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BMC Cell Biology Access Evidence for a Mitochondrial Localization of the Retinoblastoma Protein

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Abstract Background: The retinoblastoma protein (Rb) plays a central role in the regulation of cell cycle, differentiation and apoptosis. In cancer cells, ablation of Rb function or its pathway is a consequence of genetic inactivation, viral oncoprotein binding or deregulated hyperphosphorylation. Some recent data suggest that Rb relocation could also account for the regulation of its tumor suppressor activity, as is the case for other tumor suppressor proteins, Results: In this reported study, we present evidence that a fraction of the total amount of Rb protein can localize to the mitochondria in proliferative cells taken from both rodent and human cells. This result is also supported by the use of Rb siRNAs, which substantially reduced the amount of mitochondrial Rb, and by acellular assays, in which [35S]-Methionine-labeled Rb proteins bind strongly to mitochondria isolated from rat liver. Moreover, endogenous Rb is found in an internal compartment of the mitochondria, within the inner-membrane. This is consistent with the protection of Rb from alkaline treatment, which destroys any interaction of proteins that are weakly bound to mitochondria.

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Conclusion: Although a few data regarding an unspecific cytosolic localization of Rb protein have been reported for some tumor cells, our results are the first evidence of a mitochondrial localization of Rb. The mitochondrial localization of Rb is observed in parallel with its classic nuclear location and paves the way for the study of potential as-yet-unknown roles of Rb at this site. Background: The retinoblastoma protein (Rb) was the first tumor suppressor protein to be identified [1]. Its loss of function is linked to the development of numerous human cancers [2]. This protein is a major regulator of cell cycle, differentiation and apoptosis. Many of Rb's effects on cell-cycle control derive from its ability to interact with and inhibitthe E2F family of transcription factors [3]. Ablation of Rb function in both cultured cells and animals, results, as expected, in deregulated proliferation, but also, more surprisingly, in apoptosis, according to both p53-dependent and p53-independent signaling pathways [4,5]. However, some reports demonstrate that Rb can also act as an inducer of cell death and point to a controversial role for this protein in the regulation of apoptosis [6]. In normal cells, the activity of Rb predominantly depends on the level of phosphorylation of the sixteen potential cdk phosphorylable serine/threonine residues span on the protein [7,8]. It is assumed that the phosphorylation of several critical sites is required to abolish the ability of Rb to interact with E2F factors and to inhibit cell cycle progression.

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Results and discussion: Mitochondrial Rb is detected by cell fractionation studies Here, we are interested in finding out whether Rb may also be located in other cell compartments, in addition to its conventionally reported nuclear localization. To achieve this, a cellular subfractionation study was first of all conducted in order to isolate enriched mitochondria and nuclei fractions from untreated or etoposide-treated human and rodent cells, such as human primary fibroblasts (HF), human fibrosarcoma cells (HT1080), rat pheochromocytoma cells (PC12) and rat immortalized fibroblasts (FR3T3). Then the total extract (T) and subcellular fractions (N and M) were loaded on gel and analyzed using the Western Blot technique (Fig. 1A). Putative contamination of the mitochondrial fractions was monitored by detecting cytosolic (tubulin) and nuclear (PCNA or lamin A) marker proteins, and antibodies directed against COX II or cytochrome c (mitochondrial markers in living cells) confirmed the enrichment of mitochondrial fractions. Even if nuclear fractions are contaminated to varying degrees by mitochondria, which are often difficult to separate using a specific mitochondria isolation method, mitochondrial fractions are nevertheless not contaminated with either nuclei or cytosolic proteins. In this study, as already outlined, the Rb protein is detected in nuclear fractions of untreated human cells, yet, surprisingly, a fraction of Rb is also detected in the mitochondrial fractions of these cells (Fig. 1A, lane 3 and 5), suggesting that Rb may also be located at this site in parallel with the classically-described nuclear localization.

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Mitochondrial localization of Rb Consequently, in order to more accurately determine the exact mitochondrial localization of Rb, we performed a Study of the in vitro interaction of Rb with mitochondria. Afterwards, to further validate these results, we tested the in vitro interaction ability of Rb protein with isolated mitochondria in an acellular assay. For this purpose, we prepared vectors expressing the full length of Rb, together with Luciferase (used as negative control) and Bax (used as positive control, in the presence of tBid) (Fig. 3A), and analyzed the binding of the corresponding in vitro translated proteins to mitochondria in a cell-free system. The fate of [35S]Methionine-labeled proteins was tested by analyzing the interaction with fresh rat liver mitochondria from rodent cells according to the protocol previously described for Bax in the literature [14,15]. As illustrated in Fig. 3A, the Rb protein (lane 3, mitochondria-bound proteins M1) is found to bind strongly to mitochondria, similar to Bax binding (lane 8); only slight amounts of Rb and Bax remained in the supernatant (lane 2 and 7, non-mitochondria- bound proteins S1). In contrast, the non-mitochondrial protein Luciferase used as negative control barely binds to mitochondria (lane 6) and the majority of the Luciferase remains in the supernatant (lane 5). This result is consistent with the data from the subcellular fractionation study and suggests that Rb has a high affinity for mitochondrial binding. It is important to note that the interaction of Rb with isolated mitochondria was performed in a buffer, which could enable importation of protein into the mitochondria. Consequently, we cannot exclude the possibility that Rb might be imported into an internal compartment in this study. To answer this question, but also in order to determine whether Rb is only weakly bound to mitochondria or whether there is a strong interaction, mitochondria preincubated with full length Rb produced in vitro were subjected to an alkaline treatment.

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The alkaline treatment is able to destroy any interaction of proteins that are weakly bound to mitochondria. This is the case for the Bax binding previously described [16] and which we used as a positive control for the alkaline treatment (Fig. 3B, lane 9). Interestingly, the Rb protein is resistant to alkaline extraction (Fig. 3B, lane 4), implying that the interaction between Rb and mitochondria involves strong binding. These data are in agreement with the result of Rb mitoplast localization from the mitochondria subfractionation study, and suggest that Rb may be tightly bound to the mitochondria inner-membrane, either in the inter-membrane space or in the matrix side. Moreover, we cannot exclude the possibility that Rb matrix-side location may play a role in control of the expression of mitochondrial encoded genes, somehow similar to its nuclear activity. The recent discovery of Sankaran et al. concerning the contribution of Rb to the expression of genes encoding proteins of mitochondrial respiration machinery [17], together with our findings concerning the mitochondrial localization of Rb, paves the way for the possibility of Rb involvement in mitochondrial biogenesis and function. Lastly, our findings concerning the mitochondrial localization of Rb do not exclude its classically observed nuclear localization. Thus, it is important to note that the antibody most widely used in the literature to detect Rb – the G3-245 antibody – recognizes the Rb protein in both nuclear and mitochondrial fractions using the Western Blot method. Conversely, in immunofluorescence studies, the G3-245 antibody we used cannot detect any mitochondrial pattern of Rb (see Additional file 1). This may be explained by the inaccessibility of the antibody to mitochondrial located Rb, either because of a masked epitope or due to the inability of the antibody to bypass the outer mitochondrial membrane. However, when we quantified the amount of mitochondrial Rb with respect to the total Rb from the Western Blots (Fig. 1A) and then normalized to the same number of cells, we found that mitochondrial Rb level was between 1% to 3% in comparison to the total Rb depending on the cell type. These results may explain why mitochondrial Rb was not previously observed.

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Figure 3 Rb interaction with mitochondria. Rb interaction with mitochondria: A. [35S]Met-Rb, -Luciferase and -Bax proteins produced in vitro were added to purified rat liver mitochondria and then the mitochondria-bound protein fraction (M1) was separated from the non-mitochondria-bound protein fraction (S1), then subjected to gel electrophoresis and autoradiography using a phosphoimager. In vitro translated proteins, were loaded in parallel as a control (C). B. In vitro translated [35S]Met-Rb, -Luciferase and -Bax were incubated as above and then the mitochondria were alkaline treated (Alk Treat.). Input: translated proteins (C); supernatant containing non-mitochondria-bound proteins (S1); supernatant containing detached proteins after alkaline treatment (S2); mitochondria-bound proteins after alkaline treatment (M2). These data are representative for 3 independent experiments.

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Methods Cell lines, cell culture and drugs FR3T3, HF and HT1080 were grown in Dulbecco's modified Eagle's medium (DMEM-F12) supplemented with 100 ìg/ìl penicillin, 100 U/ml streptomycin, 1% Glutamax and 10% fetal bovine serum under 5% CO2 and in a humidified atmosphere. PC12 cells were supplemented with 5% horse serum. For cell death induction, etoposide at a final concentration of 50 ìg/ml (Sigma, E1383) was added to freshly plated cultures. Western Blot reagents Western Blot was performed according to the method previously described [18] and the primary antibodies used were: mouse-monoclonal anti-Rb (G3-245, BD Pharmingen), anti-cytochrome c (BD Pharmingen) and anti-F1- ATPase (â-subunit MS503, MitoScience); rabbit-polyclonal anti-Enolase (donated by N. Lamande, College de France, Paris), anti-VDAC and anti-ANT (VDAC and ANT were donated by C. Brenner, UVSQ, Versailles, France); rat monoclonal anti-Tubulin (MAS078, Sera-Lab); goat polyclonal anti-Lamin A (C-20, Santa Cruz), anti-COX II (KRb-20, Santa Cruz), anti-uMtCK (C-18, Santa Cruz), anti- Actin (sc-8432, Santa Cruz) and anti-TFIID (sc-421, Santa Cruz). The secondary antibodies (peroxidase-conjugated) were anti-mouse, anti-rabbit, anti-rat or anti-goat immunoglobulin (Biosystem). Immunoreactive bands were detected by chemiluminescence using an ECL kit (Amersham).

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Conclusion In summary, our results support the presence of a fraction of the total amount of Rb protein in the mitochondria in both rat and human cells. Although some data revealing a cytosolic location of Rb have already been reported for tumors exhibiting a high level of cdk4 activity [12,13], these results are original because, to our knowledge, there is no data in the literature concerning a mitochondrial localization of Rb, with most bibliographic data pointing to a nuclear localization. Nevertheless, this type of location is not exhaustive: we found that most of the Rb was located in nuclear fractions, as previously described. The mitochondrial localization of Rb has been visualized by both cell fractionation and in vitro assays. At mitochondrial level, Rb seems to reside inside the organelle inasmuch as it was solely detected in the mitoplast fraction. Altogether, the results present strong evidence for the mitochondrial localization of a small fraction of cellular Rb, in parallel to the nuclear localization classically described, and support the specificity of this interaction.

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