Figure 3.

Hot13 promotes oxidation of Mia40 in a direct and metal-dependent manner. (A) Mitochondria (500 μg) expressing untagged or octahistidinyl-tagged variants of Hot13 and of Erv1 were lysed with 1% Triton X-100, 300 mM NaCl, 10 mM imidazol, 1 mM phenylmethanesulphonyl fluoride and 50 mM sodium phosphate, pH 8.0. For the samples shown in the right panel, 10 mM β-mercaptoethanol was added to the extract to reduce disulphide bonds. The extract was cleared by centrifugation and applied to nickel affinity chromatography. After extensive washing, bound proteins were eluted and analysed by western blotting for the presence of Mia40. Western blots of the intermembrane space (IMS) protein cytochrome c peroxidase (CCPO) and of the matrix protein Mba1 acted as controls. Ten per cent of the extract used for the His₈-Hot13 pulldown was loaded for control (T, total). The asterisk indicates mixed disulphides of Mia40 and Erv1 due to incomplete reduction by the SDS sample buffer. (B) Radiolabelled Hot13 was incubated with mitoplasts from a GAL-MIA40 strain grown either in the presence of galactose (Mia40↑) or glucose (Mia40↓). The mitoplasts were washed, and bound Hot13 protein was detected by autoradiography. (C) Wild-type and Δhot13 mitoplasts were incubated with 67 μM recombinant MBP-Hot13 or 25 mM EDTA and 10 mM o-phenanthroline (o-Phe) in the presence or absence of 100 μM zinc acetate (ZnAc) for 10 min at 25°C. Free thiols were trapped and the redox state of Mia40 was determined. (D) The influence of glutathione (GSH) on the redox state of Mia40 was assessed as described in Fig 2B with or without 25 mM EDTA and 10 mM o-Phe. (E) The graph shows the mean values and standard deviations of four independent glutathione titration experiments. MBP, maltose-binding protein.