Fig. 5: The N-module turnover protects from increased oxidative stress.
From: A salvage pathway maintains highly functional respiratory complex I
a, b BN-PAGE followed by western blot analysis of CI in wild type (+/+) and CLPP-deficient (−/−) MEFs treated with rotenone (ROT; 200 μM), N-acetylcysteine (NAC; 2 mM) and β-nicotinamide adenine dinucleotide hydrate (NAD; 2 mM) or antimycin A (25 μM). (n = 3, biologically independent experiments). c Survival of wild type (+/+) and CLPP-deficient (−/−) MEF cells upon 16 h treatment with rotenone (ROT; 200 μM) ± NAC (2 mM). Values are calculated as a percentage of the control (CTRL) cells. Bars represent mean ± SD. (*p < 0.05). Two-way ANOVA followed by Sidak’s multiple comparison post-test was used to determine the level of statistical difference. (CTRL & NAC n = 6; ROT & ROT + NAC n = 10, biologically independent samples). d BN-PAGE followed by western blot analysis of CI profiles in wild type (+/+) and CLPP-deficient (−/−) MEFs treated with potassium cyanide (KCN; 1 mM) and myxothiazol (1 μM) for 16 h. (n = 2, biologically independent experiments). a, b, d Antibodies were raised against proteins indicated in the panels, with putative CLPP substrates shown in bold. CI depleted of N-module are indicated with the arrowheads. e Quantification of oxidized proteins in wild type and CLPP-deficient heart mitochondria by mass spectrometry. Values were normalized to mean protein abundance. Data are shown in volcano plots using permutation-based FDR calculation (FDR < 0.05), (n = 3, biologically independent experiments). Red color represents significantly more oxidized proteins in Clpp−/− mitochondria, and blue in Clpp+/+ samples. CLPP targets are indicated in bold. f Ratiometric imaging for response to hydrogen peroxide of Hela wild type and CLPP-deficient cells. Cells containing the matrix-targeted H2O2 sensor HyPer3 or as pH-control matrix-targeted SypHer sensors were analyzed for their response towards 45 µM hydrogen peroxide. The SypHer sensor was not deflected during the experiment indicating that the HyPer3 response was solely due to changes in hydrogen peroxide levels. Results were from two independent experiments (n[HeLa, HyPer3] = 141 cells, n[ClpP KO, HyPer3] = 70 cells, n[HeLa, SypHer] = 89 cells, n[ClpP KO, SypHer] = 75 cells. Shapiro–Wilk test to test for normal distribution followed by Mann–Whitney-U-test was used to determine the level of statistical difference (***p < 0.001).
