(A) Mitochondria were isolated from Mia40-depleted cells (↓) which expressed Mia40 or Mia40-SPS. The steady state levels of Mia40 substrates and control proteins were analyzed by Western blotting. (B, C) To determine the redox state of Tim10, proteins of Mia40- and Mia40-SPS-containing mitochondria were TCA-precipitated, denatured in SDS, treated with or without the reducing agent TCEP and the alkylating compounds mmPEG24 (B) or AMS (C) and visualized by SDS-PAGE and Western blotting. An inverse shift was achieved by blocking reduced thiols with N-ethylmaleimide (NEM) prior to TCA precipitation. Green arrows depict fully oxidized Tim10, red arrows the matrix chaperone Mdj1 which does not contain disulfide bonds. (D) Western blots of mitochondrial extracts of the indicated strains. Please note that expression of Mia40-SPS but not of Mia40-STOP leads to accumulation of low levels of Atp23 and Cmc1 in mitochondria. (E) Wild type and mia40-4 cells were transformed with a Cox19-HA-expressing plasmid (Bode et al., 2015) allowing detection of Cox19 with hemagglutinin (HA) antibodies in whole cell extracts. Levels of Mia40 substrates and other mitochondrial proteins were analyzed by Western blotting in the indicated cells after growth at 34°C. (F) Western blot analysis of protein levels in wild type and GAL-Mia40 cells expressing Mia40-SPS variant and high levels of Erv1. Arrows up and down indicate the expression or depletion of Mia40 in this strain, respectively. (G, H) To follow the oxidation of Cox19 in vivo, cells of the indicated strains were pulse-labeled for 3 min with [35S]-methionine and chased with cold methionine for different times (Kojer et al., 2015). TCA-precipitated protein extracts were treated with mmPEG24, immunoprecipitated with hemagglutinin-antibodies and analyzed by SDS-PAGE and autoradiography.