FIGURE 2:

Mia40 is critical and limiting for oxidative folding in the IMS of intact mammalian cells. (A) Overexpression of twin-CX₉C proteins delays Cox19 oxidation. Experiments were performed as described in Figure 1B, using an antibody against Cox19, except that cells stably expressing different twin-CX₉C proteins or Smac-HA (a protein targeted to the IMS by a mitochondrial targeting signal) were induced 1 h before the experiment with doxycycline. Reported values are the mean of two independent experiments. (B) Oxidative folding is impaired in cells depleted of Mia40. Experiments were performed as described in Figure 1B, using an antibody against Cox19, except that 72 h before experiments cells were transfected with control siRNA or two different siRNAs directed against Mia40 (#1 and #2). Mia40 depletion was verified (Supplemental Figure S2A). Reported values are the mean of at least two independent experiments. (C) Overexpression of Mia40^WT accelerates oxidative folding, and overexpression of a Mia40 active-site mutant (Mia40^SPS) impairs oxidative folding. Experiments were performed as described in Figure 1B, using an antibody against Cox19, except that cells stably expressing an empty vector (Mock), wild-type Mia40 (Mia40^WT) or an active-site mutant of Mia40 (Mia40^SPS) were induced 1 d before the experiment for 4 h with doxycycline. See Supplemental Figure S2C for localization of Mia40 variants to mitochondria. Reported values are the mean of at least two independent experiments. (D) Mia40 interacts with Cox19 via a mixed disulfide bond. Cells stably expressing Cox19-HA or an empty plasmid (Mock) were induced for 1 h and then radioactively pulse labeled for 4 h. Then thiol–disulfide exchange was inhibited by treatment with N-ethylmaleimide, cells were lysed using an SDS-containing buffer, and Mia40 was immunoprecipitated. One percent of this IP was loaded as control. Then a second IP with an antibody directed against the HA epitope was performed and analyzed by reducing and nonreducing SDS–PAGE and autoradiography. Arrowhead, disulfide-linked Mia40-Cox19 dimer; hash key, Mia40; circle, Cox19; red, reducing SDS–PAGE; non-red, nonreducing SDS–PAGE. (E) Mia40 interacts transiently with Cox19 during oxidation. Experiments were performed as in D, except that after the radioactive pulse, cells were chased with cold methionine for 0 or 20 min. Arrowhead, disulfide-linked Mia40-Cox19 dimer; hash key, Mia40; circle, Cox19; red, reducing SDS–PAGE; non-red, nonreducing SDS–PAGE. Error bars in all graphs are means ± SD.