Biogenesis of the Essential Tim9–Tim10 Chaperone Complex of Mitochondria
2007; Elsevier BV; Volume: 282; Issue: 31 Linguagem: Inglês
10.1074/jbc.m703294200
ISSN1083-351X
AutoresDusanka Milenkovic, Kipros Gabriel, Bernard Guiard, Agnes Schulze‐Specking, Nikolaus Pfanner, Agnieszka Chaciñska,
Tópico(s)Mitochondrial Function and Pathology
ResumoThe mitochondrial intermembrane space (IMS) contains an essential machinery for protein import and assembly (MIA). Biogenesis of IMS proteins involves a disulfide relay between precursor proteins, the cysteine-rich IMS protein Mia40 and the sulfhydryl oxidase Erv1. How precursor proteins are specifically directed to the IMS has remained unknown. Here we systematically analyzed the role of cysteine residues in the biogenesis of the essential IMS chaperone complex Tim9–Tim10. Although each of the four cysteines of Tim9, as well as of Tim10, is required for assembly of the chaperone complex, only the most amino-terminal cysteine residue of each precursor is critical for translocation across the outer membrane and interaction with Mia40. Mia40 selectively recognizes cysteine-containing IMS proteins in a site-specific manner in organello and in vitro. Our results indicate that Mia40 acts as a trans receptor in the biogenesis of mitochondrial IMS proteins. The mitochondrial intermembrane space (IMS) contains an essential machinery for protein import and assembly (MIA). Biogenesis of IMS proteins involves a disulfide relay between precursor proteins, the cysteine-rich IMS protein Mia40 and the sulfhydryl oxidase Erv1. How precursor proteins are specifically directed to the IMS has remained unknown. Here we systematically analyzed the role of cysteine residues in the biogenesis of the essential IMS chaperone complex Tim9–Tim10. Although each of the four cysteines of Tim9, as well as of Tim10, is required for assembly of the chaperone complex, only the most amino-terminal cysteine residue of each precursor is critical for translocation across the outer membrane and interaction with Mia40. Mia40 selectively recognizes cysteine-containing IMS proteins in a site-specific manner in organello and in vitro. Our results indicate that Mia40 acts as a trans receptor in the biogenesis of mitochondrial IMS proteins. Mitochondria must import most of their ∼1,000 different proteins from the cytosol. The classical pathway of mitochondrial protein import involves cleavable amino-terminal presequences that direct precursor proteins to the translocase of the outer membrane (TOM 4The abbreviations used are: TOM, translocase of outer membrane; TIM, translocase of inner membrane; IMS, intermembrane space; MIA, mitochondrial IMS import and assembly machinery; DTT, 1,4-dithiothreitol; Ni-NTA, nickel-nitrilotriacetic acid; MOPS, 4-morpholinepropanesulfonic acid. complex) and the presequence translocase of the inner membrane (TIM23 complex) (1Dolezal P. Likic V. Tachezy J. Lithgow T. Science. 2006; 313: 314-318Crossref PubMed Scopus (429) Google Scholar, 2Hoogenraad N.J. Ward L.A. Ryan M.T. Biochim. Biophys. Acta. 2002; 1592: 97-105Crossref PubMed Scopus (134) Google Scholar, 3Oka T. Mihara K. Mol. Cell. 2005; 18: 145-146Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 4Jensen R.E. 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The complex guides precursors of β-barrel proteins to the sorting and assembly machinery of the outer membrane (SAM complex) (20Wiedemann N. Kozjak V. Chacinska A. Schoönfisch B. Rospert S. Ryan M.T. Pfanner N. Meisinger C. Nature. 2003; 424: 565-571Crossref PubMed Scopus (301) Google Scholar, 21Wiedemann N. Truscott K.N. Pfannschmidt S. Guiard B. Meisinger C. Pfanner N. J. Biol. Chem. 2004; 279: 188-194Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 22Hoppins S.C. Nargang F.E. J. Biol. Chem. 2004; 279: 12396-12405Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), as well as the precursors of hydrophobic metabolite carriers to the carrier translocase of the inner membrane (TIM22 complex) (5Neupert W. Herrmann J.M. Annu. Rev. Biochem. 2007; 76: 723-749Crossref PubMed Scopus (1103) Google Scholar, 7Rehling P. Brandner K. Pfanner N. Nat. Rev. Mol. Cell Biol. 2004; 5: 519-530Crossref PubMed Scopus (281) Google Scholar, 11Koehler C.M. Annu. Rev. 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Mia40 binds to IMS precursor proteins upon their translocation through the TOM complex (24Chacinska A. Pfannschmidt S. Wiedemann N. Kozjak V. Sanjuán Szklarz L.K. Schulze-Specking A Truscott K.N. Guiard B Meisinger C Pfanner N. EMBO J. 2004; 23: 3735-3746Crossref PubMed Scopus (341) Google Scholar, 25Naoé M. Ohwa Y. Ishikawa D. Ohshima C. Nishikawa S. Yamamoto H. Endo T. J. Biol. Chem. 2004; 279: 47815-47821Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 26Terziyska N. Lutz T. Kozany C. Mokranjac D. Mesecke N. Neupert W. Herrmann J.M. Hell K. FEBS Lett. 2005; 579: 179-184Crossref PubMed Scopus (144) Google Scholar, 27Gabriel K. Milenkovic D. Chacinska A. Muöller J. Guiard B. Pfanner N. Meisinger C. J. Mol. Biol. 2007; 365: 612-620Crossref PubMed Scopus (128) Google Scholar, 28Terziyska N. Grumbt B. Bien M. Neupert W. Herrmann J.M. Hell K. FEBS Lett. 2007; 581: 1098-1102Crossref PubMed Scopus (54) Google Scholar). 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For Tim9 and Tim10, which contain four cysteine residues each, the high resolution structure of the hexameric complex demonstrated the formation of two intramolecular disulfide bonds in each subunit (19Webb C.T. Gorman M.A. Lazarou M. Ryan M.T. Gulbis J.M. Mol. Cell. 2006; 21: 123-133Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). Mia40 and Erv1 form a protein relay for disulfide formation in IMS precursor proteins by transferring disulfides from Erv1 via Mia40 to the substrate proteins (30Mesecke N. Terziyska N. Kozany C. Baumann F. Neupert W. Hell K. Hermann J.M. Cell. 2005; 121: 1059-1069Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar, 31Rissler M. Wiedemann N. Pfannschmidt S. Gabriel K. Guiard B. Pfanner N. Chacinska A. J. Mol. Biol. 2005; 353: 485-492Crossref PubMed Scopus (126) Google Scholar, 37Allen S. Balabanidou V. Sideris D.P. Lisowsky T. Tokatlidis K. J. Mol. Biol. 2005; 353: 937-944Crossref PubMed Scopus (193) Google Scholar). Thus, the IMS system is thought to resemble disulfide relays in the bacterial periplasm (DsbB and DsbA) (38Kadokura H. Katzen F. Beckwith J. Annu. Rev. Biochem. 2003; 72: 111-135Crossref PubMed Scopus (447) Google Scholar, 39Collet J.F. Bardwell J.C. Mol. Microbiol. 2002; 44: 1-8Crossref PubMed Scopus (178) Google Scholar) and the endoplasmic reticulum (Ero1/Erv2 and protein-disulfide isomerase) (40Sevier C.S. Kaiser C.A. Antioxid. Redox. Signal. 2006; 8: 797-811Crossref PubMed Scopus (91) Google Scholar, 41Tu B.P. Weissman J.S. J. Cell Biol. 2004; 164: 341-346Crossref PubMed Scopus (820) Google Scholar, 42Gerber J. Muöhlenhoff U. Hofhaus G. Lill R. Lisowsky T. J. Biol. Chem. 2001; 276: 23486-23491Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). However, Mia40 is not homologous to bacterial or endoplasmic reticulum proteins and performs a novel function by driving protein translocation across the outer mitochondrial membrane (24Chacinska A. Pfannschmidt S. Wiedemann N. Kozjak V. Sanjuán Szklarz L.K. Schulze-Specking A Truscott K.N. Guiard B Meisinger C Pfanner N. EMBO J. 2004; 23: 3735-3746Crossref PubMed Scopus (341) Google Scholar, 25Naoé M. Ohwa Y. Ishikawa D. Ohshima C. Nishikawa S. Yamamoto H. Endo T. J. Biol. Chem. 2004; 279: 47815-47821Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 26Terziyska N. Lutz T. Kozany C. Mokranjac D. Mesecke N. Neupert W. Herrmann J.M. Hell K. FEBS Lett. 2005; 579: 179-184Crossref PubMed Scopus (144) Google Scholar). The molecular mechanism of protein targeting to Mia40 has not been elucidated. It is unknown whether Mia40 generally interacts with cysteine-rich proteins or specifically recognizes IMS-destined proteins. For this study, we dissected the mitochondrial import steps of the precursors of Tim9 and Tim10 and analyzed the role of individual cysteine residues of the precursors in translocation across the outer membrane, binding to Mia40 and assembly into the Tim9–Tim10 chaperone complex. We report that the cysteine residues play different roles in membrane translocation and oligomeric assembly. Translocation across the outer membrane is coupled to binding to Mia40, a process that depends on one defined cysteine residue of each precursor. Mia40 distinguishes between different cysteine residues of a precursor protein in a site-specific manner in vitro and in organello. Our findings suggest that Mia40 functions as a receptor that binds precursors via transient disulfide bonds, thereby promoting substrate entry into the IMS-specific pathway and initiating the subsequent assembly process. Yeast Strains and Media—The Saccharomyces cerevisiae strain YPH499 (MATa, ade2-101, his3-Δ200, leu2-Δ1, ura3-52, trp1-Δ63, lys2-801) was used as wild type (43Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar). The mutant strains mia40-4 (MATa, ade2-101, his3-Δ200, leu2-Δ1, ura3-52, trp1-Δ63, lys2-801, mia40::ADE2 (pFL39-FOMP2–7ts/mia40-4)) and erv1-2 (YBG-0702b; MATa, ade2-101, his3-Δ200, leu2-Δ1, ura3-52, trp1-Δ63, lys2-801, erv1::ADE2 (pFL39-ERV1–2ts)) were reported previously (24Chacinska A. Pfannschmidt S. Wiedemann N. Kozjak V. Sanjuán Szklarz L.K. Schulze-Specking A Truscott K.N. Guiard B Meisinger C Pfanner N. EMBO J. 2004; 23: 3735-3746Crossref PubMed Scopus (341) Google Scholar, 31Rissler M. Wiedemann N. Pfannschmidt S. Gabriel K. Guiard B. Pfanner N. Chacinska A. J. Mol. Biol. 2005; 353: 485-492Crossref PubMed Scopus (126) Google Scholar). The mutant strain erv1-3 (YBG-0701a; MATa, ade2-101, his3-Δ200, leu2-Δ1, ura3-52, trp1-Δ63, lys2-801, erv1::ADE2 (pFL39-ERV1–1ts)) was generated by error-prone PCR and plasmid shuffling according to a published procedure (44Truscott K.N. Wiedemann N. Rehling P. Muöller H. Meisinger C. Pfanner N. Guiard B. Mol. Cell. Biol. 2002; 22: 7780-7789Crossref PubMed Scopus (86) Google Scholar). Yeast strains were grown at 19 or 30 °C in medium containing 1% (w/v) yeast extract, 2% (w/v) bacto-peptone, and 3% (v/v) glycerol (YPG). Isolation of Mitochondria—S. cerevisiae mutant strains and the corresponding wild-type strain were grown in parallel in YPG medium to an A600 nm of ∼1.5. After cell disruption by Zymolyase treatment and homogenization, mitochondria were isolated by differential centrifugation according to a standard procedure (45Meisinger C. Pfanner N. Truscott K.N. Methods Mol. Biol. 2006; 313: 33-39PubMed Google Scholar). The concentration of mitochondria was adjusted to 10 mg of protein/ml in SM buffer (250 mm sucrose, 10 mm MOPS/KOH, pH 7.2). Generation of Tim9 and Tim10 Cysteine Mutants—Single, double, and quadruple cysteine to serine mutants of Tim9 and Tim10 were generated by PCR-based site-directed mutagenesis (Stratagene QuikChange™ site-directed mutagenesis kit) from pGEM-4Z-based plasmids containing the cloned gene of interest. Primer design and PCR were performed according to the manufacturer's guidelines. The mutations were verified by sequencing (GATC Biotech). Protein Import into Mitochondria—35S-Labeled precursor proteins were synthesized using the TnT SP6 coupled transcription/translation kit (Promega) or reticulocyte lysates in the presence of [35S]methionine (GE Healthcare). After denaturation in urea buffer (8 m urea, 30 mm MOPS, 50 mm DTT, pH 7.2), 35S-labeled precursors (2% volume of import assay) were incubated with isolated yeast mitochondria (50–100 μg of protein) at 30 (for Tim9) and 35 °C (for Tim10) in import buffer (3% (w/v) fatty acid-free bovine serum albumin, 250 mm sucrose, 5 mm MgCl2, 80 mm KCl, 10 mm MOPS/KOH, 5 mm methionine, 5–10 mm KH2PO4, pH 7.2). Import reactions were stopped by placing samples on ice. Where indicated samples were treated with 50 μg/ml proteinase K. Samples were subsequently washed with SEM buffer (250 mm sucrose, 1 mm EDTA, 10 mm MOPS/KOH, pH 7.2). Mitochondria were reisolated and resuspended in Laemmli buffer (for SDS-PAGE) or digitonin solubilization buffer (1% (w/v) digitonin, 20 mm Tris-HCl, 0.5 mm EDTA, 10% (v/v) glycerol, 50 mm NaCl, 1 mm phenylmethylsulfonyl fluoride, pH 7.4) for blue native electrophoresis (24Chacinska A. Pfannschmidt S. Wiedemann N. Kozjak V. Sanjuán Szklarz L.K. Schulze-Specking A Truscott K.N. Guiard B Meisinger C Pfanner N. EMBO J. 2004; 23: 3735-3746Crossref PubMed Scopus (341) Google Scholar, 46Schaögger H. von Jagow G. Anal. Biochem. 1991; 199: 223-231Crossref PubMed Scopus (1909) Google Scholar). Reactions were separated by SDS-PAGE or blue native electrophoresis and analyzed by digital phosphorimager autoradiography (GE Healthcare). To generate mitoplasts, mitochondria were exposed to hypo-osmotic swelling in EM buffer (1 mm EDTA, 10 mm MOPS/KOH, pH 7.2) prior to the import reactions. Affinity Purification of Mia40-His10—35S-Labeled wild-type Tim9 was imported into mitochondria carrying a carboxyl-terminal His10-tagged version of Mia40 (31Rissler M. Wiedemann N. Pfannschmidt S. Gabriel K. Guiard B. Pfanner N. Chacinska A. J. Mol. Biol. 2005; 353: 485-492Crossref PubMed Scopus (126) Google Scholar) in YPH499 background. The mitochondria were lysed with 1% digitonin solubilization buffer containing 50 mm iodoacetamide and subjected to Ni-NTA-agarose affinity chromatography. After washing of the column, protein was eluted with elution buffer (20 mm Tris/HCl, 200 mm NaCl, 400 mm imidazole, 50 mm iodoacetamide, pH 7.4). Proteins were separated by nonreducing SDS-PAGE and analyzed by digital autoradiography and Western blotting with Mia40-specific antibodies. Purification of Mia40 Expressed in Escherichia coli—A fragment of DNA encoding a factor Xa cleavage site followed by Mia40 beginning at residue 95 was cloned into pET10N (47Truscott K.N. Kovermann P. Geissler A. Merlin A. Meijer M. Driessen A.J. Rassow J. Pfanner N. Wagner R. Nat. Struct. Biol. 2001; 8: 1074-1082Crossref PubMed Scopus (253) Google Scholar) for expression in E. coli cells. The resulting plasmid pN-10His-Mia40-(1–94Δ) was sequenced and transformed into the E. coli strain BL21. After growth to an A600 nm of 0.5, 1 mm isopropyl 1-thio-β-d-galactopyranoside was added to induce protein expression. After growth for a further 3 h, cells were harvested by centrifugation, resuspended in lysis buffer (300 mm NaCl, 50 mm NaH2PO4, 10 mg/ml RNase A, 5 mg/ml DNase I, pH 8.0), and exposed to sonication on ice. Cell lysates were spun at 12,000 × g at 4 °C for 30 min, and the supernatant fraction was used for Ni-NTA affinity chromatography. After washing with 10 column volumes of wash buffer (300 mm NaCl, 50 mm NaH2PO4, adjusted to pH 8.0), Mia40 was either eluted with elution buffer (20 mm Tris-HCl, 100 mm NaCl, 20% (v/v) glycerol, 250 mm imidazole, 50 mm iodoacetamide, pH 8.0) or factor Xa protease as per the manufacturer's instructions (New England Biolabs). In Vitro Binding of Proteins to Mia40—Purified Mia40 (column-immobilized or factor Xa-released) was incubated with 35S-labeled proteins in binding buffer (250 mm sucrose, 80 mm KCl, 5 mm MgCl2, 2 mm KH2PO4, 5 mm methionine, 10 mm MOPS/KOH, pH 7.2) at 4–30 °C for 4–10 min. Reactions were stopped by addition of iodoacetamide to a final concentration of 50 mm. Fractions bound to Ni-NTA columns were washed with washing buffer (30 mm imidazole, 250 mm sucrose, 80 mm KCl, 5 mm MgCl2, 2 mm KH2PO4, 5 mm methionine, 10 mm MOPS/KOH, pH 7.2), eluted with elution buffer (20 mm Tris-HCl, 100 mm NaCl, 250 mm imidazole, pH 8.0), and analyzed by SDS-PAGE. Binding of precursors to the factor Xa-cleaved Mia40 was analyzed directly by nonreducing SDS-PAGE, followed by digital phosphorimager autoradiography (GE Healthcare). Cysteine Residues Are Critical for the Biogenesis Pathway of Tim9—We used blue native electrophoresis to study the import and assembly of radiolabeled Tim9 into isolated yeast mitochondria. The native gel assay of digitonin-lysed mitochondria not only separates the two mature Tim9-containing complexes Tim9–Tim10 and TIM22 but also an import intermediate of Tim9 bound to Mia40 (Fig. 1A, lane 1) (24Chacinska A. Pfannschmidt S. Wiedemann N. Kozjak V. Sanjuán Szklarz L.K. Schulze-Specking A Truscott K.N. Guiard B Meisinger C Pfanner N. EMBO J. 2004; 23: 3735-3746Crossref PubMed Scopus (341) Google Scholar, 31Rissler M. Wiedemann N. Pfannschmidt S. Gabriel K. Guiard B. Pfanner N. Chacinska A. J. Mol. Biol. 2005; 353: 485-492Crossref PubMed Scopus (126) Google Scholar). The addition of the SH-modifying reagents N-ethylmaleimide or iodoacetamide blocked the import of Tim9 and its association with Mia40 (Fig. 1A, lanes 2 and 3), indicating that cysteine residues are crucial for formation of the Mia40 intermediate. Mature Tim9 possesses two intramolecular disulfide bonds, one formed by the two outer cysteines (Cys-35 and Cys-59) and the other formed by the two inner cysteines (Cys-39 and Cys-55) (19Webb C.T. Gorman M.A. Lazarou M. Ryan M.T. Gulbis J.M. Mol. Cell. 2006; 21: 123-133Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 32Curran S.P. Leuenberger D. Oppliger W. Koehler C.M. EMBO J. 2002; 21: 942-953Crossref PubMed Scopus (158) Google Scholar, 34Allen S. Lu H. Thornton D. Tokatlidis K. J. Biol. Chem. 2003; 278: 38505-38513Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). We asked if and which of the cysteine residues are required for the import pathway of Tim9. First, we replaced all four cysteine residues with serine residues (Fig. 1B). The resulting modified Tim9 precursor was not imported into mitochondria (Fig. 1C, lanes 1–3) (the amount of radiolabeled precursor proteins added to the import reaction was similar for each construct). Second, we generated constructs that retained two cysteines, respectively, corresponding to the disulfides formed in mature assembled Tim9. Upon replacement of the two outer cysteine residues with serine residues, the import and assembly of Tim9 was blocked (Fig. 1D, lanes 4–6). When the two inner cysteine residues were replaced, however, a partial import reaction was observed. Assembly to the mature Tim9–Tim10 and TIM22 complexes did not occur, but the precursor was able to form the Mia40 intermediate (Fig. 1D, lanes 7–9). To obtain independent evidence for the interaction of Tim9 precursors with Mia40, we used denaturing electrophoresis (SDS-PAGE) under nonreducing conditions to preserve disulfide bonds. Radiolabeled Tim9 formed a high molecular mass product in wild-type mitochondria that showed a slower gel mobility in mitochondria containing Mia40 with a deca-histidine tag (Fig. 1E, lanes 1 and 2). Upon purification of Mia40His10 by affinity chromatography, the high molecular mass product was copurified (Fig. 1E, lane 6), demonstrating that the product contained Tim9 and Mia40. In agreement with the native gel assay, formation of the Tim9-Mia40 product was inhibited when the Tim9 precursor with replaced outer cysteine residues was used, whereas the Tim9 precursor with replacement of the two inner cysteine residues formed the Tim9-Mia40 intermediate (Fig. 1F). To study the translocation of Tim9 across the outer mitochondrial membrane, we used a protease protection assay (24Chacinska A. Pfannschmidt S. Wiedemann N. Kozjak V. Sanjuán Szklarz L.K. Schulze-Specking A Truscott K.N. Guiard B Meisinger C Pfanner N. EMBO J. 2004; 23: 3735-3746Crossref PubMed Scopus (341) Google Scholar). After the import reaction, the mitochondria were treated with proteinase K to remove nonimported precursor. The mitochondria were separated by SDS-PAGE under reducing conditions, and thus all imported Tim9 molecules migrated in monomeric form (Fig. 1G). Tim9 with replaced outer cysteines was strongly inhibited in translocation to a protease-protected location, whereas Tim9 with replaced inner cysteines was imported. Thus, the result of the protease protection assay (Fig. 1G) correlated well with formation of the Mia40 intermediate (Fig. 1, D and F), whereas generation of the mature Tim9–Tim10 and TIM22 complexes was blocked with each mutant Tim9 precursor (Fig. 1D). These findings suggest that the cysteine residues of Tim9 are required at various stages of the import and assembly pathway. Differential Requirement of Cysteine Residues in Targeting and Assembly of Tim9—We asked whether both outer cysteine residues of Tim9 (Cys-35 and Cys-59) were required for translocation across the outer membrane and binding to Mia40. We thus replaced each individual cysteine residue of Tim9 with a serine residue. Each mutant precursor could not form the mature Tim9–Tim10 and TIM22 complexes (Fig. 2A). However, only the first cysteine residue Cys-35 was critical for efficient binding of the precursor to Mia40 (Fig. 2A, lanes 4–6), whereas replacement of any of the other three cysteine residues did not impair formation of the Mia40 intermediate or even enhanced it (Fig. 2A, lanes 7–15). We treated mitochondria with proteinase K after the import reaction to determine whether the mutant precursors were translocated across the outer membrane. Indeed, only replacement of the first cysteine residue impaired translocation of Tim9 into mitochondria, whereas the three other mutant precursors were imported with wild-type efficiency (Fig. 2B). A point of concern was the possibility that the replacement of cysteine residues may have resulted in misfolding or aggregation of the Tim9 precursor. Such events could alter the import competency of precursor proteins. To exclude premature folding, the precursor proteins used in this study were unfolded in 8 m urea in the presence of the reductant dithiothreitol (DTT) before the import reaction. (Additionally we observed that precursor proteins that were directly imported from reticulocyte lysates not exposed to a denaturing treatment showed the same site-specific dependence on cysteine residues as the urea unfolded precursors, indicating that misfolding of the precursors did not occur under our import conditions (data not shown).) We conclude that the first cysteine residue of Tim9 is critical for efficient binding of the precursor to Mia40, as well as translocation across the outer mitochondrial membrane. In contrast to the first cysteine residue, replacement of the third cysteine residue (C55S) led to an increased yield of the Tim9-Mia40 intermediate (Fig. 2A, lanes 4–6 versus 10–12). These two precursors were selected for a detailed analysis. The interaction of both mutant precursors with Mia40 was largely dissociated by addition of DTT like the wild-type Tim9 precursor (Fig. 2C), indicating that formation of the Tim9-Mia40 complex required a covalent linkage via disulfide bridges with each precursor. We asked if the sulfhydryl oxidase Erv1 was required for the increased yield of Mia40-bound Tim9C55S. We used yeast mutants of ERV1 that block the formation of mature Tim9–Tim10 and TIM22 complexes, whereas the initial binding to Mia40 is still possible in case of the wild-type Tim9 precursor (Fig. 2D, lanes 4–9) (31Rissler M. Wiedemann N. Pfannschmidt S. Gabriel K. Guiard B. Pfanner N. Chacinska A. J. Mol. Biol. 2005; 353: 485-492Crossref PubMed Scopus (126) Google Scholar). The enhanced accumulation of Tim9C55S at Mia40 was largely prevented in both erv1 mutant mitochondria (Fig. 2D, lanes 13–18 compared with lanes 10–12). We conclude that the mutant precursor can still bind to Mia40 in erv1 mutant mitochondria (albeit with reduced efficiency i
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