oxidation of single cysteines of Tim9 by modification with the alky- lating agent 4-acetamido-4 -maleimidylstilbene-2,2 -disulfonic acid (AMS; 500 D). Mutant precursors with a replacement of one to four cysteines were used as standards (Fig. 4 A, lanes 1–5). By in- direct thiol trapping, the number of oxidized cysteines after import into mitochondria was determined: first, free thiol groups were blocked by iodoacetamide, then the mitochondria were lysed and all disulfides were reduced, followed by modification of the origi- nally oxidized cysteines by AMS. At a low temperature (15°C), a sequential oxidation of the cysteines was observed (Fig. 4 A, lanes 6–10). At physiological temperature (30°C), three species were dissected: reduced Tim9, Tim9 with one oxidized cysteine, and fully oxidized Tim9 (Fig. 4 B). In erv1 mutant mitochondria, oxi- dation of the first cysteine was only mildly affected, but formation of the fully oxidized form was significantly inhibited (Fig. 4 B). The first cysteine residue of Tim9 is required for binding to Mia40 (Milenkovic et al., 2007; Sideris and Tokatlidis, 2007). We imported mutant precursors lacking three, two, or one of the other cysteines and observed the generation of one, two, and three oxidized cysteines, respectively (Fig. 4 C). We hypothesized that species with an odd number of oxidized cysteines may represent Mia40-linked precursors. After affinity purification of His-tagged Mia40, bound precursors were eluted, and the number of origi- nally oxidized cysteines was determined. The precursors lacking three or two cysteines contained one oxidized cysteine in the Mia40-bound form (Fig. 4 D), which reflects the mixed disulfide with Mia40. Of particular importance was the precursor Tim9C55S. Here, two forms, one with a single oxidized cysteine and one with all three cysteines oxidized, were bound to Mia40 (Fig. 4 D). Thus, in addition to the mixed disulfide with Mia40, a further disulfide bond was transferred to the precursor bound to Mia40. In erv1 mutant mitochondria, the oxidation of Tim9C55S was im- paired with a particular defect in generation of the third oxidized cysteine (Fig. 4, E and F). We determined the number of oxidized cysteines in the Mia40-bound precursor of wild-type and erv1 mutant mitochondria. Full oxidation of the precursor strongly de- pended on the activity of Erv1, whereas oxidation of the first cysteine (mixed disulfide with Mia40) was only moderately af- fected in the erv1 mutant (Fig. 4 G). Thus, active Erv1 is critical for the efficient transfer of a further disulfide bond to the precur- sor, which is bound to Mia40 via a mixed disulfide, demonstrat- ing the function of Erv1 in the ternary complex. We conclude that the association of Erv1 with the precursor–Mia40 complex per- mits an efficient transfer of disulfides in a ternary complex. Conclusions We report that the interactions of Mia40 with Erv1 and precur- sor are not mutually exclusive, but all three components associ- ate in a ternary complex. This intermediate complex is a transient step in precursor oxidation and thus has escaped experimental detection in previous studies. We found two means to trap pre- cursors in a ternary complex. First, a trap mutant form of Mia40 accumulated precursors and Erv1. Second, mitochondria were swollen, leading to a release of IMS components, including approximately half of Erv1, whereas the amount of membrane- bound Mia40 was unchanged. Erv1 was rate-limiting for dissocia- tion of the precursor–Mia40 complex because overexpression as well as addition of purified Erv1 promoted substrate release from Mia40. The ternary complex thus accumulated in mitoplasts containing limiting amounts of Erv1. Figure 4. Erv1 promotes disulﬁde transfer to Mia40-bound precursors. (A) 35S-Tim9 precursors were treated with AMS (left) or imported into mitochondria at 15°C, and subjected to indirect thiol trapping (right). Samples were analyzed by Tricine–SDS-PAGE. (B) 35S-Tim9 was imported into mitochondria at 30°C and treated as described in A. (C) 35S-Tim9 precursors were imported into WT mitochondria. (D) 35S-Tim9 precursors were imported into Mia40His mitochondria and subjected to afﬁnity puriﬁcation and indirect thiol trapping. (E) 35S-Tim9C55S was imported into mitochondria for the indicated times or for 24 min in the presence of iodoacetamide (IA). Samples were treated as described in A. (F) 35S-Tim9C55S with one or three oxidized cysteines were quanti- ﬁed; error bars indicate SEM (n = 5). (G) 35S-Tim9C55S was imported into Mia40His mitochondria and analyzed as in D. 200 JCB • VOLUME 183 • NUMBER 2 • 2008