all three partners in a ternary complex to promote the flow of electrons. However, such a ternary complex has not been shown experimentally. We addressed the cooperation of sulfhydryl oxidase and disulfide carrier during oxidation of mitochondrial IMS precur- sors. Mia40 (Tim40), which contains three essential pairs of cysteines, forms a mixed disulfide with precursors in transloca- tion across the outer membrane, drives their import into the IMS, and transfers disulfides to the proteins (Chacinska et al., 2004; Naoé et al., 2004; Terziyska et al., 2005, 2007; Grumbt et al., 2007; Milenkovic et al., 2007; Müller et al., 2008). Mesecke et al. (2005) found that upon release of the oxidized substrate, reduced Mia40 was reoxidized by Erv1 (also containing three pairs of cysteines), thus permitting new rounds of precursor import. Here, we report that, contrary to the view that Erv1 acts only on sub- strate-free Mia40, we have identified a ternary complex of Erv1, Mia40, and precursor. This ternary complex assists in the transfer of disulfide bonds to IMS precursors by spatially connecting sulf- hydryl oxidase, carrier protein, and substrate. Results and discussion Disulﬁde-linked precursor–Mia40 intermediate in erv1 mutant mitochondria The import and assembly stages of the 35S-labeled precursors of the small translocase of the inner mitochondrial membrane (Tim) proteins of the IMS were monitored by blue native electrophore- sis. After disulfide-mediated interaction with Mia40, the oxidized proteins, which contain two disulfides each, assemble to the ma- ture hexameric complexes (Tim9–Tim10 or Tim8–Tim13); Tim9 and Tim10 additionally assemble into the TIM22 complex of the inner membrane (Fig. 1 A; Chacinska et al., 2004; Curran et al., 2004; Milenkovic et al., 2007; Sideris and Tokatlidis, 2007; Müller et al., 2008). Blue native analysis of a yeast mutant of Erv1 re- vealed that precursors were efficiently bound to Mia40 in the mu- tant mitochondria, whereas the subsequent assembly into mature complexes was inhibited (Rissler et al., 2005; Müller et al., 2008), shown here for Tim10 with three different erv1 mutants (Fig. 1 A). These results questioned the prevailing model that Erv1 was only needed for reoxidation of Mia40 to activate it for substrate binding, and suggested that Erv1 may play a role in later steps of substrate maturation. To analyze the precursor–Mia40 conjugate under denaturing conditions, we used nonreducing SDS-PAGE (Milenkovic et al., 2007). Formation of the conjugate was impaired in mia40-3 mutant mitochondria, and its gel migration was faster (Fig. 1 B, lanes 4–6) because of a C-terminal truncation of Mia40-3 (Chacinska et al., 2004). The conjugate was observed in similar amounts in erv1 mutant mitochondria and wild-type mitochondria (Fig. 1 B, lanes 7–10), as well as after purification of tagged Mia40 from lysed mitochondria (Fig. 1 B, lanes 11–16). The total amount of imported precursor, determined by treatment of the mitochondria with protease, was decreased in erv1 mutant mitochondria (Fig. 1 B, lanes 9 and 10), in agreement with the requirement of Erv1 for the Mia40-driven import reaction (Allen et al., 2005; Mesecke et al., 2005; Rissler et al., 2005). We hypothesized that in erv1 mutants, the MIA reaction cycle was impaired such that imported 196 JCB • VOLUME 183 • NUMBER 2 • 2008 precursors remained bound to Mia40, preventing assembly of the proteins and further rounds of precursor import. A ternary complex of Erv1, Mia40, and precursor protein The hypothesis raised the possibility that Erv1 may associate with the substrate–Mia40 complex. To capture a putative ternary complex, we used the trap mutant form Mia40-4, which ef- ficiently binds substrate but is impaired in its release (Chacinska et al., 2004; Milenkovic et al., 2007; Müller et al., 2008). In mia40-4 mitochondria, formation of the mature complexes (Tim9–Tim10, Tim8–13, and TIM22) is impaired, and precursor proteins accumulate at Mia40-4. We noticed an additional com- plex in the high molecular weight range of blue native gels upon import of 35S-precursors (Fig. 1 C, Mia40–intermediate II). Mito- chondria carrying His-tagged versions of Mia40 and Mia40-4 were lysed in digitonin and subjected to affinity purification. The amount of Erv1 bound to Mia40-4 was increased approximately twofold in the mutant (Fig. 1 D). The yield of copurification of the imported precursor of Tim9 with Mia40-4 was also enhanced (Fig. 1 D), and nonreducing SDS-PAGE revealed a precursor conjugate in the higher molecular weight range of mia40-4 mito- chondria (Fig. 1 E, lane 2). We conclude that both substrate and Erv1 were accumulated at the mutant protein Mia40-4. In wild-type mitochondria, a ternary complex of Erv1, Mia40, and precursor would represent a short-lived intermedi- ate in the reaction cycle for precursor oxidation. We searched for conditions to slow down the reaction and stabilize an inter- mediate complex with wild-type Mia40. Upon rupturing the outer membrane by swelling (mitoplasting), IMS components like the small Tim proteins were released, and the amount of Erv1 was reduced by approximately half (Erv1 was found in two fractions, soluble and peripherally membrane associated; Fig. 2 A, lanes 10 and 12; and Fig. S1, available at http://www .jcb.org/cgi/content/full/jcb.200804095/DC1). Upon import of Tim9 into mitoplasts, the formation of mature TIM complexes is impaired, whereas precursors accumulate at the membrane- bound Mia40 (Milenkovic et al., 2007). In addition to increased amounts of the Mia40–intermediate I, we observed the high molecular weight intermediate II (Fig. 2 A, lanes 5–8). Forma- tion of the intermediate depended on the presence of precursors, as immunodecoration of the mature complexes did not reveal the high molecular weight complex (Fig. 2 A, lanes 9 and 10). Mia40–intermediate I disappeared with time, whereas the amount of intermediate II increased (Fig. 2 A, lanes 5–8), which indicates that the 35S-precursor moved from intermediate I to intermediate II. To analyze the composition of the intermediate, we used antibody-shift blue native electrophoresis upon accumulation of 35S-precursors in mitoplasts. Antibodies against Mia40 as well as antibodies against Erv1 shifted the intermediate II complex, whereas intermediate I was shifted only by anti-Mia40 (Fig. 2 B, lanes 1 and 5; Erv1-specific antibodies also depleted intermedi- ate II formed in the mia40-4 mutant; Fig. S1). To exclude that intermediate II was associated with the membrane translocases (translocase of the outer mitochondrial membrane [TOM] and TIM22), we used antibodies shifting those complexes (Rehling