Figure 1. Accumulation of precursor proteins at Mia40. (A) Isolated mitochondria were incubated with 35S-Tim10 and analyzed by native electrophoresis. WT, wild-type. (B) 35S-Tim9 was imported into mitochondria (samples 1–10). Tim9-Mia40 was isolated by afﬁnity puriﬁcation from Mia40His mitochondria (right). The samples were analyzed by nonreducing SDS-PAGE. (C) 35S-precursors were incubated with mitochondria and analyzed by native electropho- resis. Asterisk, nonspeciﬁc band. (D) Mia40 or Mia40His mitochondria were subjected to afﬁnity puriﬁcation, SDS-PAGE, and autoradiography (35S-Tim9) or immunoblotting. Load, 2.5%; eluate, 100%. (E) 35S-Tim9 was imported and analyzed by nonreducing SDS-PAGE. et al., 2003; Wiedemann et al., 2003). Neither anti-Tom22 nor anti-Tim54 shifted intermediate II. Anti-Tim54 shifted the TIM22 complex as expected, whereas neither anti-Mia40 nor anti-Erv1 shifted the TIM22 complex or the Tim9–Tim10 com- plex (Fig. 2 B). The dicarboxylate carrier (DIC), which is im- ported independently of Mia40/Erv1 (Rehling et al., 2003; Chacinska et al., 2004), was not shifted by the antibodies (Fig. 2 B). We conclude that Erv1, Mia40, and precursor are present in the intermediate II complex. The precursor–Mia40 conjugates were dissociated by ad- dition of the reductant DTT (Fig. 2 C), which indicates that they were linked by disulfide bonds. To compare the conjugates formed in mitoplasts and mia40-4 mutants, we performed affin- ity purifications. Upon separation by nonreducing SDS-PAGE, the migration of the precursor–Mia40 conjugates was indistin- guishable between mitochondria, mitoplasts, and mia40-4 mutants (Fig. 2 D). To obtain independent evidence for a ternary complex, we used coimmunoprecipitation. 35S-Tim9 imported into mitoplasts was immunoprecipitated with antibodies against Mia40, as well as Erv1, but not control antibodies (Fig. 2 E, lanes 1–5). To dis- tinguish if 35S-Tim9 was directly coprecipitated with Erv1 or via binding to Mia40, we used the mutant form Mia40-3, which is impaired in precursor binding (Chacinska et al., 2004; Müller et al., 2008). The coprecipitation of 35S-Tim9 with anti-Erv1 was impaired in the mia40-3 mutant, like that with anti-Mia40 (Fig. 2 E, lanes 6 and 8). Because in mitoplasts the precursor would have direct access to Erv1 without a need for Mia40-dependent trans- location across the outer membrane (Milenkovic et al., 2007), we conclude that Mia40 is required for the interaction of Erv1 with the precursor. In mia40-4 mitochondria, Tim9 was efficiently coprecipitated with anti-Erv1 as well as anti-Mia40 (Fig. 2 E), confirming the findings of the blue native analysis. Collectively, we conclude that Erv1, Mia40, and precursor are present in a ternary complex, representing a transient inter- mediate stage during precursor oxidation, which is slowed down in mitoplasts and mia40-4 mitochondria. Erv1 is required for resolving the ternary complex In erv1 mutant mitochondria, the Mia40–intermediate II formed with an efficiency close to that of wild-type mitochondria (Fig. 3 A). Although the original erv1-1 mutant showed only a minor ef- fect on assembly of small Tim proteins, as described previously, the other erv1 mutants were strongly impaired in the formation of the mature Tim9–Tim10 and TIM22 complexes (Figs. 1 A and 3 A; Allen et al., 2005; Rissler et al., 2005; Milenkovic et al., 2007; Müller et al., 2008). We reasoned that Erv1 may be rate-limiting for the turnover of intermediate II. When Erv1 was overexpressed, the amount of intermediate II was indeed decreased (Fig. 3 B). Moreover, overexpression of Erv1 in mia40-4 cells restored growth at elevated temperatures (Fig. 3 C) and decreased the OXIDATIVE PROTEIN FOLDING IN MITOCHONDRIA • Stojanovski et al. 197