Figure 3. Inﬂuence of Erv1 on the ternary complex. (A) 35S-Tim9 was incubated with mitochondria and mitoplasts for 30 min and analyzed by native electrophoresis. (B) Mitoplasts were incubated with 35S-Tim9. (C) Growth of mia40-4 cells overproducing Erv1. (D) 35S-Tim9 was incubated with mitochon- dria and analyzed by nonreducing SDS-PAGE. (E–G) Mitoplasts were incubated with recombinant Erv1 forms. Pellets (E and G) and supernatants (F) were analyzed by nonreducing SDS-PAGE. amount of intermediate II (Fig. 3 D). To directly test if Erv1 was needed for dissociation of intermediate II, we generated the inter- mediate in mitoplasts and added recombinant Erv1. Thereby, the amount of intermediate II was significantly decreased (Fig. 3 E), and released precursor was found in the supernatant fraction (Fig. 3 F). Mutant forms of Erv1 (Hofhaus et al., 2003) did not change the amount of intermediate II (Fig. 3 G). We conclude that Erv1 promotes a dissociation of the ternary complex. Disulﬁde transfer in the ternary complex Each small Tim protein contains two CysX3Cys motifs, forming two intramolecular disulfide bonds in the mature protein (Curran et al., 2002; Koehler, 2004; Webb et al., 2006). The fully oxidized proteins are released from Mia40 and assemble to hexameric com- plexes (Lu et al., 2004; Müller et al., 2008). To determine why Erv1 was needed for precursor release, we asked if it promoted disul- fide formation on a Mia40-bound precursor. We monitored the OXIDATIVE PROTEIN FOLDING IN MITOCHONDRIA • Stojanovski et al. 199