Fig. 4: Mitochondrial import defects after depletion of DNAJC15.
From: Stress adaptation of mitochondrial protein import by OMA1-mediated degradation of DNAJC15
a, WT HeLa cells were labeled with different stable isotopes through SILAC for a minimum of five splits, to adapt cellular pools of stable-isotope-labeled lysine and arginine. Differently labeled cells were depleted of DNAJC19 (siJC19) and DNAJC15 (siJC15) for 48 h, and an equal number of cells corresponding to the three conditions were pooled before subcellular fractionation, to minimize experimental variation. Samples were analyzed by LC–MS/MS (n = 5, biologically independent samples). b, Boxplot visualizing the distribution of mitochondrial proteins (MitoCarta 3.0) in the whole cell and in the mitochondrial fractions, which were treated with Proteinase K (PK) where indicated. A two-sided Mann–Whitney U-test was performed. Quantile box plot showing the median, 25th and 75th percentiles; whiskers show minimum and maximum values after outlier removal (defined as distance from median 0.5 × IQR, outliers not shown) (n = 5, biologically independent samples). c, Significantly altered mitochondrial proteins (MitoCarta 3.0) in the different fractions (FDR < 0.05) after depletion of DNAJC19 (siJC19, top) and DNAJC15 (siJC15; bottom), separated according to their SILAC ratio compared with WT (SILAC ratio > 0 for upregulation and SILAC ratio < 0 for downregulation). d,e, The indicated radiolabeled proteins, COQ10B (d) and TIMMDC1 (e), were incubated with the indicated mitochondria for the indicated times at 30 °C. Import was blocked with a 5 min incubation of 8 µM antimycin A, 1 µM valinomycin and 20 µM oligomycin. After treatment with Proteinase K (20 µg ml−1) for 20 min on ice, mitochondria were subjected to SDS–PAGE and radioimaging. Imported proteins were quantified, and the amounts of the radiolabeled proteins were normalized to the control at time point 1 h. Values are means ± s.d. (n = 3, biologically independent samples). The area under the curve for each replicate and P values were calculated using a two-sided unpaired t-test. f, Mitochondrial pathways enrichment analysis (MitoCarta 3.0) of significantly changed mitochondrial proteins after DNAJC15 depletion relative to WT (Fisher exact test, FDR < 0.02). g, Heat map of the two-way ANOVA significant protein groups (–log10(P) > 2.9). Proteins corresponding to the significantly enriched Gene Ontology term ‘mitochondrial ribosome’ are highlighted. The dataset was filtered for minimum required values of more than 3 (n = 5, biologically independent samples). LFQ, label-free quantification. h, Boxplot visualizing the half-life distribution of the mostly affected proteins (log2FC < –0.58) in siRNA-depleted DNAJC15 HeLa cells compared with MitoCoP-annotated proteins. The quantile box plot shows the median (center line) and 25th and 75th percentiles, and whiskers show the minimum and maximum values after outlier removal (0.5 × IQR distance from median, outliers not shown). A two-sided Mann–Whitney U-test was performed (n = 5, biologically independent samples, including outliers). The half-life was extracted from Morgenstern et al.31. i, Boxplot visualizing the distribution of the half-life of significantly downregulated and mitochondrial proteins (log2(FC) < –0.58, FDR < 0.05) in DNAJC15−/− HeLa cells after siRNA-mediated depletion of TIMM17A compared with all MitoCop-annotated proteins. Quantile box plots show the median (center line) and 25th and 75th percentiles, and whiskers show minimum and maximum values after outlier removal (0.5 × IQR distance from median, outliers not shown). Mann–Whitney U-test was performed (n = 5, biologically independent samples, including outliers). The half-life was extracted from Morgenstern et al.31.
