Fig. 4

MIA mutant shows increased levels of ROS. a Wild-type yeast and mia40-4int mutant were grown in respiratory medium at 19 °C and shifted to 37 °C. Samples were collected at the indicated time points and analysed by immunoblotting. b Mitochondria were isolated from wild-type cells and mia40-4int mutant and analysed by Blue Native gel electrophoresis followed by immunoblotting. c Endogenous levels of H₂O₂ and superoxide of wild-type cells and mia40-4int. Mean ± SEM, n = 3. *P-value < 0.04; **P-value < 0.02; two-sided, paired t-test. d Carbonylation of proteins in wild-type cells and mia40-4int mutant was analysed by immunoblotting with anti-DNP antibody or quantified by spectrophotometry. Mean ± SEM, n = 3. *P-value < 0.05; two-sided, paired t-test. DNPH, 2,4-dinitrophenylhydrazine. e Volcano plot showing difference in average % oxidation (mia40-4int minus wild-type) for 2,099 unique cysteine-containing peptides plotted against the −log₁₀P-value (ANOVA test, n = 4). Cysteine-containing peptides with significantly increased oxidation levels are highlighted in dark grey (% oxidation ≥ 7, P-value < 0.05, n = 4). Lines indicate a difference in average % oxidation of ≥ 7 and a P-value of 0.05 (−log₁₀ = 1.3). WT, wild-type