Figure 3.

The combination of Mia40 and Erv1 directly oxidize Tim13 in vitro. (A) Reduced Tim13 (15 μM) was incubated with combinations of Mia40 (0.5 μM), Erv1 (0.5 μM), and mutant Erv1 (C133S or C30S, 0.5 μM) in a time course assay as indicated. Aliquots were removed at the indicated times and free thiols on Tim13 were blocked with AMS. Oxidized and reduced (Tim13-AMS₄) Tim13 were detected by nonreducing SDS-PAGE followed by immunoblotting with antibodies against Tim13. (B) The amount of oxidized Tim13 was quantitated using VersaDoc and associated Quantity One software (Bio-Rad Laboratories) (p < 0.001; n = 4). (C) H₂O₂ production was monitored over a 50-min time period during Tim13 oxidation. H₂O₂ production was detected with the indicator Amplex Red and displayed as relative fluorescence units (n = 3). (D) As in A, Mia40 was incubated with Erv1, reduced Tim13, and the combination of reduced Tim13 and Erv1. Aliquots were removed at 5, 15, 30, and 120 min, and the Mia40 oxidation state was monitored by thiol trapping with AMS and immunoblotting with an antibody against Mia40. Note that the addition of two AMS molecules was detected (Mia40-AMS₂; n = 3).