes that had the protein immunoglobulin G IgG firmly attached to their surfaces the peptides were then chemically cross-linked to the IgG. Subsequently cultured normal rat kidney cells were added to the dishes and incubated for 30 minutes to allow adhesion. After the nonbound cells were washed away the relative amounts of cells that had adhered firmly were determined by staining the bound cells with a dye and measuring the intensity of the staining with a spectrophotometer. The plots shown here indicate that cell adhesion increased above the background level with increasing peptide concentration for those peptides containing the RGD sequence but not for the variants lacking this sequence modification underlined. From M. D. Pierschbacher and E. Ruoslahti 1984 Proc. Nat’l. Acad. Sci. USA 81:5985.

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A three-dimensional model of fibronectin binding to integrin based on structures of parts of both fibronectin and integrin has been assembled Figure 6-25a. In a high- resolution structure of the integrin-binding fibronectin type III repeat and its neighboring type III domain the RGD se- quence is at the apex of a loop that protrudes outward from the molecule in a position facilitating binding to integrins Figure 6-25a b. Although the RGD sequence is required for binding to several integrins its affinity for integrins is substantially less than that of intact fibronectin or of the en- tire cell-binding region in fibronectin. Thus structural fea- tures near to the RGD sequence in fibronectins e.g. parts of adjacent repeats such as the synergy region see Figure 6-25b and in other RGD-containing proteins enhance their binding to certain integrins. Moreover the simple soluble dimeric forms of fibronectin produced by the liver or fibro- blasts are initially in a nonfunctional closed conformation that binds poorly to integrins because the RGD sequence is not readily accessible. The adsorption of fibronectin to a col- 222 CHAPTER 6 • Integrating Cells into Tissues EIIIB EIIIA IIICS Type I repeat Type II repeat Type III repeat Fibrin heparan sulfate binding Collagen binding Integrin Fibrin binding Heparan sulfate binding NH 2 COOH SS RGD a b Synergy region RGD sequence ▲ FIGURE 6-25 Model of fibronectin binding to integrin through its RGD-containing type III repeat. a Scale model of fibronectin is shown docked by two type III repeats to the extracellular domains of integrin. Structures of fibronectin’s domains were determined from fragments of the molecule. The EIIIA EIIIB and IIICS domains not shown see Figure 6-23 are variably spliced into the structure at locations indicated by arrows. b A high-resolution structure shows that the RGD binding sequence red extends outward in a loop from its compact type III domain on the same side of fibronectin as the synergy region blue which also contributes to high-affinity binding to integrins. Adapted from D. J. Leahy et al. 1996 Cell 84:161 . a b 0.5 m Cell exterior Fibronectin fibrils Plasma membrane Actin-containing microfilaments Cell interior EXPERIMENTAL FIGURE 6-26 Integrins mediate linkage between fibronectin in the extracellular matrix and the cytoskeleton. a Immunofluorescent micrograph of a fixed cultured fibroblast showing colocalization of the 5 1 integrin and actin-containing stress fibers. The cell was incubated with two types of monoclonal antibody: an integrin-specific antibody linked to a green fluorescing dye and an actin-specific antibody linked to a red fluorescing dye. Stress fibers are long bundles of actin microfilaments that radiate inward from points where the cell contacts a substratum. At the distal end of these fibers near the plasma membrane the coincidence of actin red and fibronectin-binding integrin green produces a yellow fluorescence. b Electron micrograph of the junction of fibronectin and actin fibers in a cultured fibroblast. Individual actin-containing 7-nm microfilaments components of a stress fiber end at the obliquely sectioned cell membrane. The microfilaments appear in close proximity to the thicker densely stained fibronectin fibrils on the outside of the cell. Part a from J. Duband et al. 1988 J. Cell Biol. 107:1385. Part b from I. J. Singer 1979 Cell 16:675 courtesy of I. J. Singer copyright 1979 MIT.

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lagen matrix or the basal lamina or experimentally to a plas- tic tissue-culture dish results in a conformational change that enhances its ability to bind to cells. Most likely this confor- mational change increases the accessibility of the RGD se- quence for integrin binding. Microscopy and other experimental approaches e.g. bio- chemical binding experiments have demonstrated the role of integrins in cross-linking fibronectin and other ECM compo- nents to the cytoskeleton. For example the colocalization of cytoskeletal actin filaments and integrins within cells can be visualized by fluorescence microscopy Figure 6-26a. The binding of cell-surface integrins to fibronectin in the matrix induces the actin cytoskeleton–dependent movement of some integrin molecules in the plane of the membrane. The ensu- ing mechanical tension due to the relative movement of dif- ferent integrins bound to a single fibronectin dimer stretches the fibronectin. This stretching promotes self-association of the fibronectin into multimeric fibrils. The force needed to unfold and expose functional self- association sites in fibronectin is much less than that needed to disrupt fibronectin–integrin binding. Thus fi- bronectin molecules remain bound to integrin while cell- generated mechanical forces induce fibril formation. In ef- fect the integrins through adapter proteins transmit the in- tracellular forces generated by the actin cytoskeleton to extracellular fibronectin. Gradually the initially formed fibronectin fibrils mature into highly stable matrix compo- nents by covalent cross-linking. In some electron micro- graphic images exterior fibronectin fibrils appear to be aligned in a seemingly continuous line with bundles of actin fibers within the cell Figure 6-26b. These observations and the results from other studies provided the first example of a molecularly well defined adhesion receptor i.e. an inte- grin forming a bridge between the intracellular cytoskeleton and the extracellular matrix components—a phenomenon now known to be widespread. KEY CONCEPTS OF SECTION 6.4 The Extracellular Matrix of Nonepithelial Tissues ■ Connective tissue such as tendon and cartilage differs from other solid tissues in that most of its volume is made up of extracellular matrix ECM rather than cells. ■ The synthesis of fibrillar collagen e.g. types I II and III begins inside the cell with the chemical modification of newly made chains and their assembly into triple- helical procollagen within the endoplasmic reticulum. Af- ter secretion procollagen molecules are cleaved associate laterally and are covalently cross-linked into bundles called fibrils which can form larger assemblies called fibers see Figure 6-20. ■ The various collagens are distinguished by the ability of their helical and nonhelical regions to associate into fib- rils to form sheets or to cross-link other collagen types see Table 6-1. ■ Hyaluronan a highly hydrated GAG is a major com- ponent of the ECM of migrating and proliferating cells. Certain cell-surface adhesion receptors bind hyaluronan to cells. ■ Large proteoglycan aggregates containing a central hyaluronan molecule noncovalently bound to the core pro- tein of multiple proteoglycan molecules e.g. aggrecan contribute to the distinctive mechanical properties of the matrix see Figure 6-22. ■ Fibronectins are abundant multiadhesive matrix proteins that play a key role in migration and cellular differentia- tion. They contain binding sites for integrins and ECM components collagens proteoglycans and can thus attach cells to the matrix see Figure 6-23. ■ The tripeptide RGD sequence Arg-Gly-Asp found in fibronectins and some other matrix proteins is recognized by several integrins. Adhesive Interactions and Nonepithelial Cells After adhesive interactions in epithelia form during differen- tiation they often are very stable and can last throughout the life span of epithelial cells or until the cells undergo differen- tiation into loosely associated nonpolarized mesenchymal cells the epithelial–mesenchymal transition. Although such long-lasting nonmotile adhesion also exists in nonepithelial tissues some nonepithelial cells must be able to crawl across or through a layer of extracellular matrix or other cells. In this section we describe various cell-surface structures in nonepithelial cells that mediate long-lasting adhesion and transient adhesive interactions that are especially adapted for the movement of cells. The detailed intracellular mechanisms used to generate the mechanical forces that propel cells and modify their shapes are covered in Chapter 19. Integrin-Containing Adhesive Structures Physically and Functionally Connect the ECM and Cytoskeleton in Nonepithelial Cells As already discussed in regard to epithelia integrin-containing hemidesmosomes connect epithelial cells to the basal lamina and through adapter proteins to intermediate filaments of the cytoskeleton see Figure 6-1. In nonepithelial cells integrins in the plasma membrane also are clustered with other molecules in various adhesive structures called focal adhesions focal contacts focal complexes 3D adhesions and fibrillar adhe- sions and in circular adhesions called podosomes Chapter 14. These structures are readily observed by fluorescence mi- croscopy with the use of antibodies that recognize integrins or other coclustered molecules Figure 6-27. Like cell–matrix anchoring junctions in epithelial cells the various adhesive structures attach nonepithelial cells to the extracellular matrix 6.5 6.5 • Adhesive Interactions and Nonepithelial Cells 223

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they also contain dozens of intracellular adapter and associ- ated proteins that mediate attachment to cytoskeletal actin fil- aments and activate adhesion-dependent signals for cell growth and cell motility. Although found in many nonepithelial cells integrin- containing adhesive structures have been studied most fre- quently in fibroblasts grown in cell culture on flat glass or plastic surfaces substrata. These conditions only poorly ap- proximate the three-dimensional ECM environment that nor- mally surrounds such cells in vivo. When fibroblasts are cultured in three-dimensional ECM matrices derived from cells or tissues they form adhesions to the three-dimensional ECM substratum called 3D adhesions. These structures dif- fer somewhat in composition shape distribution and activ- ity from the focal or fibrillar adhesions seen in cells growing on the flat substratum typically used in cell-culture experi- ments see Figure 6-27. Cultured fibroblasts with these “more natural” anchoring junctions display greater adhesion and mobility increased rates of cell proliferation and spindle- shaped morphologies more like those of fibroblasts in tissues than do cells cultured on flat surfaces. These observations in- dicate that the topological compositional and mechanical e.g. flexibility properties of the extracellular matrix all play a role in controlling the shape and activity of a cell. Tissue- specific differences in these matrix characteristics probably contribute to the tissue-specific properties of cells. 224 CHAPTER 6 • Integrating Cells into Tissues a Focal adhesion b 3D adhesion EXPERIMENTAL FIGURE 6-27 Integrins cluster into adhesive structures with various morphologies in nonepithelial cells. Immunofluorescence methods were used to detect adhesive structures green on cultured cells. Shown here are focal adhesions a and 3D adhesions b on the surfaces of human fibroblasts. Cells were grown directly on the flat surface of a culture dish a or on a three-dimensional matrix of ECM components b. The shape distribution and composition of the integrin-based adhesions formed by cells vary depending on culture conditions. Part a from B. Geiger et al. 2001 Nature Rev. Mol. Cell Biol. 2:793. Part b courtesy of K. Yamada and E. Cukierman see E. Cukierman et al. 2001 Science 294:1708–12. TABLE 6-2 Selected Vertebrate Integrins Subunit Primary Cellular Composition Distribution Ligands 1 1 Many types Mainly collagens also laminins 2 1 Many types Mainly collagens also laminins 41Hematopoietic cells Fibronectin VCAM-1 5 1 Fibroblasts Fibronectin L2T lymphocytes ICAM-1 ICAM-2 M 2 Monocytes Serum proteins e.g. C3b fibrinogen factor X ICAM-1 IIb 3 Platelets Serum proteins e.g. fibrinogen von Willebrand factor vitronectin fibronectin 6 4 Epithelial cells Laminin The integrins are grouped into subfamilies having a common subunit. Ligands shown in red are CAMs all others are ECM or serum proteins. Some subunits can have multiply spliced isoforms with different cytosolic domains. SOURCE: R. O. Hynes 1992 Cell 69:11.

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Diversity of Ligand–Integrin Interactions Contributes to Numerous Biological Processes Although most cells express several distinct integrins that bind the same ligand or different ligands many integrins are expressed predominantly in certain types of cells. Table 6-2 lists a few of the numerous integrin-mediated interactions with ECM components or CAMs or both. Not only do many integrins bind more than one ligand but several of their lig- ands bind to multiple integrins. All integrins appear to have evolved from two ancient general subgroups: those that bind RGD-containing mole- cules e.g. fibronectin and those that bind laminin. For ex- ample 5 1 integrin binds fibronectin whereas the widely expressed 1 1 and 2 1 integrins as well as the 6 4 in- tegrin expressed by epithelial cells bind laminin. The 1 2 and several other integrin subunits contain a distinctive in- serted domain the I-domain. The I-domain in some integrins e.g. 1 1 and 2 1 mediates binding to various collagens. Other integrins containing subunits with I-domains are ex- pressed exclusively on leukocytes and hematopoietic cells these integrins recognize cell-adhesion molecules on other cells including members of the Ig superfamily e.g. ICAMs VCAMs and thus participate in cell–cell adhesion. The diversity of integrins and their ECM ligands enables integrins to participate in a wide array of key biological processes including the migration of cells to their correct locations in the formation the body plan of an embryo morphogenesis and in the inflammatory response. The importance of integrins in diverse processes is highlighted by the defects exhibited by knockout mice engineered to have mutations in each of almost all of the integrin subunit genes. These defects include major abnormalities in development blood vessel formation leukocyte function the response to infection inflammation bone remodeling and hemostasis. Cell–Matrix Adhesion Is Modulated by Changes in the Binding Activity and Numbers of Integrins Cells can exquisitely control the strength of integrin- mediated cell–matrix interactions by regulating the ligand- binding activity of integrins or their expression or both. Such regulation is critical to the role of these interactions in cell migration and other functions. Many if not all integrins can exist in two conformations: a low-affinity inactive form and a high-affinity active form Figure 6-28. The results of structural studies and ex- periments investigating the binding of ligands by integrins have provided a model of the changes that take place when integrins are activated. In the inactive state the het- erodimer is bent the conformation of the ligand-binding site at the tip of the molecule allows only low-affinity ligand binding and the cytoplasmic C-terminal tails of the two sub- units are closely bound together. In the “straight” active state alterations in the conformation of the domains that form the binding site permit tighter high-affinity ligand binding and the cytoplasmic tails separate. These structural models also provide an attractive expla- nation for the ability of integrins to mediate outside-in and inside-out signaling. The binding of certain ECM molecules or CAMs on other cells to the bent low-affinity structure would force the molecule to straighten and consequently separate the cytoplasmic tails. Intracellular adapters could “sense” the separation of the tails and as a result either bind or dissociate from the tails. The changes in these adapters 6.5 • Adhesive Interactions and Nonepithelial Cells 225 FIGURE 6-28 Model for integrin activation. Left The molecular model is based on the x-ray crystal structure of the extracellular region of v 3 integrin in its inactive low-affinity “bent” form with the subunit in shades of blue and the subunit in shades of red. The major ligand- binding sites are at the tip of the molecule where the propeller domain dark blue and A domain dark red interact. An RGD peptide ligand is shown in yellow. Right Activation of integrins is thought to be due to conformational changes that include straightening of the molecule key movements near the propeller and A domains which increases the affinity for ligands and separation of the cytoplasmic domains resulting in altered interactions with adapter proteins. See text for further discussion. Adapted from M. Arnaout et al. 2002 Curr. Opin. Cell Biol. 14:641 and R. O. Hynes 2002 Cell 110:673. Activation α β Ligand β propeller β propeller βA domain βA domain

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could then alter the cytoskeleton and activate or inhibit in- tracellular signaling pathways. Conversely changes in the metabolic state of the cells e.g. changes in the platelet cy- toskeleton that accompany platelet activation see Figure 19-5 could cause intracellular adapters to bind to the tails or to dissociate from them and thus force the tails to either sep- arate or associate. As a consequence the integrin would ei- ther bend inactivate or straighten activate thereby altering its interaction with the ECM or other cells. Platelet function provides a good example of how cell–matrix interactions are modulated by controlling inte- grin binding activity. In its basal state the IIb 3 integrin present on the plasma membranes of platelets normally cannot bind tightly to its protein ligands e.g. fibrinogen fibronectin all of which participate in the formation of a blood clot because it is in the inactive bent conformation. The binding of a platelet to collagen or thrombin in a form- ing clot induces from the cytoplasm an activating conforma- tional change in IIb 3 integrin that permits it to tightly bind clotting proteins and participate in clot formation. Per- sons with genetic defects in the 3 integrin subunit are prone to excessive bleeding attesting to the role of this integrin in the formation of blood clots. The attachment of cells to ECM components can also be modulated by altering the number of integrin molecules ex- posed on the cell surface. The 4 1 integrin which is found on many hematopoietic cells precursors of red and white blood cells offers an example of this regulatory mechanism. For these hematopoietic cells to proliferate and differenti- ate they must be attached to fibronectin synthesized by sup- portive “stromal” cells in the bone marrow. The 4 1 integrin on hematopoietic cells binds to a Glu-Ile-Leu-Asp- Val EILDV sequence in fibronectin thereby anchoring the cells to the matrix. This integrin also binds to a sequence in a CAM called vascular CAM-1 VCAM-1 which is present on stromal cells of the bone marrow. Thus hematopoietic cells directly contact the stromal cells as well as attach to the matrix. Late in their differentiation hematopoietic cells de- crease their expression of 4 1 integrin the resulting reduc- tion in the number of 4 1 integrin molecules on the cell surface is thought to allow mature blood cells to detach from the matrix and stromal cells in the bone marrow and subse- quently enter the circulation. Molecular Connections Between the ECM and Cytoskeleton Are Defective in Muscular Dystrophy The importance of the adhesion receptor–mediated linkage between ECM components and the cyto- skeleton is highlighted by a set of hereditary muscle-wasting diseases collectively called muscular dystro- phies. Duchenne muscular dystrophy DMD the most com- mon type is a sex-linked disorder affecting 1 in 3300 boys that results in cardiac or respiratory failure in the late teens or early twenties. The first clue to understanding the molec- ular basis of this disease came from the discovery that per- sons with DMD carry mutations in the gene encoding a protein named dystrophin. This very large protein was found to be a cytosolic adapter protein binding to actin filaments and to an adhesion receptor called dystroglycan. ❚ Dystroglycan is synthesized as a large glycoprotein pre- cursor that is proteolytically cleaved into two subunits. The subunit is a peripheral membrane protein and the sub- unit is a transmembrane protein whose extracellular domain associates with the subunit Figure 6-29. Multiple O- linked oligosaccharides are attached covalently to side-chain hydroxyl groups of serine and threonine residues in the subunit. These O-linked oligosaccharides bind to various basal lamina components including the multiadhesive ma- trix protein laminin and the proteoglycans perlecan and 226 CHAPTER 6 • Integrating Cells into Tissues Agrin Neurexin Perlecan Laminin α β γα β δ Actin α-Dystrobrevin NOS Syntrophins GRB2 Dystrophin Sarcospan Sarcoglycan complex αβ-Dystroglycan Cytosol O-linked sugar Basal lamina N-linked sugar ▲ FIGURE 6-29 Schematic model of the dystrophin glycoprotein complex DGC in skeletal muscle cells. The DGC comprises three subcomplexes: the dystroglycan subcomplex the sarcoglycan/sarcospan subcomplex of integral membrane proteins and the cytosolic adapter subcomplex comprising dystrophin other adapter proteins and signaling molecules. Through its O-linked sugars -dystroglycan binds to components of the basal lamina such as laminin. Dystrophin— the protein defective in Duchenne muscular dystrophy—links -dystroglycan to the actin cytoskeleton and -dystrobrevin links dystrophin to the sarcoglycan/sarcospan subcomplex. Nitric oxide synthase NOS produces nitric oxide a gaseous signaling molecule and GRB2 is a component of signaling pathways activated by certain cell-surface receptors Chapter 14. See text for further discussion. Adapted from S. J. Winder 2001 Trends Biochem. Sci. 26:118 and D. E. Michele and K. P . Campbell 2003 J. Biol. Chem.

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agrin. The neurexins a family of adhesion molecules ex- pressed by neurons also are bound by the subunit. The transmembrane segment of the dystroglycan sub- unit associates with a complex of integral membrane pro- teins its cytosolic domain binds dystrophin and other adapter proteins as well as various intracellular signaling proteins. The resulting large heterogeneous assemblage the dystrophin glycoprotein complex DGC links the extracel- lular matrix to the cytoskeleton and signaling pathways within muscle cells see Figure 6-29. For instance the sig- naling enzyme nitric oxide synthase NOS is associated through syntrophin with the cytosolic dystrophin subcom- plex in skeletal muscle. The rise in intracellular Ca 2 during muscle contraction activates NOS to produce nitric oxide NO which diffuses into smooth muscle cells surrounding nearby blood vessels. By a signaling pathway described in Chapter 13 NO promotes smooth muscle relaxation lead- ing to a local rise in the flow of blood supplying nutrients and oxygen to the skeletal muscle. Mutations in dystrophin other DGC components laminin or enzymes that add the O-linked sugars to dystro- glycan disrupt the DGC-mediated link between the exterior and the interior of muscle cells and cause muscular dystro- phies. In addition dystroglycan mutations have been shown to greatly reduce the clustering of acetylcholine receptors on muscle cells at the neuromuscular junctions Chapter 7 which also is dependent on the basal lamina proteins laminin and agrin. These and possibly other effects of DGC defects apparently lead to a cumulative weakening of the mechanical stability of muscle cells as they undergo contraction and re- laxation resulting in deterioration of the cells and muscular dystrophy. Ca 2 -Independent Cell–Cell Adhesion in Neuronal and Other Tissues Is Mediated by CAMs in the Immunoglobulin Superfamily Numerous transmembrane proteins characterized by the presence of multiple immunoglobulin domains repeats in their extracellular regions constitute the Ig superfamily of CAMs or IgCAMs. The Ig domain is a common protein motif containing 70–110 residues that was first identified in antibodies the antigen-binding immunoglobulins. The human D. melanogaster and C. elegans genomes include about 765 150 and 64 genes respectively that encode pro- teins containing Ig domains. Immunoglobin domains are found in a wide variety of cell-surface proteins including T- cell receptors produced by lymphocytes and many proteins that take part in adhesive interactions. Among the IgCAMs are neural CAMs intercellular CAMs ICAMs which func- tion in the movement of leukocytes into tissues and junc- tion adhesion molecules JAMs which are present in tight junctions. As their name implies neural CAMs are of particular im- portance in neural tissues. One type the NCAMs primarily mediate homophilic interactions. First expressed during mor- phogenesis NCAMs play an important role in the differen- tiation of muscle glial and nerve cells. Their role in cell adhesion has been directly demonstrated by the inhibition of adhesion with anti-NCAM antibodies. Numerous NCAM isoforms encoded by a single gene are generated by alter- native mRNA splicing and by differences in glycosylation. Other neural CAMs e.g. L1-CAM are encoded by different genes. In humans mutations in different parts of the L1-CAM gene cause various neuropathologies e.g. mental retardation congenital hydrocephalus and spasticity. An NCAM comprises an extracellular region with five Ig repeats and two fibronectin type III repeats a single mem- brane-spanning segment and a cytosolic segment that inter- acts with the cytoskeleton see Figure 6-2. In contrast the extracellular region of L1-CAM has six Ig repeats and four fi- bronectin type III repeats. As with cadherins cis intracellular interactions and trans intercellular interactions probably play key roles in IgCAM-mediated adhesion see Figure 6-3. The covalent attachment of multiple chains of sialic acid a negatively charged sugar derivative to NCAMs alters their adhesive properties. In embryonic tissues such as brain poly- sialic acid constitutes as much as 25 percent of the mass of NCAMs. Possibly because of repulsion between the many negatively charged sugars in these NCAMs cell–cell contacts are fairly transient being made and then broken a property necessary for the development of the nervous system. In con- trast NCAMs from adult tissues contain only one-third as much sialic acid permitting more stable adhesions. Movement of Leukocytes into Tissues Depends on a Precise Sequence of Combinatorially Diverse Sets of Adhesive Interactions In adult organisms several types of white blood cells leuko- cytes participate in the defense against infection caused by foreign invaders e.g. bacteria and viruses and tissue dam- age due to trauma or inflammation. To fight infection and clear away damaged tissue these cells must move rapidly from the blood where they circulate as unattached relatively quiescent cells into the underlying tissue at sites of infection inflammation or damage. We know a great deal about the movement into tissue termed extravasation of four types of leukocytes: neutrophils which release several antibacter- ial proteins monocytes the precursors of macrophages which can engulf and destroy foreign particles and T and B lymphocytes the antigen-recognizing cells of the immune system. Extravasation requires the successive formation and breakage of cell–cell contacts between leukocytes in the blood and endothelial cells lining the vessels. Some of these contacts are mediated by selectins a family of CAMs that mediate leukocyte–vascular cell interactions. A key player in these interactions is P-selectin which is localized to the blood-facing surface of endothelial cells. All selectins contain 6.5 • Adhesive Interactions and Nonepithelial Cells 227

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P-selectin ICAM-2 Endothelial cell Vesicle containing P-selectin Extravasation PAF receptor Selectin ligand specific carbohydrate PAF Leukocyte resting state Endothelial activation and leukocyte attachment and rolling Leukocyte activation PAF activates integrin αLβ2 integrin 1 ICAM-1 2 3 Firm adhesion via integrin/ICAM binding 4 5 a Ca 2 -dependent lectin domain which is located at the distal end of the extracellular region of the molecule and recognizes oligosaccharides in glycoproteins or glyco- lipids see Figure 6-2. For example the primary ligand for P- and E-selectins is an oligosaccharide called the sialyl Lewis-x antigen a part of longer oligosaccharides present in abundance on leukocyte glycoproteins and glycolipids. Figure 6-30 illustrates the basic sequence of cell–cell in- teractions leading to the extravasation of leukocytes. Various inflammatory signals released in areas of infection or in- flammation first cause activation of the endothelium. P-selectin exposed on the surface of activated endothelial cells mediates the weak adhesion of passing leukocytes. Be- cause of the force of the blood flow and the rapid “on” and “off” rates of P-selectin binding to its ligands these “trapped” leukocytes are slowed but not stopped and liter- ally roll along the surface of the endothelium. Among the signals that promote activation of the endothelium are chemokines a group of small secreted proteins 8–12 kDa produced by a wide variety of cells including endothelial cells and leukocytes. For tight adhesion to occur between activated endothelial cells and leukocytes 2-containing integrins on the surfaces of leukocytes also must be activated by chemokines or other local activation signals such as platelet-activating factor PAF. Platelet-activating factor is unusual in that it is a phospholipid rather than a protein it is exposed on the sur- face of activated endothelial cells at the same time that P-selectin is exposed. The binding of PAF or other activators to their receptors on leukocytes leads to activation of the leukocyte integrins to their high-affinity form see Figure 6-28. Most of the receptors for chemokines and PAF are members of the G protein–coupled receptor superfamily dis- cussed in Chapter 13. Activated integrins on leukocytes then bind to each of two distinct IgCAMs on the surface of en- 228 CHAPTER 6 • Integrating Cells into Tissues ▲ FIGURE 6-30 Sequence of cell–cell interactions leading to tight binding of leukocytes to activated endothelial cells and subsequent extravasation.Step : In the absence of inflammation or infection leukocytes and endothelial cells lining blood vessels are in a resting state. Step : Inflammatory signals released only in areas of inflammation or infection or both activate resting endothelial cells to move vesicle-sequestered selectins to the cell surface. The exposed selectins mediate loose binding of leukocytes by interacting with carbohydrate ligands on leukocytes. Activation of the endothelium also causes 2 1 synthesis of platelet-activating factor PAF and ICAM-1 both expressed on the cell surface. PAF and other usually secreted activators including chemokines then induce changes in the shapes of the leukocytes and activation of leukocyte integrins such as L 2 which is expressed by T lymphocytes . The subsequent tight binding between activated integrins on leukocytes and CAMs on the endothelium e.g. ICAM-2 and ICAM-1 results in firm adhesion and subsequent movement extravasation into the underlying tissue .See text for further discussion. Adapted from R. O. Hynes and A. Lander 1992 Cell 68:303. 5 4 3 MEDIA CONNECTIONS Focus Animation: Cell–Cell Adhesion in Leukocyte Extravasation

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dothelial cells: ICAM-2 which is expressed constitutively and ICAM-1. ICAM-1 whose synthesis along with that of E-selectin and P-selectin is induced by activation does not usually contribute substantially to leukocyte endothelial cell adhesion immediately after activation but rather participates at later times in cases of chronic inflammation. The resulting tight adhesion mediated by the Ca 2 -independent integrin– ICAM interactions leads to the cessation of rolling and to the spreading of leukocytes on the surface of the endothelium soon the adhered cells move between adjacent endothelial cells and into the underlying tissue. The selective adhesion of leukocytes to the endothelium near sites of infection or inflammation thus depends on the sequential appearance and activation of several different CAMs on the surfaces of the interacting cells. Different types of leukocytes express specific integrins containing the 2 subunit: for example L 2 by T lymphocytes and M 2 by monocytes the circulating precursors of tissue macrophages. Nonetheless all leukocytes move into tissues by the same general mechanism depicted in Figure 6-30. Many of the CAMs used to direct leukocyte adhesion are shared among different types of leukocytes and target tissues. Yet often only a particular type of leukocyte is directed to a particular tissue. A three-step model has been proposed to account for the cell-type specificity of such leukocyte– endothelial cell interactions. First endothelium activation promotes initial relatively weak transient and reversible binding e.g. the interaction of selectins and their carbohy- drate ligands. Without additional local activation signals the leukocyte will quickly move on. Second cells in the im- mediate vicinity of the site of infection or inflammation re- lease or express on their surfaces chemical signals e.g. chemokines PAF that activate only special subsets of the transiently attached leukocytes. Third additional activation- dependent CAMs e.g. integrins engage their binding part- ners leading to strong sustained adhesion. Only if the proper combination of CAMs binding partners and activation sig- nals are engaged in the right order at a specific site will a given leukocyte adhere strongly. This additional example of combinatorial diversity and cross talk allows parsimonious exploitation of a small set of CAMs for diverse functions throughout the body. Leukocyte-adhesion deficiency is caused by a ge- netic defect in the synthesis of the integrin 2 sub- unit. Persons with this disorder are susceptible to repeated bacterial infections because their leukocytes can- not extravasate properly and thus fight the infection within the tissue. Some pathogenic viruses have evolved mechanisms to ex- ploit for their own purposes cell-surface proteins that par- ticipate in the normal response to inflammation. For example many of the RNA viruses that cause the common cold rhinoviruses bind to and enter cells through ICAM-1 and chemokine receptors can be important entry sites for human immunodeficiency virus HIV the cause of AIDS. ❚ Gap Junctions Composed of Connexins Allow Small Molecules to Pass Between Adjacent Cells Early electron micrographs of virtually all animal cells that were in contact revealed sites of cell–cell contact with a char- acteristic intercellular gap Figure 6-31a. This feature prompted early morphologists to call these regions gap junc- tions. In retrospect the most important feature of these junc- tions is not the gap itself but a well-defined set of cylindrical particles that cross the gap and compose pores connecting the cytoplasms of adjacent cells—hence their alternate name of intercytoplasmic junctions. In epithelia gap junctions are distributed along the lateral surfaces of adjacent cells see Figures 6-1 and 6-5. In many tissues e.g. the liver large numbers of individ- ual cylindrical particles cluster together in patches. This prop- erty has enabled researchers to separate gap junctions from other components of the plasma membrane. When the plasma membrane is purified and then sheared into small fragments some pieces mainly containing patches of gap junctions are generated. Owing to their relatively high pro- tein content these fragments have a higher density than that of the bulk of the plasma membrane and can be purified on an equilibrium density gradient see Figure 5-37. When these 6.5 • Adhesive Interactions and Nonepithelial Cells 229 Gap junction 50 nm b 50 nm a ▲ EXPERIMENTAL FIGURE 6-31 Gap junctions have a characteristic appearance in electron micrographs. a In this thin section through a gap junction connecting two mouse liver cells the two plasma membranes are closely associated for a distance of several hundred nanometers separated by a “gap” of 2–3 nm. b Numerous roughly hexagonal particles are visible in this perpendicular view of the cytosolic face of a region of plasma membrane enriched in gap junctions. Each particle aligns with a similar particle on an adjacent cell forming a channel connecting the two cells. Part a courtesy of D. Goodenough. Part b courtesy of N. Gilula.

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preparations are viewed in cross section the gap junctions appear as arrays of hexagonal particles that enclose water- filled channels Figure 6-31b. Such pure preparations of gap junctions have permitted the detailed biophysical and func- tional analysis of these structures. The effective pore size of gap junctions can be measured by injecting a cell with a fluorescent dye covalently linked to molecules of various sizes and observing with a fluores- cence microscope whether the dye passes into neighboring cells. Gap junctions between mammalian cells permit the passage of molecules as large as 1.2 nm in diameter. In in- sects these junctions are permeable to molecules as large as 2 nm in diameter. Generally speaking molecules smaller than 1200 Da pass freely and those larger than 2000 Da do not pass the passage of intermediate-sized molecules is variable and limited. Thus ions many low-molecular-weight precur- sors of cellular macromolecules products of intermediary metabolism and small intracellular signaling molecules can pass from cell to cell through gap junctions. In nervous tissue some neurons are connected by gap junctions through which ions pass rapidly thereby allow- ing very rapid transmission of electrical signals. Impulse transmission through these connections called electrical synapses is almost a thousandfold as rapid as at chemical synapses Chapter 7. Gap junctions are also present in many non-neuronal tissues where they help to integrate the electrical and metabolic activities of many cells. In the heart for instance gap junctions rapidly pass ionic signals among muscle cells and thus contribute to the electrically stimu- lated coordinate contraction of cardiac muscle cells during a beat. As discussed in Chapter 13 some extracellular hor- monal signals induce the production or release of small in- tracellular signaling molecules called second messengers e.g. cyclic AMP and Ca 2 that regulate cellular metabo- lism. Because second messengers can be transferred between cells through gap junctions hormonal stimulation of one cell can trigger a coordinated response by that same cell and many of its neighbors. Such gap junction–mediated signal- ing plays an important role for example in the secretion of digestive enzymes by the pancreas and in the coordinated muscular contractile waves peristalsis in the intestine. An- other vivid example of gap junction–mediated transport is the phenomenon of metabolic coupling or metabolic coop- eration in which a cell transfers nutrients or intermediary metabolites to a neighboring cell that is itself unable to syn- thesize them. Gap junctions play critical roles in the devel- opment of egg cells in the ovary by mediating the movement of both metabolites and signaling molecules between an oocyte and its surrounding granulosa cells as well as be- tween neighboring granulosa cells. A current model of the structure of the gap junction is shown in Figure 6-32. Vertebrate gap junctions are composed 230 CHAPTER 6 • Integrating Cells into Tissues CM E M C Gap- junction channel Cytosol Intercellular gap a b 2 nm Connexon hemichannel FIGURE 6-32 Molecular structure of gap junctions. a Schematic model of a gap junction which comprises a cluster of channels between two plasma membranes separated by a gap of about 2–3 nm. Both membranes contain connexon hemichannels cylinders of six dumbbell- shaped connexin molecules. Two connexons join in the gap between the cells to form a gap-junction channel 1.5–2.0 nm in diameter that connects the cytosols of the two cells. b Electron density of a recombinant gap-junction channel determined by electron crystallography. Shown here are side views of the complete structure top and the same structure with several chains removed to show the channel’s interior center on the bottom are perpendicular cross sections through the gap junction within and between the membrane bilayers. There appear to be 24 transmembrane helices per connexon hemichannel consistent with each of the six connexin subunits having four helices. The narrowest part of the channel is ≈1.5 nm in diameter. M membrane bilayer E extracellular gap C cytosol. Part b from V. M. Unger et al. 1999 Science 283:1176.

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of connexins a family of structurally related transmembrane proteins with molecular weights between 26000 and 60000. A completely different family of proteins the innexins forms the gap junctions in invertebrates. Each vertebrate hexago- nal particle consists of 12 connexin molecules: 6 of the mol- ecules are arranged in a connexon hemichannel—a hexagonal cylinder in one plasma membrane—and joined to a connexon hemichannel in the adjacent cell membrane forming the con- tinuous aqueous channel between the cells. Each connexin molecule spans the plasma membrane four times one con- served transmembrane helix from each subunit apparently lines the aqueous channel. There are probably more than 20 different connexin genes in vertebrates and different sets of connexins are ex- pressed in different cell types. Some cells express a single con- nexin consequently their gap-junction channels are homotypic consisting of identical connexons. Most cells however express at least two connexins these different pro- teins assemble into hetero-oligomeric connexons which in turn form heterotypic gap-junction channels. This diversity in channel composition leads to differences in the permeabil- ity of channels to various molecules. For example channels made from a 43-kDa connexin isoform Cx43 are more than a hundredfold as permeable to ADP and ATP as those made from Cx32 32 kDa. Moreover the permeability of gap junctions can be altered by changes in the intracellular pH and Ca 2 concentration as well as by the phosphorylation of connexin providing numerous mechanisms for regulat- ing transport through them. The generation of mutant mice with inactivating muta- tions in connexin genes has highlighted the importance of connexins in a wide variety of cellular systems. For instance Cx43-defective mice exhibit numerous defects including de- fective oocyte maturation due to decreased gap-junctional communication between granulosa cells in the ovary. Mutations in several connexin genes are related to human diseases including neurosensory deafness Cx26 and Cx31 cataract or heart malformations Cx43 Cx46 and Cx50 and the X-linked form of Charcot- Marie-Tooth disease Cx32 which is marked by progressive degeneration of peripheral nerves. ❚ KEY CONCEPTS OF SECTION 6.5 Adhesive Interactions and Nonepithelial Cells ■ Many nonepithelial cells have integrin-containing aggre- gates e.g. focal adhesions 3D adhesions podosomes that physically and functionally connect cells to the extracellu- lar matrix and facilitate inside-out and outside-in signaling. ■ Integrins exist in two conformations that differ in the affinity for ligands and interactions with cytosolic adapter proteins see Figure 6-28. ■ Dystroglycan an adhesion receptor expressed by muscle cells forms a large complex with dystrophin other adapter proteins and signaling molecules see Figure 6-29. This complex links the actin cytoskeleton to the surrounding ma- trix providing mechanical stability to muscle. Mutations in various components of this complex cause different types of muscular dystrophy. ■ Neural cell-adhesion molecules CAMs which belong to the immunoglobulin Ig family of CAMs mediate Ca 2 -independent cell–cell adhesion predominantly in neural tissue and muscle. ■ The combinatorial and sequential interaction of several types of CAMs e.g. selectins integrins and ICAMs is crit- ical for the specific and tight adhesion of different types of leukocytes to endothelial cells in response to local signals induced by infection or inflammation see Figure 6-30. ■ Gap junctions are constructed of multiple copies of con- nexin proteins assembled into a transmembrane channel that interconnects the cytoplasm of two adjacent cells see Figure 6-32. Small molecules and ions can pass through gap junctions permitting metabolic and electrical coupling of adjacent cells. Plant Tissues We turn now to the assembly of plant cells into tis- sues. The overall structural organization of plants is generally simpler than that of animals. For in- stance plants have only four broad types of cells which in mature plants form four basic classes of tissue: dermal tis- sue interacts with the environment vascular tissue transports water and dissolved substances e.g. sugars ions space- filling ground tissue constitutes the major sites of metabolism and sporogenous tissue forms the reproductive organs. Plant tissues are organized into just four main organ systems: stems have support and transport functions roots provide anchorage and absorb and store nutrients leaves are the sites of photosynthesis and flowers enclose the repro- ductive structures. Thus at the cell tissue and organ levels plants are generally less complex than most animals. Moreover unlike animals plants do not replace or repair old or damaged cells or tissues they simply grow new or- gans. Most importantly for this chapter and in contrast with animals few cells in plants directly contact one another through molecules incorporated into their plasma mem- branes. Instead plant cells are typically surrounded by a rigid cell wall that contacts the cell walls of adjacent cells Figure 6-33. Also in contrast with animal cells a plant cell rarely changes its position in the organism relative to other cells. These features of plants and their organization have de- termined the distinctive molecular mechanisms by which their cells are incorporated into tissues. ❚ 6.6 6.6 • Plant Tissues 231

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The Plant Cell Wall Is a Laminate of Cellulose Fibrils in a Matrix of Glycoproteins The plant cell wall is ≈0.2 m thick and completely coats the outside of the plant cell’s plasma membrane. This structure serves some of the same functions as those of the extracellu- lar matrix produced by animal cells even though the two structures are composed of entirely different macromolecules and have a different organization. Like the extracellular ma- trix the plant cell wall connects cells into tissues signals a plant cell to grow and divide and controls the shape of plant organs. Just as the extracellular matrix helps define the shapes of animal cells the cell wall defines the shapes of plant cells. When the cell wall is digested away from plant cells by hydrolytic enzymes spherical cells enclosed by a plasma membrane are left. In the past the plant cell wall was viewed as an inanimate rigid box but it is now recognized as a dynamic structure that plays important roles in control- ling the differentiation of plant cells during embryogenesis and growth. Because a major function of a plant cell wall is to with- stand the osmotic turgor pressure of the cell the cell wall is built for lateral strength. It is arranged into layers of cellulose microfibrils—bundles of long linear extensively hydrogen- bonded polymers of glucose in glycosidic linkages. The cel- lulose microfibrils are embedded in a matrix composed of pectin a polymer of D-galacturonic acid and other mono- saccharides and hemicellulose a short highly branched polymer of several five- and six-carbon monosaccharides. The mechanical strength of the cell wall depends on cross- linking of the microfibrils by hemicellulose chains see Figure 6-33. The layers of microfibrils prevent the cell wall from stretching laterally. Cellulose microfibrils are synthesized on the exoplasmic face of the plasma membrane from UDP- glucose and ADP-glucose formed in the cytosol. The polymer- izing enzyme called cellulose synthase moves within the plane of the plasma membrane as cellulose is formed in directions determined by the underlying microtubule cytoskeleton. Unlike cellulose pectin and hemicellulose are synthesized in the Golgi apparatus and transported to the cell surface where they form an interlinked network that helps bind the walls of adjacent cells to one another and cushions them. When purified pectin binds water and forms a gel in the presence of Ca 2 and borate ions—hence the use of pectins in many processed foods. As much as 15 percent of the cell wall may be composed of extensin a glycoprotein that con- tains abundant hydroxyproline and serine. Most of the hy- droxyproline residues are linked to short chains of arabinose a five-carbon monosaccharide and the serine residues are linked to galactose. Carbohydrate accounts for about 65 per- cent of extensin by weight and its protein backbone forms an extended rodlike helix with the hydroxyl or O-linked car- bohydrates protruding outward. Lignin—a complex insol- uble polymer of phenolic residues—associates with cellulose and is a strengthening material. Like cartilage proteoglycans lignin resists compression forces on the matrix. The cell wall is a selective filter whose permeability is controlled largely by pectins in the wall matrix. Whereas water and ions diffuse freely across cell walls the diffusion of large molecules including proteins larger than 20 kDa is limited. This limitation may account for why many plant hormones are small water-soluble molecules which can dif- fuse across the cell wall and interact with receptors in the plasma membrane of plant cells. Loosening of the Cell Wall Permits Elongation of Plant Cells Because the cell wall surrounding a plant cell prevents the cell from expanding its structure must be loosened when the cell grows. The amount type and direction of plant cell growth are regulated by small-molecule hormones e.g. in- doleacetic acid called auxins. The auxin-induced weakening of the cell wall permits the expansion of the intracellular vacuole by uptake of water leading to elongation of the cell. We can grasp the magnitude of this phenomenon by consid- ering that if all cells in a redwood tree were reduced to the size of a typical liver cell the tree would have a maximum height of only 1 meter. The cell wall undergoes its greatest changes at the meri- stem of a root or shoot tip. These sites are where cells divide and expand. Young meristematic cells are connected by thin primary cell walls which can be loosened and stretched to 232 CHAPTER 6 • Integrating Cells into Tissues Primary wall Plasma membrane 50 nm Pectin Cellulose microfibril Hemicellulose ▲ FIGURE 6-33 Schematic representation of the cell wall of an onion. Cellulose and hemicellulose are arranged into at least three layers in a matrix of pectin polymers. The size of the polymers and their separations are drawn to scale. To simplify the diagram most of the hemicellulose cross-links and other matrix constituents e.g. extensin lignin are not shown. Adapted from M. McCann and K. R. Roberts 1991 in C. Lloyd ed. The Cytoskeletal Basis of Plant Growth and Form Academic Press p. 126.

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allow subsequent cell elongation. After cell elongation ceases the cell wall is generally thickened either by the se- cretion of additional macromolecules into the primary wall or more usually by the formation of a secondary cell wall composed of several layers. Most of the cell eventually de- generates leaving only the cell wall in mature tissues such as the xylem—the tubes that conduct salts and water from the roots through the stems to the leaves see Figure 8-45. The unique properties of wood and of plant fibers such as cotton are due to the molecular properties of the cell walls in the tissues of origin. Plasmodesmata Directly Connect the Cytosols of Adjacent Cells in Higher Plants Plant cells can communicate directly through specialized cell–cell junctions called plasmodesmata which extend through the cell wall. Like gap junctions plasmodesmata are open channels that connect the cytosol of a cell with that of an adjacent cell. The diameter of the cytosol-filled channel is about 30–60 nm and plasmodesmata can traverse cell walls as much as 90 nm thick. The density of plasmodesmata varies depending on the plant and cell type and even the smallest meristematic cells have more than 1000 intercon- nections with their neighbors. Molecules smaller than about 1000 Da including a va- riety of metabolic and signaling compounds generally can diffuse through plasmodesmata. However the size of the channel through which molecules pass is highly regulated. In some circumstances the channel is clamped shut in oth- ers it is dilated sufficiently to permit the passage of mole- cules larger than 10000 Da. Among the factors that affect the permeability of plasmodesmata is the cytosolic Ca 2 concentration with an increase in cytosolic Ca 2 reversibly inhibiting movement of molecules through these structures. Although plasmodesmata and gap junctions resemble each other functionally their structures differ in two significant ways Figure 6-34. The plasma membranes of the adjacent plant cells merge to form a continuous channel the annulus at each plasmodesma whereas the membranes of cells at a gap junction are not continuous with each other. In addition an extension of the endoplas- mic reticulum called a desmotubule passes through the annulus which connects the cytosols of adjacent plant cells. Many types of molecules spread from cell to cell through plasmodesmata including proteins nucleic acids metabolic products and plant viruses. Soluble molecules pass through the cytosolic annulus whereas membrane- bound molecules can pass from cell to cell through the desmotubule. 6.6 • Plant Tissues 233 Endoplasmic reticulum Cell 1 Cell 2 Plasma membrane Cell wall Desmotubule Annulus Plasmodesma a ▲ FIGURE 6-34 Structure of a plasmodesma. a Schematic model of a plasmodesma showing the desmotubule an extension of the endoplasmic reticulum and the annulus a plasma membrane–lined channel filled with cytosol that interconnects the cytosols of adjacent cells. Not shown is a gating complex that fills the channel and controls the transport of materials through the plasmodesma. b Electron micrograph of thin section of plant cell and cell wall containing multiple plasmodesmata. E. H. Newcomb and W. P . Wergin/Biological Photo Service. Plasmodesmata Plasma membrane Cell wall ER ER b

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Only a Few Adhe