Fig. 5: Impaired mitochondrial import after loss of DNAJC15 limits OXPHOS activity.
From: Stress adaptation of mitochondrial protein import by OMA1-mediated degradation of DNAJC15
a, Oxygen flux was measured in intact mitochondria isolated from DNAJC15-depleted HeLa cells and WT cells, using an Oroboros respirometer. Oxygen flux was assessed in the presence of mitochondrial complex I substrates (pyruvate 10 mM, glutamate 5 mM, malate 5 mM), under phosphorylating conditions (ADP+Pi), under non-phosphorylating conditions (oligomycin, 10 µM) and after uncoupling with CCCP (n = 5, biologically independent samples). Values are means ± s.d. b, Oxygen flux was measured in intact mitochondria isolated from DNAJC15-depleted HeLa WT cells and those re-expressing DNAJC15 (JC15), using an Oroboros respirometer. Oxygen flux was assessed in the presence of mitochondrial complex I substrates (pyruvate 10 mM, glutamate 5 mM, malate 5 mM), under phosphorylating conditions (ADP+Pi), under non-phosphorylating conditions (oligomycin 10 µM) and after uncoupling with CCCP (n = 4, biologically independent samples). Data were normalized to the average of oxygen flux of WT HeLa cells under phosphorylating conditions. Values are means ± s.d. c, Oxygen flux was measured in intact mitochondria isolated from DNAJC15-depleted HeLa WT cells and those re-expressing uncleavable DNAJC15 (JC15Δ2), using an Oroboros respirometer. Oxygen flux was assessed in the presence of mitochondrial complex I substrates (pyruvate 10 mM, glutamate 5 mM, malate 5 mM), under phosphorylating conditions (ADP+Pi), non-phosphorylating conditions (oligomycin 10 µM) and after uncoupling with CCCP (n = 4, biologically independent samples). Data were normalized to the average of oxygen flux of wild-type HeLa cells under phosphorylating conditions. Values are means ± s.d. d, Oxygen flux was measured in intact mitochondria isolated from DNAJC15-depleted HeLa cells and WT cells, using an Oroboros respirometer. Oxygen flux was assessed in the presence of mitochondrial complex II substrate (succinate 10 mM) and rotenone (140 nM) under phosphorylating and non-phosphorylating conditions and after uncoupling (n = 4, biologically independent samples). Values are means ± s.d. e, Oxygen flux was measured in DNAJC15-depleted HeLa cells and WT cells under phosphorylating and non-phosphorylating conditions and after uncoupling (n = 4, biologically independent samples). Values are means ± s.d. f, Volcano plot showing the interaction between mitochondrial proteins and DNAJC15 weakening after the OMA1-cleavage site was mutated (JC15Δ2) (according to MitoCarta 3.0), compared to DNAJC15−/− cells expressing DNAJC155 (JC15Δ2 − JC15 in JC15−/−). Four significant mitochondrial interactors are highlighted in green (FDR < 0.05; n = 4, biologically independent samples). P values (y axis) were calculated using an unpaired two-sided t-test. g, Volcano plot showing mitochondrial S-DNAJC15 (S-JC15) interactors, identified by DNAJC15 bait pulldown, compared with the IgG control in AFG3L2−/−SPG7−/− cells (S-JC15 − IgG in AFG3L2−/− SPG7−/−). Proteins that were also significantly enriched in wild-type DNAJC15 pulldown samples relative to IgG were excluded (FDR < 0.05, n = 4 biologically independent samples). P values (y axis) were calculated using an unpaired two-sided t-test. Significantly changed CoQ proteins were highlighted in dark green. h, CoQ9 abundance, normalized to the protein content, in cells expressing siRNA-resistant DNAJC15 (JC15) and DNAJC15Δ2 (JC15Δ2), after siRNA-mediated depletion of DNAJC15 (siJC15). P values were calculated using a two-sided unpaired t-test. Quantile box plots show the median (center line) and 25th and 75th percentiles, and whiskers show minimum and maximum values (1.5 × IQR distance from median, outliers not shown, n = 5, biologically independent samples). i, CoQ10 abundance, normalized to the protein content, in cells expressing siRNA-resistant DNAJC15 (JC15) and DNAJC15Δ2 (JC15Δ2), after siRNA-mediated depletion of DNAJC15 (siJC15). P values were calculated using a two-sided unpaired t-test. Quantile box plots show the median (center line) and 25th and 75th percentiles, and whiskers show minimum and maximum values (1.5 × IQR distance from median, outliers not shown, n = 5, biologically independent samples).
