Two possibilities are conceivable for the organization of the ternary complex. Erv1 may bind to the precursor or to Mia40. Our findings support the scenario that Erv1 does not directly bind the precursor but acts on Mia40 because the coprecipita- tion of precursor with Erv1 requires the presence of functional Mia40, and is enhanced by the Mia40-trap form. We suggest that Erv1 promotes maturation (oxidation) of Mia40-bound substrate in the ternary complex through the transfer of disul- fide bonds to precursors via Mia40, and this serves as a prereq- uisite for substrate release. Most disulfide relay components of mitochondria, bacteria, and ER share only limited homology on the primary structure level; however, basic functional mechanisms of disulfide shut- tling may be related (Gerber et al., 2001; Sevier et al., 2001; Gross et al., 2002; Kadokura et al., 2004; Tu and Weissman, 2004; Inaba et al., 2006; Sevier and Kaiser, 2006). By splitting bacterial DsbB into two domains, disulfide transfer in a complex of three polypeptides, DsbA with the two DsbB domains was shown (Kadokura and Beckwith, 2002). Here, the central poly- peptide contained one redox-active pair of cysteines; in the ternary complex, each cysteine was bound to a different partner polypeptide. Mia40 may function in a similar manner with one redox-active pair of cysteines (Grumbt et al., 2007). Alternatively, more than one cysteine residue of precursor and Mia40 may be involved in forming transient disulfides in the ternary complex. Several components of the ER and bacterial systems contain more than one redox-active pair of cysteines (Tsai and Rapoport, 2002; Kadokura et al., 2004; Kulp et al., 2006; Sevier and Kaiser, 2006). Moreover, more than one molecule of Mia40 may partici- pate in the reaction, as human Mia40 forms a homo-oligomer linked by noncovalent interactions (Hofmann et al., 2005). In ad- dition to disulfide-mediated interactions, noncovalent interactions and conformational changes likely contribute to the cooperation of the partners in the ternary complex. The findings presented here change the view that the disul- fide carrier (Mia40) strictly alternates between binding to Erv1 and precursor in a sequential relay system (Mesecke et al., 2005; Herrmann and Köhl, 2007). The identification of a ternary com- plex suggests that the flow of electrons between oxidase, carrier, and substrate does not require sequential association and disso- ciation of the components, but rather their concerted action within a single complex. Thus, two distinct redox reactions are spatially coupled, which results in a channeling mechanism for disulfide transfer. Moreover, for substrates that receive more than one disulfide, the binding of Erv1 to the Mia40–precursor complex may permit an efficient transfer of multiple disulfides in a ternary complex. We speculate that the concept of mitochondrial di- sulfide channeling, comprising oxidase, disulfide carrier, and substrate, provides mechanistic implications for the ER and bacterial systems. Materials and methods Strains Strains generated and used in this study are derivatives of the wild-type YPH499 strain of yeast Saccharomyces cerevisiae (MATa, ade2-101, his3- 200, leu2- 1, ura3-52, trp1- 63, and lys2-801). The temperature- sensitive erv1-1 (YPH-BG-Erv1ts-F124S), erv1-2 (YBG-0702b), erv1-3 (YBG- 0701a), and erv1-5 (YPH-BGErv1ts-C159S) mutants have been described previously (Rissler et al., 2005; Milenkovic et al., 2007; Müller et al., 2008). The mutant strain erv1-6 was generated by site-directed mutagenesis followed by plasmid shufﬂing in the background of the genomic deletion of ERV1 (YPH- BG-Erv1ts-C176S; MATa, ade2-101, his3- 200, leu2- 1, ura3-52, trp1- 63, lys2-801, and erv1::ADE2 [pFL39-erv1-C176S]). The erv1-6 mutant possesses the amino acid substitution Cys176Ser (Hofhaus et al., 2003). To overproduce Erv1, the yeast YPH499 strain and mia40-4 mutant strain were transformed with the plasmid p9014-8 harboring the ERV1 gene under the control of its endogenous promoter and terminator in the multicopy Yep352 yeast vector. The suppression by p9014-8 was analyzed on YPD plates (1% [wt/vol] yeast extract, 2% [wt/vol] bactopeptone, and 2% [wt/vol] glucose). The C-terminal His10-tagged versions of Mia40 were gener- ated by genomic integration in a wild-type background, resulting in Mia40His (yAC53; MATa, ade2-101, his3- 200, leu2- 1, ura3-52, trp1- 63, lys2- 801, and MIA40His10; Milenkovic et al., 2007), and in the erv1-2 mutant background, resulting in erv1-2 Mia40His (yAC55-81; MATa, ade2-101, his3- 200, leu2- 1, ura3-52, trp1- 63, lys2-801, MIA40His10, and erv1::ADE2 [pFL39-erv1-2]). The Mia40-4int strain (BG-fomp2-7-int-4-12; MATa, ade2-101, his3- 200, leu2- 1, ura3-52, trp1- 63, lys2-801, and mia40-4) was constructed by integration of the mia40-4 allele (amino acids substitutions T313A, I316M, and F342L according to original open reading frame assignment in the Saccharomyces Genome Database [Chacinska et al., 2004]; T289A, I292M, and F318L according to the current open read- ing frame assignment) into the genome of YPH499. Subsequently, a His10 tag was introduced at the 3 end of the mia40-4 genomic coding sequence, giving rise to Mia40-4His (yAC64-1; MATa, ade2-101, his3- 200, leu2- 1, ura3-52, trp1- 63, lys2-801, and mia40-4His10). Mitochondrial procedures Mitochondria were isolated from S. cerevisiae strains grown on YPG me- dium (1% [wt/vol] yeast extract, 2% [wt/vol] bactopeptone, and 3% [wt/ vol] glycerol) at 19, 24, or 30°C by differential centrifugation, as de- scribed previously, and mitoplasts were generated by hypoosmotic swell- ing (Stojanovski et al., 2007). Mitochondrial precursor proteins were synthesized in rabbit reticulocyte lysate in the presence of [35S]methionine and subjected to in vitro import assays (Stojanovski et al., 2007). Import reactions were performed at 25°C (dicarboxylate carrier and Tim9), 30°C (Tim8, Tim9, and Tim13), or 35°C (Tim10 and Tim22). Samples analyzed by nonreducing SDS-PAGE were solubilized in Laemmli buffer (minus re- ducing agent) containing 50 mM iodoacetamide. Carbonate extractions were performed as described previously (Stojanovski et al., 2007). For coimmunoprecipitation experiments, 35S-labeled Tim9 was im- ported into mitochondria or mitoplasts, and the reaction was stopped by the addition of 50 mM iodoacetamide. Mitochondria were isolated and solubilized in digitonin-containing buffer (1% digitonin, 20 mM Tris-Cl, pH 7.4, 50 mM NaCl, 10% [wt/vol] glycerol, and 50 mM iodoacetimide) for 20 min on ice. After clariﬁcation of the solubilized material, supernatants were isolated and incubated with IgGs coupled to protein–A Sepharose for 1 h at 4°C. Antibody-bound material was washed three times in digitonin- containing buffer, upon which Laemmli buffer was added to the samples. Samples were heated at 95°C for 5 min and analyzed by SDS-PAGE. Recombinant Erv1His and its mutant versions (C130S and C133S) were puriﬁed via nickel–nitrilotriacetic acid–agarose as described previ- ously (Hofhaus et al., 2003; Milenkovic et al., 2007). For release assays, recombinant Erv1 (1 and 2 μg per 50-μl reaction volume) was added, and samples were incubated at 30°C for 20 min. The reaction was stopped by the addition of 50 mM iodoacetamide. Analysis of protein complexes by blue native electrophoresis and antibody-shift/depletion analysis was per- formed as described previously (Wiedemann et al., 2003; Chacinska et al., 2004; Stojanovski et al., 2007). Afﬁnity puriﬁcation of Mia40His and Mia40-4His was performed as described previously (Rissler et al., 2005; Milenkovic et al., 2007). Radiolabeled proteins were detected by digital autoradiography (Storm imaging system; GE Healthcare) and analyzed by ImageQuant software (GE Healthcare). Western blot analysis was per- formed using enhanced chemiluminescence. Thiol-trapping experiments Thiol modiﬁcations were performed as described previously (Jakob et al., 1999; Curran et al., 2002). Oxidation reactions of 35S-precursors during their import into isolated mitochondria were blocked by modiﬁcation of their free sulfhydryl groups with 50 mM iodoacetamide. After reisolation, mito- chondria were solubilized in digitonin-containing buffer (1% digitonin, 20 mM Tris-Cl, pH 7.4, 50 mM NaCl, and 10% [wt/vol] glycerol) supplemented with 10 mM of the reducing agent Tris(2-carboxyethyl) phosphine hydrochloride OXIDATIVE PROTEIN FOLDING IN MITOCHONDRIA • Stojanovski et al. 201