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• W2000008518 abstract "Proteins with multiple cellular functions provide biological diversity to eukaryotic cells. In the current studies, we identified the mitochondrial functions of human prohibitin 2 (PHB2), which was initially identified as a repressor of estrogen-dependent transcriptional activity. The mitochondrial complex of PHB2 consists of PHB1, voltage-dependent anion channel 2, adenine nucleotide translocator 2, and the anti-apoptotic Hax-1, which is a novel binding partner for PHB2. RNA interference-mediated knockdown of PHB2 in HeLa cells resulted in caspase-dependent apoptosis through down-regulation of Hax-1 and fragmentation of mitochondria. We also found that, although PHB2 is predominantly expressed in the mitochondria of HeLa cells, it translocates to nucleus in the presence of estrogen receptor α and estradiol. Here, we first demonstrated the roles of mammalian PHB2 in mitochondria and the molecular mechanism of its nuclear targeting and showed that PHB2 is a possible molecule directly coupling nuclear-mitochondrial interaction. Proteins with multiple cellular functions provide biological diversity to eukaryotic cells. In the current studies, we identified the mitochondrial functions of human prohibitin 2 (PHB2), which was initially identified as a repressor of estrogen-dependent transcriptional activity. The mitochondrial complex of PHB2 consists of PHB1, voltage-dependent anion channel 2, adenine nucleotide translocator 2, and the anti-apoptotic Hax-1, which is a novel binding partner for PHB2. RNA interference-mediated knockdown of PHB2 in HeLa cells resulted in caspase-dependent apoptosis through down-regulation of Hax-1 and fragmentation of mitochondria. We also found that, although PHB2 is predominantly expressed in the mitochondria of HeLa cells, it translocates to nucleus in the presence of estrogen receptor α and estradiol. Here, we first demonstrated the roles of mammalian PHB2 in mitochondria and the molecular mechanism of its nuclear targeting and showed that PHB2 is a possible molecule directly coupling nuclear-mitochondrial interaction. In eukaryotes, biological diversity is acquired from a limited number of genes due to the existence of proteins with multiple cellular functions. A key factor is the dynamic regulation of these diverse cellular functions. One such multifunctional protein, prohibitin (PHB) 2The abbreviations used are: PHB, prohibitin; ANT, adenine nucleotide translocator; ERα, estrogen receptor alpha; GAPDH, glyceraldehydes 3-phosphate dehydrogenase; Hax-1, HS1-asociated protein X-1; MTS, mitochondrial targeting sequence; OPA1, optic atrophy 1; PTP, permeability transition pore; RNAi, RNA interference; siRNA, small interfering RNA; VDAC, voltage-dependent anion channel; GFP, green fluorescent protein; CMV, cytomegalovirus; GST, glutathione S-transferase; Z, benzyloxycarbonyl; FMK, fluoromethyl ketone; CHX, cycloheximide; PBS, phosphate-buffered saline; RT, reverse transcription; siPHB, siRNA for PHB.2The abbreviations used are: PHB, prohibitin; ANT, adenine nucleotide translocator; ERα, estrogen receptor alpha; GAPDH, glyceraldehydes 3-phosphate dehydrogenase; Hax-1, HS1-asociated protein X-1; MTS, mitochondrial targeting sequence; OPA1, optic atrophy 1; PTP, permeability transition pore; RNAi, RNA interference; siRNA, small interfering RNA; VDAC, voltage-dependent anion channel; GFP, green fluorescent protein; CMV, cytomegalovirus; GST, glutathione S-transferase; Z, benzyloxycarbonyl; FMK, fluoromethyl ketone; CHX, cycloheximide; PBS, phosphate-buffered saline; RT, reverse transcription; siPHB, siRNA for PHB. 1, was originally identified in mammals as a putative negative regulator of cell proliferation (1Nuell M.J. Stewart D.A. Walker L. Friedman V. Wood C.M. Owens G.A. Smith J.R. Schneider E.L. Dell'Orco R. Lumpkin C.K. Danner D.B. McClung J.K. Mol. Cell. Biol. 1991; 11: 1372-1381Crossref PubMed Scopus (228) Google Scholar). PHB1 and PHB2 are closely related proteins and are highly conserved among yeast (2McClung J.K. Jupe E.R. Liu X.T. Dell'Orco R.T. Exp. Gerontol. 1995; 30: 99-124Crossref PubMed Scopus (171) Google Scholar), plants (3Snedden W.A. Fromm H. Plant Mol. Biol. 1997; 33: 753-756Crossref PubMed Scopus (59) Google Scholar), worms (4Loukas A. Maizels R.M. DNA Seq. 1998; 9: 323-328Crossref PubMed Scopus (8) Google Scholar), flies (5Eveleth Jr., D.D. Marsh J.L. Nucleic Acids Res. 1986; 14: 6169-6183Crossref PubMed Scopus (70) Google Scholar), and mammals (6Sato T. Saito H. Swensen J. Olifant A. Wood C. Danner D. Sakamoto T. Takita K. Kasumi F. Miki Y. Cancer Res. 1992; 52: 1643-1646PubMed Google Scholar). Subcellular localization of PHBs has been confined to mitochondria in a variety of these species, although they also localize in nucleus in some mammalian cell lines (7Fusaro G. Dasgupta P. Rastogi S. Joshi B. Chellappan S. J. Biol. Chem. 2003; 278: 47853-47861Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 8Kurtev V. Margueron R. Kroboth K. Ogris E. Cavailles V. Seiser C. J. Biol. Chem. 2004; 279: 24834-24843Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). To date, it is known that PHBs are localized in the mitochondrial inner membrane where they form a large protein complex (9Nijtmans L.G. Artal S.M. Grivell L.A. Coates P.J. Cell Mol. Life Sci. 2002; 59: 143-155Crossref PubMed Scopus (251) Google Scholar). In addition, variety functions of PHBs have been suggested, including a role in cell cycle regulation (1Nuell M.J. Stewart D.A. Walker L. Friedman V. Wood C.M. Owens G.A. Smith J.R. Schneider E.L. Dell'Orco R. Lumpkin C.K. Danner D.B. McClung J.K. Mol. Cell. Biol. 1991; 11: 1372-1381Crossref PubMed Scopus (228) Google Scholar, 10Wang S. Nath N. Adlam M. Chellappan S. Oncogene. 1999; 18: 3501-3510Crossref PubMed Scopus (204) Google Scholar, 11Wang S. Nath N. Fusaro G. Chellappan S. Mol. Cell. Biol. 1999; 19: 7447-7460Crossref PubMed Scopus (142) Google Scholar), transmembrane signal transduction (12Terashima M. Kim K.M. Adachi T. Nielsen P.J. Reth M. Kohler G. Lamers M.C. EMBO J. 1994; 13: 3782-3792Crossref PubMed Scopus (206) Google Scholar, 13Montano M.M. Ekena K. Delage-Mourroux R. Chang W. Martini P. Katzenellenbogen B.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6947-6952Crossref PubMed Scopus (244) Google Scholar), and control of life span (14Coates P.J. Jamieson D.J. Smart K. Prescott A.R. Hall P.A. Curr. Biol. 1997; 7: 607-610Abstract Full Text Full Text PDF PubMed Google Scholar).In yeast, PHBs have been shown to exist in mitochondria and to function as chaperones that stabilize newly synthesized mitochondrial protein, possibly by negative regulation of the AAA protease (15Steglich G. Neupert W. Langer T. Mol. Cell. Biol. 1999; 19: 3435-3442Crossref PubMed Google Scholar, 16Nijtmans L.G. de Jong L. Artal Sanz M. Coates P.J. Berden J.A. Back J.W. Muijsers A.O. van der Spek H. Grivell L.A. EMBO J. 2000; 19: 2444-2451Crossref PubMed Scopus (445) Google Scholar). Even in Caenorhabditis elegans, PHBs are essential for embryonic viability and germ line differentiation, and deficiency of these proteins results in altered mitochondrial biogenesis (17Artal-Sanz M. Tsang W.Y. Willems E.M. Grivell L.A. Lemire B.D. van der Spek H. Nijtmans L.G. J. Biol. Chem. 2003; 278: 32091-32099Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar).Alternatively, in mammals, knowledge of human PHBs focused on their transcriptional regulatory functions has been accumulated. Human PHB1 has also been shown to interact with the retinoblastoma protein, which results in the inhibition of the transcriptional activity of E2F (10Wang S. Nath N. Adlam M. Chellappan S. Oncogene. 1999; 18: 3501-3510Crossref PubMed Scopus (204) Google Scholar). In a B-cell lymphoma line, stable overexpression of PHB1 protects the cells from camptothecin-induced apoptosis, possibly via down-regulation of E2F activity (18Fusaro G. Wang S. Chellappan S. Oncogene. 2002; 21: 4539-4548Crossref PubMed Scopus (94) Google Scholar). Furthermore, human PHB2, also known as a repressor of estrogen receptor (ER) activity, has been shown to interact with and inhibit the transcriptional activity of the ER (13Montano M.M. Ekena K. Delage-Mourroux R. Chang W. Martini P. Katzenellenbogen B.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6947-6952Crossref PubMed Scopus (244) Google Scholar). These findings suggest the involvement of mammalian PHB proteins in the regulation of transcription in the nucleus.On the contrary, in fibroblasts, mammalian PHB proteins mainly localize in mitochondria, and their expression is up-regulated by mitochondrial stress and down-regulated during cellular senescence (19Coates P.J. Nenutil R. McGregor A. Picksley S.M. Crouch D.H. Hall P.A. Wright E.G. Exp. Cell Res. 2001; 265: 262-273Crossref PubMed Scopus (167) Google Scholar). Therefore, it is thought that mammalian PHB proteins also have crucial roles in the mitochondria. Recently, human PHB1 was reported to associate with mitochondrial complex I, suggesting that PHB1 plays a role in complex assembly (20Bourges I. Ramus C. Mousson de Camaret B. Beugnot R. Remacle C. Cardol P. Hofhaus G. Issartel J.P. Biochem. J. 2004; 383: 491-499Crossref PubMed Scopus (108) Google Scholar). Thus, mammalian PHBs have been suggested to be involved not only in the regulation of transcription but also cellular senescence, apoptosis, and mitochondrial respiratory activity (9Nijtmans L.G. Artal S.M. Grivell L.A. Coates P.J. Cell Mol. Life Sci. 2002; 59: 143-155Crossref PubMed Scopus (251) Google Scholar, 18Fusaro G. Wang S. Chellappan S. Oncogene. 2002; 21: 4539-4548Crossref PubMed Scopus (94) Google Scholar). The molecular functions of PHBs in mitochondria, especially PHB2, however, remain unclear.In this study, to clarify the dynamic regulatory mechanism of the pleiotropic PHB2 in mammalian cells, we examined mitochondrial function of human PHB2 and then investigated its targeting mechanism to the nucleus. First, we identified the binding partners for PHB2 in the mitochondria by immunoprecipitation and mass spectrometric analysis. Hax-1, which was initially identified as an HS1-binding protein (21Suzuki Y. Demoliere C. Kitamura D. Takeshita H. Deuschle U. Watanabe T. J. Immunol. 1997; 158: 2736-2744PubMed Google Scholar) and as an inhibitor of apoptosis (22Cilenti L. Soundarapandian M.M. Kyriazis G.A. Stratico V. Singh S. Gupta S. Bonventre J.V. Alnemri E.S. Zervos A.S. J. Biol. Chem. 2004; 279: 50295-50301Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar), was found to directly associate with PHB2 in mitochondria. RNA interference (RNAi)-mediated knockdown of PHB2 resulted in a reduction of the level of Hax-1 protein. In addition, caspase-mediated apoptotic cell death was enhanced in PHB2 knockdown cells. This induction of cell death was likely due to the down-regulation of Hax-1, because its knockdown also caused the same manner of cell death without a reduction of PHB2 protein level. Furthermore, the knockdown of PHB2 caused the fragmentation of mitochondria by a mechanism independent of Hax-1.Secondly, we found that, although PHB2 is predominantly expressed in the mitochondria of HeLa cells, it translocates to the nucleus in the presence of ERα and estradiol (E2). We further found that human PHB2 contains both an uncleavable mitochondrial targeting sequence (MTS) at its N terminus and an ERα-dependent nuclear localization sequence at its C terminus, suggesting that it is shuttled from the nucleus to mitochondria.Taken together, our results show that human PHB2 has pleiotropic functions in the mitochondria, including inhibition of apoptosis via the PHB2·Hax-1 complex and regulation of the mitochondrial morphology. We also demonstrated that, in the presence of ERα and E2, PHB2 is translocated into the nucleus where it functions as a repressor of transcription. PHB2 is a versatile molecule that couples transcription in the nucleus and regulation of mitochondrial function in the mitochondria, suggesting that it plays a role in communication between these two organelles.EXPERIMENTAL PROCEDURESPlasmid Construction—The coding regions of PHB1, PHB2, and Hax-1 were amplified by PCR from a human heart or HeLa cell cDNA library. Amino acids 1-50 and 45-272 of PHB1 and 1-50 and 51-299 of PHB2 were also amplified by PCR. The PCR products with 3× FLAG tag sequences or GFP-coding sequences at their 3′ termini were introduced into mammalian expression vector pCMV-SPORT (Invitrogen). Amino acids 1-100 and the full-length (1-299) of PHB2 were amplified by PCR and introduced into mammalian expression vector pEF4/Myc-His B (Invitrogen). The coding sequence of PHB2 was also inserted into the pGEX-4T-3 vector (Amersham Biosciences) to express it as a GST fusion protein. For RNAi, siRNA sequences for PHB1 (5′-AACACAGCCTTCCTTCTGCTC-3′) (23Gamble S.C. Odontiadis M. Waxman J. Westbrook J.A. Dunn M.J. Wait R. Lam E.W. Bevan C.L. Oncogene. 2004; 23: 2996-3004Crossref PubMed Scopus (90) Google Scholar), PHB2 (5′-AAGAACCCTGGCTACATCAAA-3′), and Hax-1 (5′-AACCAGAGAGGACAATGATCT-3′) were introduced into the pSilencer 3.1-H1 Puro vector (Ambion, Austin, TX).Cell Culture and Transfection—HeLa and MCF7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C in an atmosphere containing 5% CO2. Transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Mitochondria were stained with rhodamine 123 (0.25 μg/ml) or MitoTracker Red CM-H2XRos (250 nm, Molecular Probes, Eugene, OR) for 30 min at 37 °C. Cells expressing pSilencer 3.1-H1 Puro constructs were selected with 2 μg/ml puromycin (Sigma). Caspase activity was inhibited with 50 μm Z-VAD-FMK (Calbiochem, San Diego, CA). Cells expressing the ER-expression plasmid were cultured in Opti-MEM I without phenol red (Invitrogen) supplemented with 10 nm ICI 182,780 (Tocris), and treated with 1 μm of E2 for 2 h. For inhibition of cytoplasmic translation, the cells were simultaneously cultured with 50 or 100 μg/ml cycloheximide (CHX).Immunocytochemistry—HeLa cells were plated on 35-mm poly-l-lysine-coated glass-bottomed dishes (Matsunami Glass Ind.) and fixed for 20 min at room temperature with 4% paraformaldehyde and 0.4% Triton X-100 in PBS. The cells were incubated with antibodies against FLAG (Sigma, rabbit polyclonal), cytochrome c (BD Pharmingen, mouse monoclonal), ERα (Upstate Biotechnology, rabbit polyclonal), or Myc (BD Biosciences, mouse monoclonal) in PBST (PBS with 0.05% Tween 20) containing 2% horse serum. After washing three times with PBS, the cells were incubated with Alexa 488-conjugated anti-rabbit IgG (Molecular Probes) and Cy3-conjugated anti-mouse IgG (Amersham Biosciences) in PBST containing 2% horse serum for 1 h at room temperature. Fluorescent images were captured and analyzed with a μRadiance™ Laser Scanning Confocal Microscope System (Bio-Rad).Preparation and Fractionation of Mitochondria—Mitochondria were prepared from HeLa cells as previously described (24Satoh M. Hamamoto T. Seo N. Kagawa Y. Endo H. Biochem. Biophys. Res. Commun. 2003; 300: 482-493Crossref PubMed Scopus (130) Google Scholar). To assess membrane association, mitochondria suspended in sucrose solution (0.25 m sucrose supplemented with 0.2 mm EDTA) were sonicated on ice. Intact mitochondria were removed by centrifugation at 4 °C for 10 min at 10,000 × g, and the supernatant containing sonicated mitochondria was further centrifuged at 4 °C for 30 min at 100,000 × g. The pellets were collected as the mitochondrial membrane fraction. The mitochondrial membrane fraction was then treated for 30 min on ice with 0.1 m Na2CO3 in sucrose solution. The solution was centrifuged at 4 °C for 30 min at 100,000 × g to separate the soluble proteins from the membranes.For the protease protection assay, mitochondria were treated for 20 min at room temperature with 0.25 mg/ml trypsin (Sigma) in sucrose solution containing the indicated concentrations of digitonin (Sigma) or 1% Triton X-100. The reaction was stopped by adding trichloroacetic acid.The submitochondrial fraction was prepared as follows: sonicated mitochondria in 0.45 m sucrose were layered onto a linear sucrose gradient (11 ml, 0.6-1.6 m sucrose in 10 mm HEPES-KOH, pH 7.4, and 0.2 mm EDTA) and centrifuged at 4 °C for 16 h at 100,000 × g. The gradient was collected in 25 0.5-ml fractions and then analyzed by Western blotting.Cell Counting—The living cell number in a 6-well plate was determined by trypan blue staining. Cells were trypsinized and collected by centrifugation. The cell pellets were suspended in 0.4% trypan blue solution (Invitrogen), and living cells were counted.Mass Spectrometry—Protein bands on acrylamide gels were stained with Coomassie Brilliant Blue R-250 and cut out. In-gel digestion with trypsin was carried out as described by Shevchenko et al. (25Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7772) Google Scholar). Proteins were identified using a Finnigan LTQ liquid chromatography-tandem mass spectrometry system (Thermo Electron Corp.).Western Blotting—Samples were separated by electrophoresis on SDS-polyacrylamide gels (10% or 12% acrylamide) and then electrophoretically transferred to nitrocellulose membranes (Hybond ECL, Amersham Biosciences). The membranes were probed with primary and horseradish peroxidase-conjugated secondary antibodies, and immunoreactive bands were visualized with enhanced chemiluminescence reagents (Amersham Biosciences). The following primary antibodies were used: anti-PHB1 (1:200, NeoMarkers), anti-PHB2 (1:1000, Upstate Biotechnology), anti-Hax-1 (1:250, BD Transduction Laboratories), anti-cytochrome c (1:100, BD Pharmingen), anti-GAPDH (1:3000, Chemicon), anti-VDAC (1:2000, Calbiochem), anti-FLAG (1:2000, Sigma), anti-PDHE2 (1:1000, Molecular Probes), anti-OPA1 (1:1000 (24Satoh M. Hamamoto T. Seo N. Kagawa Y. Endo H. Biochem. Biophys. Res. Commun. 2003; 300: 482-493Crossref PubMed Scopus (130) Google Scholar)), and anti-Mfn1/hFzo1 (1:100, rabbit anti-sera against the peptide fragment, CVQLENELENFTKQFLPSSNEES, corresponding to the C-terminal portion of the protein).Immunoprecipitation—The mitochondrial pellet from cells expressing PHB2-FLAG or Hax-1-FLAG was extracted with radioimmune precipitation assay buffer (20 mm Tris-HCl, pH 8, 150 mm NaCl, 1% sodium deoxycholate, and 1% Triton X-100). After sonication for 4 min on ice, the solution was centrifuged at 4 °C for 15 min at 10,000 × g. Immunoprecipitation was carried out by incubation of the supernatant with 5 μg of anti-FLAG antibody (Sigma, mouse monoclonal) and protein G-Sepharose (Amersham Biosciences) at 4 °C for overnight.In Vitro Binding Assay—Using Escherichia coli XL2-blue, expression of GST-PHB2 was induced with 1 mm isopropyl 1-thio-β-d-galactopyranoside for 3 h. The fusion protein was then affinity purified with glutathione-Sepharose 4B (Amersham Biosciences). FLAG-tagged Hax-1 and PHB1 were synthesized in vitro using the TnT SP6 Coupled Reticulocyte Lysate System (Promega). The in vitro-translated products were mixed with GST or GST-PHB2 proteins in TNE buffer (20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 2 mm EDTA, and 1% Nonidet P-40) and then affinity purified with glutathione-Sepharose 4B.RT-PCR—Total RNA was isolated from HeLa cells using TRIzol (Invitrogen) according to the manufacturer's instructions. Two micrograms of the total RNA was subjected to RT-PCR (SuperScript II, Invitrogen) using random hexamer primers for the RT reaction.RESULTSMammalian PHB Proteins Mainly Localize in the Mitochondria of HeLa Cells—In fibroblasts, mammalian PHB proteins are found mainly in the mitochondria (14Coates P.J. Jamieson D.J. Smart K. Prescott A.R. Hall P.A. Curr. Biol. 1997; 7: 607-610Abstract Full Text Full Text PDF PubMed Google Scholar), whereas they are also localized in the nucleus in MCF7 cells (7Fusaro G. Dasgupta P. Rastogi S. Joshi B. Chellappan S. J. Biol. Chem. 2003; 278: 47853-47861Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 8Kurtev V. Margueron R. Kroboth K. Ogris E. Cavailles V. Seiser C. J. Biol. Chem. 2004; 279: 24834-24843Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). We expressed human PHB proteins with a FLAG tag at their C termini in HeLa cells and found that they are mainly present in the mitochondria where they colocalize with mitochondrial cytochrome c (Fig. 1A, left panel). When we expressed GFP fusion proteins of PHBs in MCF-7 or HeLa cells, like the FLAG-tagged proteins, PHB1-GFP and PHB2-GFP were expressed in the mitochondria of HeLa cells (Fig. 1A, right panel) along with cytochrome c (data not shown). We found that a portion of PHB2-GFP was also present in the nucleus of MCF7 cells, whereas PHB1-GFP was only present in the mitochondria in these cells (Fig. 1A, right panel). These results demonstrate that, in HeLa cells, PHB proteins mainly localize in the mitochondria.Association of PHB2 with Hax-1 in Mitochondria—To clarify the function of human PHB2 in mitochondria, we next performed immunoprecipitation studies using a mitochondrial extract from HeLa cells expressing PHB2-FLAG. Immunoprecipitation with an anti-FLAG antibody (FLAG-IP) revealed that PHB2-FLAG specifically and reproducibly coprecipitates four proteins with molecular masses of 30-37 kDa (Fig. 1B), which were not detected in untransfected control extract (data not shown). Mass spectrometric analysis identified these proteins as PHB1, HS-associated protein X-1 (Hax-1) (21Suzuki Y. Demoliere C. Kitamura D. Takeshita H. Deuschle U. Watanabe T. J. Immunol. 1997; 158: 2736-2744PubMed Google Scholar), voltage-dependent anion channel 2 (VDAC2), and adenine nucleotide translocator 2 (ANT2), respectively (Fig. 1B). We expected that PHB1 would coprecipitate with PHB2, because they form stable complex in mitochondria (9Nijtmans L.G. Artal S.M. Grivell L.A. Coates P.J. Cell Mol. Life Sci. 2002; 59: 143-155Crossref PubMed Scopus (251) Google Scholar). ANT and VDAC are components of the permeability transition pore (PTP), and the association of PHB2 with ANT or VDAC has been reported as a possible mitochondrial nucleoid complex in Xenopus (26Bogenhagen D.F. Wang Y. Shen E.L. Kobayashi R. Mol. Cell. Proteomics. 2003; 2: 1205-1216Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Therefore, Hax-1 appears to be a novel binding partner of PHB2. The interaction between PHB2-FLAG and Hax-1 in the mitochondrial extract was confirmed by Western blotting with an antiHax-1 antibody following to FLAG-IP (Fig. 1C, upper panel). We further confirmed that Hax-1-FLAG coimmunoprecipitates with PHB2, PHB1, and VDAC (Fig. 1C, lower panel).Next, to determine whether the interaction between PHB2 and Hax-1 is direct, we performed in vitro pull-down assays. GST-PHB2 directly bound to Hax-1 (Fig. 1D, lane 6), but PHB1, which was believed to directly interact with PHB2 (14Coates P.J. Jamieson D.J. Smart K. Prescott A.R. Hall P.A. Curr. Biol. 1997; 7: 607-610Abstract Full Text Full Text PDF PubMed Google Scholar), was not pulled down by GST-PHB2 (Fig. 1D, lane 4). In addition, the presence of PHB1 did not disturb the interaction between GST-PHB2 and Hax-1 (Fig. 1D, lane 7).Hax-1 Is an Integral Protein of the Outer Mitochondrial Membrane—Hax-1, originally isolated as an HS-1 binding protein, is known to localize mainly in mitochondria (21Suzuki Y. Demoliere C. Kitamura D. Takeshita H. Deuschle U. Watanabe T. J. Immunol. 1997; 158: 2736-2744PubMed Google Scholar). We confirmed that FLAG-tagged Hax-1 is mainly localized in the mitochondria of HeLa cells (Fig. 2A).FIGURE 2Mitochondrial Hax-1 integrated in the outer membrane localized in the intermembrane space. A, C-terminally FLAG-tagged Hax-1 was expressed in HeLa cells. The HeLa cells were fixed and immunostained with anti-FLAG (α-FLAG) and anti-cytochrome c antibodies (α-cyt. c). As shown in the merged image (Merge), Hax-1 colocalizes with mitochondrial cytochrome c. Scale bars, 10 μm. B, mitochondria from HeLa cells were sonicated (sonic.) and separated into supernatants (sup.) and membrane-pellets (ppt.) by centrifugation at 100,000 × g. The membrane pellets were further treated with Na2CO3 (alkali) and separated into “sup.” and “ppt.” by centrifugation at 100,000 × g. Samples were analyzed by Western blotting using antibodies (α) against PHB1, PHB2, Hax-1, and cytochrome c (cyt. c). C, the outer membrane (OM) and the inner membrane (IM) fractions were prepared by sucrose density gradient centrifugation of submitochondrial particles. Proteins were analyzed by Western blotting with indicated antibodies. The Western blot analysis revealed that Hax-1 and the outer membrane protein VDAC are present in the outer membrane fraction. D, mitochondria were treated with various concentrations of trypsin, digitonin, and Triton X-100 and then analyzed by Western blotting with indicated antibodies. The protease protection assay showed that Hax-1 and cytochrome c are localized in the intermembrane space.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To clarify the submitochondrial localization of Hax-1, we initially examined whether Hax-1 is a membrane-integrated protein. Following sonication of HeLa cell mitochondria, most of the Hax-1 protein remained in the mitochondrial membrane pellets as did the integral inner membrane protein PHB1 and the inner membrane-associated protein cytochrome c (Fig. 2B, lane 3). As expected, alkali treatment of the mitochondrial membrane pellets released cytochrome c, an inner membrane-associated protein, from the membrane pellets (Fig. 2B, lanes 4 and 5). In contrast, Hax-1 as well as PHB1 and PHB2 remained in alkali-washed membrane pellets, indicating that they are integral mitochondrial membrane proteins (Fig. 2B, lane 5).We further examined whether Hax-1 is integrated in the outer or inner mitochondrial membrane. Submitochondrial particles were fractionated by sucrose density gradient centrifugation, and the outer membrane and inner membrane fractions were collected. The effectiveness of the separation of these two membrane types was confirmed by Western blotting with antibodies to the outer membrane protein VDAC and the inner membrane protein PHB1. We found that Hax-1 was present in the outer membrane fraction as along with VDAC (Fig. 2C), indicating that Hax-1 is integrated in the outer mitochondrial membrane.Protease protection assay was performed to further define the submitochondrial localization of Hax-1. Hax-1 as well as the mitochondrial matrix protein pyruvate dehydrogenase E2 subunit and the intermembrane space protein cytochrome c or optic atrophy 1 (OPA1) remained in intact mitochondria after trypsin treatment (Fig. 2D, lane 2). Selective disruption of the outer membrane with digitonin decreased Hax-1, cytochrome c, and OPA1 levels following trypsin treatment but not effect the level of pyruvate dehydrogenase E2 (Fig. 2D, lane 4), demonstrating that, like cytochrome c and OPA1, Hax-1 localizes in the intermembrane space. PHB proteins have been reported to be mitochondrial inner membrane proteins, and it has been suggested that their C termini are exposed to the intermembrane space in yeast (15Steglich G. Neupert W. Langer T. Mol. Cell. Biol. 1999; 19: 3435-3442Crossref PubMed Google Scholar). However, PHB proteins are known to be resistant to protease treatment after disruption of the outer membrane (19Coates P.J. Nenutil R. McGregor A. Picksley S.M. Crouch D.H. Hall P.A. Wright E.G. Exp. Cell Res. 2001; 265: 262-273Crossref PubMed Scopus (167) Google Scholar, 27Berger K.H. Yaffe M.P. Mol. Cell. Biol. 1998; 18: 4043-4052Crossref PubMed Scopus (161) Google Scholar), which may be due to their tight folding. Our experiments are consistent with these previous studies that PHB proteins are resistant to proteolytic degradation following disruption of the outer membrane.Knockdown of PHB2 Induces Reduction of Hax-1 Protein and Apoptosis—To clarify the function of human PHB2 in mitochondria and the significance of its interaction with Hax-1, we performed RNAi-mediated knockdown studies. Small interfering RNA (siRNA) for PHB1 or PHB2 based on a short hairpin RNA expression vector containing a puromycin-resistant gene was expressed in HeLa cells. Semiquantitative RT-PCR showed that the expression levels of PHB1 and PHB2 mRNAs were specifically reduced by siRNAs for PHB1 (siPHB1) and PHB2 (siPHB2), respectively (Fig. 3A). Western blotting confirmed that siPHB1 and siPHB2 decreased the levels of PHB1 and PHB2 but did not affect the expression of other mitochondrial proteins (cytochrome c, VDAC, and pyruvate dehydrogenase E2 subunit) or cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Fig. 3B). Although the siR-NAs were specific for each PHB protein at the mRNA level (Fig. 3A), the protein levels of PHB1 and PHB2 were reduced by siPHB2 and siPHB1, respectively. This is not surprising because the two PHB proteins are interdependent in yeast and C. elegans so that deletion of one decreases the protein level of the other (17Artal-Sanz M. Tsang W.Y. Willems E.M. Grivell L.A. Lemire B.D. van der Spek H. Nijtmans L.G. J. Biol. Chem. 2003; 278: 32091-32099Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 27Berger K.H. Yaffe M.P. Mol. Cell. Biol. 1998; 18: 4043-4052Crossref PubMed Scopus (161) Google Scholar).FIGURE 3RNAi-mediated knockdown of PHB2 down-regulates Hax-1. Five days after transfection, protein or mRNA expression in HeLa cells transfected with siRNA for PHB1 (siPHB1), PHB2 (siPHB2), or Hax-1 (siHax-1) was compared with untransfected cells (-) and/or cells expressi" @default.
• W2000008518 created "2016-06-24" @default.
• W2000008518 creator A5001663062 @default.
• W2000008518 creator A5005374458 @default.
• W2000008518 creator A5053216951 @default.
• W2000008518 creator A5078109225 @default.
• W2000008518 date "2006-11-01" @default.
• W2000008518 modified "2023-10-14" @default.
• W2000008518 title "Mitochondrial Functions and Estrogen Receptor-dependent Nuclear Translocation of Pleiotropic Human Prohibitin 2" @default.
• W2000008518 cites W1551597777 @default.
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