Identification of a Novel Mitochondrial Complex Containing Mitofusin 2 and Stomatin-like Protein 2

A reverse genetics approach was utilized to discover new proteins that interact with the mitochondrial fusion mediator mitofusin 2 (Mfn2) and that may participate in mitochondrial fusion. In particular, in vivo formaldehyde cross-linking of whole HeLa cells and immunoprecipitation with purified Mfn2 antibodies of SDS cell lysates were used to detect an ∼42-kDa protein. This protein was identified by liquid chromatography and tandem mass spectrometry as stomatin-like protein 2 (Stoml2), previously described as a peripheral plasma membrane protein of unknown function associated with the cytoskeleton of erythrocytes (Wang, Y., and Morrow, J. S. (2000) J. Biol. Chem. 275, 8062–8071). Immunoblot analysis with anti-Stoml2 antibodies showed that Stoml2 could be immunoprecipitated specifically with Mfn2 antibody either from formaldehyde-cross-linked and SDS-lysed cells or from cells lysed with digitonin. Subsequent immunocytochemistry and cell fractionation experiments fully supported the conclusion that Stoml2 is indeed a mitochondrial protein. Furthermore, demonstration of mitochondrial membrane potential-dependent import of Stoml2 accompanied by proteolytic processing, together with the results of sublocalization experiments, suggested that Stoml2 is associated with the inner mitochondrial membrane and faces the intermembrane space. Notably, formaldehyde cross-linking revealed a "ladder" of high molecular weight protein species, indicating the presence of high molecular weight Stoml2-Mfn2 hetero-oligomers. Knockdown of Stoml2 by the short interfering RNA approach showed a reduction of the mitochondrial membrane potential, without, however, any obvious changes in mitochondrial morphology. A reverse genetics approach was utilized to discover new proteins that interact with the mitochondrial fusion mediator mitofusin 2 (Mfn2) and that may participate in mitochondrial fusion. In particular, in vivo formaldehyde cross-linking of whole HeLa cells and immunoprecipitation with purified Mfn2 antibodies of SDS cell lysates were used to detect an ∼42-kDa protein. This protein was identified by liquid chromatography and tandem mass spectrometry as stomatin-like protein 2 (Stoml2), previously described as a peripheral plasma membrane protein of unknown function associated with the cytoskeleton of erythrocytes (Wang, Y., and Morrow, J. S. (2000) J. Biol. Chem. 275, 8062–8071). Immunoblot analysis with anti-Stoml2 antibodies showed that Stoml2 could be immunoprecipitated specifically with Mfn2 antibody either from formaldehyde-cross-linked and SDS-lysed cells or from cells lysed with digitonin. Subsequent immunocytochemistry and cell fractionation experiments fully supported the conclusion that Stoml2 is indeed a mitochondrial protein. Furthermore, demonstration of mitochondrial membrane potential-dependent import of Stoml2 accompanied by proteolytic processing, together with the results of sublocalization experiments, suggested that Stoml2 is associated with the inner mitochondrial membrane and faces the intermembrane space. Notably, formaldehyde cross-linking revealed a "ladder" of high molecular weight protein species, indicating the presence of high molecular weight Stoml2-Mfn2 hetero-oligomers. Knockdown of Stoml2 by the short interfering RNA approach showed a reduction of the mitochondrial membrane potential, without, however, any obvious changes in mitochondrial morphology. Mammalian mitofusins Mfn1 and Mfn2 are large GTPases of the mitochondrial outer membrane that mediate mitochondrial fusion (1Santel A. Fuller M.T. J. Cell Sci. 2001; 114: 867-874Crossref PubMed Google Scholar, 2Santel A. Frank S. Gaume B. Herrler M. Youle R.J. Fuller M.T. J. Cell Sci. 2003; 116: 2763-2774Crossref PubMed Scopus (305) Google Scholar, 3Chan D.C. Annu. Rev. Cell Dev. Biol. 2006; 22: 79-99Crossref PubMed Scopus (720) Google Scholar). They also contain two coiled-coil domains or heptad repeats (HR1 and HR2) 2The abbreviations used are: HR, heptad repeat; DMEM, Dulbecco's modified Eagle's medium; FA, formaldehyde; FACS, fluorescence-activated cell sorting; LC/MS/MS, liquid chromatography and tandem mass spectrometry; MEF, mouse embryonic fibroblasts; PBS, phosphate-buffered saline; GSPBS, goat serum in PBS; siRNA, short interfering RNA; TMRE, tetramethylrhodamine ethyl ester perchlorate; IP, immunoprecipitation. 2The abbreviations used are: HR, heptad repeat; DMEM, Dulbecco's modified Eagle's medium; FA, formaldehyde; FACS, fluorescence-activated cell sorting; LC/MS/MS, liquid chromatography and tandem mass spectrometry; MEF, mouse embryonic fibroblasts; PBS, phosphate-buffered saline; GSPBS, goat serum in PBS; siRNA, short interfering RNA; TMRE, tetramethylrhodamine ethyl ester perchlorate; IP, immunoprecipitation. (3Chan D.C. Annu. Rev. Cell Dev. Biol. 2006; 22: 79-99Crossref PubMed Scopus (720) Google Scholar). The major portion of each of the proteins, including the N terminus-proximal GTPase domain and HR1 and the C terminus-proximal HR2, is exposed to the cytosol (4Rojo M. Legros F. Chateau D. Lombes A. J. Cell Sci. 2002; 115 (Pt 8): 1663-1674Crossref PubMed Google Scholar). The attachment to the outer membrane is mediated by two membrane-spanning segments that are separated by a small intermembrane space loop and that are located between the HR1 and HR2 repeats. Human Mfn1 and Mfn2 are highly homologous proteins with 62% identity at the amino acid level with both, however, being essential proteins. The homozygous -/- Mfn1-null and -/- Mfn2-null mice die during embryonic development (5Chen H. Detmer S.A. Ewald A.J. Griffin E.E. Fraser S.E. Chan D.C. J. Cell Biol. 2003; 160: 189-200Crossref PubMed Scopus (1743) Google Scholar). The mouse embryonic fibroblasts (MEF), derived from the -/- Mfn1-null or -/- Mfn2-null embryos, have distinct mitochondrial morphology defects, and exhibit severe reduction in mitochondrial fusion activity (5Chen H. Detmer S.A. Ewald A.J. Griffin E.E. Fraser S.E. Chan D.C. J. Cell Biol. 2003; 160: 189-200Crossref PubMed Scopus (1743) Google Scholar). Thus, Mfn1 and Mfn2 may have different functions in mitochondrial fusion. Unlike MEF lacking Mfn1 or Mfn2, MEF lacking both mitofusins completely lack mitochondrial fusion capacity and show severe cellular dysfunction (6Chen H. Chomyn A. Chan D.C. J. Biol. Chem. 2005; 280: 26185-26192Abstract Full Text Full Text PDF PubMed Scopus (989) Google Scholar). Functions of Mfn2 that are independent of its fusion activity, i.e. controlling mitochondrial metabolism and repressing vascular smooth muscle cell proliferation, have also been reported (7Pich S. Bach D. Briones P. Liesa M. Camps M. Testar X. Palacin M. Zorzano A. Hum. Mol. Genet. 2005; 14: 1405-1415Crossref PubMed Scopus (332) Google Scholar, 8Chen K.H. Guo X. Ma D. Guo Y. Li Q. Yang D. Li P. Qiu X. Wen S. Xiao R.P. Tang J. Nat. Cell Biol. 2004; 6: 872-883Crossref PubMed Scopus (318) Google Scholar).Mfn2 and Mfn1 have the capacity to form homo- and hetero-oligomers, as demonstrated by co-immunoprecipitation of tagged proteins (5Chen H. Detmer S.A. Ewald A.J. Griffin E.E. Fraser S.E. Chan D.C. J. Cell Biol. 2003; 160: 189-200Crossref PubMed Scopus (1743) Google Scholar, 9Ishihara N. Eura Y. Mihara K. J. Cell Sci. 2004; 117: 6535-6546Crossref PubMed Scopus (499) Google Scholar). At least one of the mitofusins is required on each of the adjacent mitochondria to promote mitochondrial fusion (10Koshiba T. Detmer S.A. Kaiser J.T. Chen H. McCaffery J.M. Chan D.C. Science. 2004; 305: 858-862Crossref PubMed Scopus (642) Google Scholar). The initial step of this process is characterized by tethering of two adjacent mitochondria via assembly of a mitofusin complex, which is mediated by HR2 forming a dimeric, antiparallel coiled-coil (10Koshiba T. Detmer S.A. Kaiser J.T. Chen H. McCaffery J.M. Chan D.C. Science. 2004; 305: 858-862Crossref PubMed Scopus (642) Google Scholar). The subsequent steps of mitochondrial fusion, including the exact function of the GTPase domains, are less well understood. In yeast, an interaction of the mitofusin homolog Fzo1, involving the GTPase domain and the HR domains, is essential for fusion (11Griffin E.E. Chan D.C. J. Biol. Chem. 2006; 281: 16599-16606Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). In the same organism, mitochondrial fusion has been reconstituted in vitro. Mitochondrial fusion proceeds through discreet and sequential mitochondrial outer and inner membrane fusion events, with both steps requiring GTP hydrolysis (12Meeusen S. McCaffery J.M. Nunnari J. Science. 2004; 305: 1747-1752Crossref PubMed Scopus (342) Google Scholar). A third GTPase, Opa1, appears also to be involved in the process of mitochondrial fusion (13Olichon A. Emorine L.J. Descoins E. Pelloquin L. Brichese L. Gas N. Guillou E. Delettre C. Valette A. Hamel C.P. Ducommun B. Lenaers G. Belenguer P. FEBS Lett. 2002; 523: 171-176Crossref PubMed Scopus (325) Google Scholar). This GTPase is synthesized as a precursor, which is processed by a matrix-processing peptidase to a mature form, large Opa1 (l-Opa1). l-Opa1 is anchored to the inner mitochondrial membrane and can undergo another proteolytic processing, which cleaves off the transmembrane segment and forms the small form (s-Opa1) (14Ishihara N. Fujita Y. Oka T. Mihara K. EMBO J. 2006; 25: 2966-2977Crossref PubMed Scopus (651) Google Scholar). In both yeast and mammals, the mitochondrial morphology is regulated through this proteolytic processing of Opa1 (Mgm1) in an ATP or mitochondrial membrane potential-dependent manner (14Ishihara N. Fujita Y. Oka T. Mihara K. EMBO J. 2006; 25: 2966-2977Crossref PubMed Scopus (651) Google Scholar, 15Herlan M. Vogel F. Bornhovd C. Neupert W. Reichert A.S. J. Biol. Chem. 2003; 278: 27781-27788Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 16McQuibban G.A. Saurya S. Freeman M. Nature. 2003; 423: 537-541Crossref PubMed Scopus (313) Google Scholar, 17Herlan M. Bornhovd C. Hell K. Neupert W. Reichert A.S. J. Cell Biol. 2004; 165: 167-173Crossref PubMed Scopus (187) Google Scholar). Interestingly, Opa1-mediated fusion was reported to depend on Mfn1 but not on Mfn2 (18Cipolat S. Martins de Brito O. Dal Zilio B. Scorrano L. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 15927-15932Crossref PubMed Scopus (884) Google Scholar). The detailed differences in Mfn1 and Mfn2 function are, however, unclear at present. In yeast, an additional protein, Ugo1, which participates in mitochondrial fusion, has been identified (19Sesaki H. Jensen R.E. J. Cell Biol. 2001; 152: 1123-1134Crossref PubMed Scopus (189) Google Scholar, 20Sesaki H. Jensen R.E. J. Biol. Chem. 2004; 279: 28298-28303Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). This protein spans the outer mitochondrial membrane and interacts, via its N terminus-proximal cytosolic domain, with Fzo1 and, via its C terminus-proximal intermembrane space domain, with Mgm1, thus linking the outer and the inner membrane fusion machinery. However, neither an Ugo1 homolog nor additional proteins that facilitate mitochondrial fusion have been identified in mammals.In this study, we have identified a novel complex containing Mfn2 and Stoml2. Our results suggest that Stoml2 is a novel mitochondrial intermembrane space/inner membrane-localized protein that forms a large hetero-oligomeric complex with Mfn2.EXPERIMENTAL PROCEDURESPlasmid Construction and in Vitro Transcription/Translation—To construct a plasmid encoding a His-tagged protein corresponding to amino acids 1–405 of Mfn2 (His-Mfn2-(1–405)), a 1215-nucleotide-long fragment of an MFN2 cDNA was amplified by PCR from pBSKIAA0214, which carries a human heart cDNA of MFN2 (21Nagase T. Seki N. Ishikawa K. Ohira M. Kawarabayasi Y. Ohara O. Tanaka A. Kotani H. Miyajima N. Nomura N. DNA Res. 1996; 3 (341-354): 321-329Crossref PubMed Scopus (205) Google Scholar), and cloned into the NdeI-BamHI sites of pET-15b. The resulting pET-15b/Mfn2-(1–405) construct was verified by sequencing. To prepare an Mfn2 mammalian expression plasmid, a ∼4550-nucleotidelong HindIII-NotI fragment of pBSKIAA0214 (21Nagase T. Seki N. Ishikawa K. Ohira M. Kawarabayasi Y. Ohara O. Tanaka A. Kotani H. Miyajima N. Nomura N. DNA Res. 1996; 3 (341-354): 321-329Crossref PubMed Scopus (205) Google Scholar), carrying the entire MFN2 cDNA, was cloned into the HindIII and NotI sites of pcDNA3 (Invitrogen).To prepare a Stoml2 expression plasmid, poly(A)+ RNA isolated from HeLa cells was reverse-transcribed and PCR-amplified using the Superscript One-Step reverse transcriptase-PCR system (Invitrogen). The resulting 1107-nucleotide-long fragment containing the entire open reading frame of STOML2 was subcloned into pGEM-Easy T (Promega) and verified by sequencing. An EcoRI fragment containing the entire open reading frame of STOML2 was further cloned into pcDNA3, and a clone of pcDNA3/Stoml2 with the correct orientation with respect to the cytomegalovirus promoter was selected.Cell Lines, Transfection, and siRNA Treatment—The HeLa human cell line (ATCC CCL-2) was grown in high glucose Dulbecco's modified Eagle's medium (DMEM; containing 4.5 g/liter glucose and 110 mg/liter sodium pyruvate, Invitrogen), supplemented with 10% fetal bovine serum. HeLa S3 (ATCC CCL-2.2), suspension-adapted HeLa cells, were grown as described (22Chomyn A. Methods Enzymol. 1996; 264: 197-211Crossref PubMed Google Scholar). For transient expression experiments, cells were transfected with pcDNA3-derived plasmids using FuGENE 6 (Roche Applied Science) and incubated for ∼36 h to allow transgene expression. For RNA interference-mediated knockdown experiments, cells were transfected with 20 nm siRNA directed against STOML2 mRNA or MFN2 mRNA (siGENOME SMART pool, Dharmacon) using Dharmafect I (Dharmacon), according the manufacturer's protocol, and incubated for 4 days to reduce Stoml2 or Mfn2 protein levels. In some experiments involving siRNASTOML2, cells were also transfected for a second time, on the third day, and incubated for a total of 6 days.Antibodies—His-Mfn2-(1–405) protein was expressed in the Escherichia coli strain BL21(DE3), and the recombinant protein was purified on nickel-nitrilotriacetic acid-agarose according to the manufacturer's protocol (Qiagen). The purified protein was used to generate antibodies in rabbits (Covance). The anti-Mfn2 antibodies were then blot-purified as described (23Tang W-J.Y. Methods Cell Biol. 1993; 37: 95-104Crossref PubMed Scopus (50) Google Scholar). The preparation and purification of anti-Mfn1 antibodies has been described (2Santel A. Frank S. Gaume B. Herrler M. Youle R.J. Fuller M.T. J. Cell Sci. 2003; 116: 2763-2774Crossref PubMed Scopus (305) Google Scholar). The following antibodies were also used: mouse anti-Bcl-2 monoclonal antibodies (Santa Cruz Biotechnology), goat anti-enolase polyclonal antibodies (Santa Cruz Biotechnology), rabbit anti-COXII serum (24Mariottini P. Chomyn A. Doolittle R.F. Attardi G. J. Biol. Chem. 1986; 261: 3355-3362Abstract Full Text PDF PubMed Google Scholar), mouse anti-COXIV monoclonal antibodies (Molecular Probes), mouse anti-F1-ATPase β-subunit monoclonal antibodies (Mitosciences), mouse anti-porin monoclonal antibodies (Calbiochem), rabbit anti-prohibitin polyclonal antibodies (Lab Vision), mouse anti-Opa1 monoclonal antibodies (BD Transduction Laboratories), chicken anti-Stoml2 (N-180) polyclonal IgY antibodies (Genway), chicken anti-Stoml2 (C-186) polyclonal IgY antibodies (Genway), mouse anti-α-tubulin monoclonal antibodies (Oncogene), and rabbit anti-actin polyclonal antibodies (Sigma).Isolation and Purification of Mitochondria—Adherent cells were harvested from 20 T-175 flasks by trypsinization, washed once in PBS (140 mm NaCl, 3.8 mm NaH2PO4, 16.2 mm Na2HPO4) containing 10% calf serum and twice in NKM buffer (0.13 m NaCl, 5 mm KCl, 7.5 mm MgCl2, 10 mm Tris-HCl, pH 7.4 (25 °C)). Cells in suspension culture, exponentially growing in a 3-liter volume, were harvested and washed twice in NKM buffer. The washed cells were resuspended in six volumes (relative to the packed cells) of TKM buffer (10 mm Tris-HCl, pH 6.7 (25 °C), 10 mm KCl, 0.15 mm MgCl2) containing a Complete Mini protease inhibitor mixture (Roche Applied Science) and, after 2 min, were disrupted with a motor-driven Potter-Elvehjem glass-Teflon homogenizer until ∼60–75% of the nuclei had been released (22Chomyn A. Methods Enzymol. 1996; 264: 197-211Crossref PubMed Google Scholar). The remainder of the preparation of the mitochondria-enriched fraction was as described (22Chomyn A. Methods Enzymol. 1996; 264: 197-211Crossref PubMed Google Scholar). The mitochondria were further purified by centrifugation through a discontinuous Percoll/metrizamide gradient as described (25Madden E.A. Storrie B. Anal. Biochem. 1987; 163: 350-357Crossref PubMed Scopus (36) Google Scholar, 26Storrie B. Madden E.A. Methods Enzymol. 1990; 182: 203-225Crossref PubMed Scopus (495) Google Scholar) but using an SW41 rotor. The purified mitochondria were washed and resuspended in STKM buffer (0.25 m sucrose in TKM) and stored at -80 °C.Submitochondrial Localization and Import of Stoml2—For membrane association studies, purified mitochondria (1 mg/ml) were incubated in a buffer (0.05 mm EDTA, Complete Mini protease inhibitor mixture, EDTA-free (Roche Applied Science), 20 mm HEPES, pH 7.5 (25 °C)) for 15 min on ice. Then the same volume of distilled H2O, 3.0 m NaCl, 0.2 m Na2CO3, or 2% Triton X-100 was added, and incubation was continued for 30 min. Samples were centrifuged at 100,000 × gav for 30 min, and both pellet and supernatant fractions were analyzed by Western blotting.For protease sensitivity studies, import-competent mitochondria (2 mg/ml) were incubated with 0.5 mg/ml proteinase K in a buffer (25 mm sucrose, 75 mm sorbitol, 100 mm KCl, 10 mm KH2PO4, 0.05 mm EDTA, 5 mm MgCl2, 10 mm Tris-HCl, pH 7.4 (25 °C)) in the presence of digitonin, as specified in Fig. 4, for 30 min on ice. The reaction was stopped by the addition of phenylmethylsulfonyl fluoride (5 mm final) and an equal volume of 2× Sample buffer (20% glycerol, 10% β-mercaptoethanol, 4% SDS, 125 mm Tris-HCl, pH 6.8 (25 °C)). The samples were boiled for 5 min and analyzed by Western blotting.Preparation of import-competent mitochondria and import of protein into isolated mitochondria were carried out as described (27Fernandez-Silva P. Martinez-Azorin F. Micol V. Attardi G. EMBO J. 1997; 16: 1066-1079Crossref PubMed Scopus (139) Google Scholar). The Stoml2 was synthesized in the rabbit reticulocyte lysate-based TNT T7-coupled transcription/translation system (Promega), in the presence of [35S]methionine (Amersham Biosciences), according the manufacturer's protocol.[35S]Met Labeling, Formaldehyde (FA) Cross-linking, and Immunoprecipitation—To label cells with [35S]methionine, 1.6 × 106 cells were grown in a 55-cm2 plate containing 4 ml of low methionine (2 × 10-2 mm methionine)-DMEM supplemented with 10% dialyzed fetal bovine serum in the presence of 0.5 mCi Expre35S35S protein labeling mixture (1000 Ci/mmol) for 16 h. After the labeling period, the cells were washed twice in regular culture medium and processed for FA cross-linking.35S-Labeled or unlabeled cells were scraped with a rubber policeman and resuspended in culture medium (∼2 × 106/10 ml). The cell suspension was mixed with a one-tenth volume of 11% FA (Polysciences) in PBS and incubated under mixing for 1–15 min at room temperature. Then, an equal volume of 250 mm glycine in PBS was added, and incubation was continued for 15 min to quench the unreacted FA. The cells were recovered by centrifugation and washed sequentially in PBS containing 1% bovine serum albumin and PBS.For denaturing immunoprecipitation, ∼1 × 106 cells were lysed in 0.05 ml of buffer A (1% SDS, 1 mm EDTA, 50 mm Tris-HCl, pH 7.5 (25 °C)) with vortexing in the presence of acid-washed glass beads. Then 0.85 ml of Tween-IP buffer (0.5% Tween 20, 150 mm NaCl, 0.1 mm EDTA, 50 mm Tris-HCl, pH 7.5 (25 °C)) containing the Complete Mini protease inhibitor mixture was added, and insoluble debris were removed by centrifugation at 16,000 × gav for 10 min. The supernatants (same amounts of total proteins) were incubated with the appropriate purified rabbit antibodies for 3 h at 4 °C. Then, 0.05 ml of the protein A-Sepharose suspension (100 mg of protein A-Sepharose (Sigma)/2.8 ml of a buffer (1 mg/ml bovine serum albumin, 1 mm NaN3, 10 mm Tris-HCl, pH 7.5)) was added, and the incubation was continued for 30 min. The immune complexes captured on Sepharose beads were centrifuged through a sucrose cushion (3.42 g of sucrose/10 ml of Tween-IP buffer), washed twice with Tween-IP buffer and once with 1% 2-mercaptoethanol, and solubilized in Sample buffer (10% glycerol, 5% β-mercaptoethanol, 2% SDS, 62.5 mm Tris-HCl, pH 6.8 (25 °C)). To reverse the cross-linking, the resulting samples were heated to 95 °C for 25 min. The proteins were separated by SDS-PAGE and detected by autoradiography or, after the transfer to a membrane, by immunoblotting as described below.In the case of the large scale experiment (Fig. 2B), mitochondria, prepared by differential centrifugation from 21 g of HeLa S3 cells, were treated with 1% FA in buffer B (250 mm Sucrose, 10 mm KCl, 0.15 mm MgCl2, 20 mm HEPES, pH 7.2 (25 °C)) for 15 min. The FA was quenched by the addition of an equal volume of 250 mm glycine in buffer B. The mitochondrial pellet was then lysed in 15 ml of buffer A, insoluble debris were removed by centrifugation, and the resulting supernatant was diluted 18-times with Tween-IP buffer. Protein complexes were captured using purified anti-Mfn2 antibodies covalently attached to Dynabeads-protein A (Dynal) with dimethyl pimelimidate dihydrochloride, according to the manufacturer's protocol, and were eluted with a buffer (500 mm NaCl, 100 mm glycine, pH 2.8 (25 °C)). The eluate was neutralized and concentrated on an Amicon Ultra centrifugal device (Millipore). Proteins were heat-treated, separated by SDS-PAGE, and detected by silver staining, and selected protein bands were excised. The further sample preparation and analysis was done by the Protein/Peptide Microanalytical Laboratory at the California Institute of Technology. Briefly, gel slices were treated with sequencing grade trypsin, and the resulting peptides were analyzed by liquid chromatography and tandem mass spectrometry (LC/MS/MS). The identified peptide sequences were matched to the National Center for Biotechnology Information sequence data base using MASCOT software. The probabilities-based Mowse scores and the coverage of the proteins with the identified peptides were as follows (probability Mowse score/sequence coverage in percent): Stoml2, 42A (430/26), Stoml2, 42B (430/26), Mfn1 (500/28), Mfn2 (570/33).FIGURE 2Stoml2 is specifically immunoprecipitated with Mfn2 antibodies from FA-cross-linked cells. A,[35S]Met-labeled HeLa CCL2 cells were treated with FA, lysed, immunoprecipitated with either preimmune serum (pre-i) or anti-Mfn2 antibodies, heat-treated to 95 °C to reverse cross-linked products (indicated as revers.), and analyzed by SDS-PAGE and autoradiography. Time (in minutes) of FA treatment is indicated. The nonspecific ∼47- and ∼52-kDa bands were absent in the HeLa S3 immunoprecipitates (not shown). B, the mitochondrial fraction, prepared by differential centrifugation from HeLa S3 cells, was treated with FA for 15 min, lysed with SDS, and immunoprecipitated with anti-Mfn2 antibodies attached to magnetic beads. The resulting immunoprecipitate was heat-treated and analyzed by SDS-PAGE and silver staining. C, HeLa CCL2 cells were treated as indicated with FA for 15 min, lysed with SDS, immunoprecipitated with either preimmune serum or anti-Mfn2 antibodies, heat-treated, and analyzed by Western blotting with Stoml2 antibodies. In parallel, 1% of total cell lysate used for immunoprecipitation (1% of input) was analyzed.View Large Image Figure ViewerDownload Hi-res image Download (PPT)For nondenaturing immunoprecipitation, ∼1 × 106 cells were lysed in 0.8 ml of a co-IP buffer (0.5% digitonin, 120 mm NaCl, Complete Mini protease inhibitor mixture, 50 mm HEPES, pH 7.5 (25 °C)) for 15 min at 4 °C. The insoluble debris were removed by centrifugation at 16,000 × gav for 10 min. The resulting supernatants were incubated with the appropriate antibodies and protein A-Sepharose as described above. The immune complexes captured on Sepharose beads were centrifuged through a sucrose cushion (3.42 g of sucrose/10 ml of co-IP buffer), washed three times with co-IP buffer and once with co-IP buffer lacking digitonin, and further analyzed by Western blotting.Sample Preparation and Western Blot Analysis—To remove contaminating rabbit IgGs present in the rabbit reticulocyte lysate-based TNT T7-coupled transcription/translation system, which could interfere, in the Western blot analysis, with detection of in vitro synthesized Stoml2, samples of in vitro synthesized Stoml2 were incubated with protein A-Sepharose beads in an appropriate buffer (50 mm NaCl, 0.1 mm EDTA, 0.1% Tween 20, 25 mm Tris-HCl, pH 7.5 (25 °C)) for 1 h at 4°C, and the Sepharose beads were then removed by centrifugation.The FA-cross-linked cells or FA-cross-linked purified mitochondria were incubated with buffer containing 2% SDS, 100 mm NaCl, and 10 mm Tris-HCl, pH 8.0 (25 °C) for 15 min and then centrifuged to remove insoluble debris, and the resulting extracts were used for Western blot analysis.The total protein concentration in samples of whole cells, cell extracts, or purified mitochondria was determined by the Bradford method or by the bicinchoninic acid method (28Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213377) Google Scholar, 29Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18445) Google Scholar). Samples (whole cells, subcellular fractions, purified mitochondria, protein A-Sepharose-treated in vitro synthesized proteins, or immunoprecipitated proteins) were mixed with an equal volume of 2× Sample buffer and analyzed by SDS-PAGE. Rainbow (Amersham Biosciences RPN756), Magic Mark XP (Invitrogen), or Kaleidoscope (Bio-Rad) size markers were analyzed in parallel. Proteins were then electrophoretically transferred to a Hybond ECL nitrocellulose membrane (Amersham Biosciences) in a previously described transfer buffer (30Bossy-Wetzel E. Newmeyer D.D. Green D.R. EMBO J. 1998; 17: 37-49Crossref PubMed Scopus (1105) Google Scholar) modified to contain 0.037% SDS. Blocking, first and second antibody incubations, and washes were carried out in PBSTw (PBS plus 0.1% Tween 20) or PBSTw containing 5% nonfat milk. The specific protein complexes were identified by autoradiography using the Super-Signal West Pico chemiluminescence reagent (Pierce).Fluorescence Microscopy and FACS Analysis—Cells were grown in a chambered coverglass (Lab-Tek), and the medium was replaced ∼12 h before an experiment. Cells were stained with 25 nm tetramethylrhodamine ethyl ester perchlorate (TMRE, Molecular Probes) or 50 nm Mitotracker Red CMXRos (Molecular Probes) in DMEM without serum for 30 min and washed again with culture medium. Microscopy was performed using an inverted Nikon Diaphot microscope equipped with a Nikon G2A filter cube, a Zeiss Planapo 63×/1.4 oil objective, and a Nikon D50 digital camera. For FACS analysis, cells were stained with 10 nm TMRE, trypsin-harvested, resuspended in PBS containing 10 nm TMRE, and analyzed by a FACSCalibur analyzer (Bio-Rad). Oxygen consumption measurements were done as described previously (31Hajek P. Villani G. Attardi G. J. Biol. Chem. 2001; 276: 606-615Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar).Confocal Immunofluorescence Microscopy—Cells grown on glass coverslips were sequentially incubated in 2% FA in PBS, PBS, anhydrous methanol, PBS, and 2% goat serum in PBS (GSPBS) containing 0.5% Triton X-100. The coverslips were then incubated with anti-Stoml2 antibodies (C-186; diluted 1:50 in GSPBS) and anti-COXIV antibodies (diluted to 5 μg/ml in GSPBS) for 3 h at 37°Cina humidified chamber. As a negative control, nonimmune chicken IgY antibodies (Aves Labs) were used instead of the anti-Stoml2 antibodies. After four washes in GSPBS, the coverslips were incubated with 1:50-diluted fluorescein isothiocyanate-conjugated donkey antichicken IgY and 1:50-diluted rhodamine Red-X-conjugated goat anti-mouse IgG (both from Jackson ImmunoResearch Laboratories) for 1 h at room temperature. After four washes in PBS, the coverslips were mounted onto microscope slides in FluoroGuard antifade reagent (Bio-Rad) and analyzed on a Zeiss 410 laser-scanning microscope equipped with a 488-nm argon and a 543-nm helium neon laser, a Zeiss 63×/1.25 oil objective, and Zeiss LSM software.RESULTSWe used in vivo FA cross-linking of HeLa cells to preserve Mfn2 protein complexes and to identify possible Mfn2-interacting proteins. FA is a highly specific cross-linker that is reactive with primary amines within 2 Aå of one another, is easily reversible, and is used to preserve protein-protein, protein-DNA, or protein-RNA complexes (32Orlando V. Strutt H. Paro R. Methods (Amst.). 1997; 11: 205-214Google Scholar). To detect Mfn2, immunoblotting with antibodies generated against a large portion of the Mfn2 protein was used. These antibodies detected a major immunoreactive protein in the untreated cell lysates (Fig. 1A, left panel, first lane), identified as Mfn2, as it was specifically increased in amount when HeLa cells were transfected with an Mfn2 expression vector (see Fig. 7B) and was specifically reduced when HeLa cells were treated with siRNAMFN2