Fig. 1
Impact of CHCHD4 on the mitochondrial proteome. a Schematic of SILAC procedure. HCT116 cells expressing control (non-targeting) shRNA or expressing CHCHD4-targeting shRNA and control U2OS or CHCHD4 (WT)-expressing U2OS cells (WT.cl1) cells were incubated in either l-lysine and l-arginine (light) or l-lysine-13C6, 15N2 (Lys-8) and l-arginine-13C6, 15N4 (Arg-10) (heavy)-containing media for at least 5 cell divisions. Mitochondrial fractions were prepared, and sample pairs were combined prior to LC-MS analysis. b Volcano plot showing log2 expression ratios of all detected proteins (1050) in enriched mitochondrial fractions from U2OS cells expressing CHCHD4-targeting shRNA compared to control (non-targeting) shRNA expressing cells n = 2 independent SILAC experiments each involving parallel double labelling of cells. c Volcano plot showing log2 expression ratios of all detected proteins (1450) in enriched mitochondrial fractions from CHCHD4 (WT.cl1) expressing U2OS cells compared to control U2OS cells. CHCHD4 highlighted (red dot) n = 3 independent SILAC experiments each involving parallel double labelling of cells. d Volcano plot showing log2 expression ratios of known CHCHD4 substrates containing a twin-CxnC motif identified from our SILAC analyses described in a. SAMM50 is highlighted (yellow) as a mitochondrial protein that does not significantly change and is not a predicted CHCHD4 substrate. Dashed line denotes significance threshold, calculated by Student’s t test, expressed as -log10 of calculated p value. e Volcano plots, showing changes in respiratory chain CI (left) and CIV (right) protein subunits from our SILAC analyses described in a and c. Log2 expression ratios in WT.cl1 vs control U2OS (blue circles) and CHCHD4 shRNA1 vs control shRNA (red circles) are shown. f Western blots show NDUFS3, SDHA, UQCRC2, COXIV and CHCHD4 protein levels in whole cell lysates (WCL) and mitochondrial fractions (mitochondria) prepared from control HCT116 (NT) and HCT116 cells stably expressing an shRNA control vector (shRNA Ctrl1) or shRNA(1) targeting CHCHD4 (CH shRNA). PHB1 was used as a mitochondrial load control, and α-tubulin was used as total load control. Densitometric ratio of each mitochondrial protein relative to PHB1 load is indicated, n = 3. g Western blots show NDUFS3, SDHA, UQCRC2, COXIV and CHCHD4 protein levels in whole cell lysates (WCL) and mitochondrial fractions (mitochondria) prepared from control U2OS (Ctrl), CHCHD4 (WT)-expressing (WT.cl1, WT.cl3) and CHCHD4 (C66A/C68A)-expressing cells. PHB1 was used as a mitochondrial load control and α-tubulin was used as total load control. Densitometric ratio of each mitochondrial protein relative to PHB1 load is indicated. n = 3. h, i Graphs show mtDNA copy number calculated as ratio of mt-ND1 and B2M expression analysed by Q-PCR using total DNA isolated from cells described in f and g, respectively. n = 3. mean ± SD, n.s. not significant. j Graph shows basal OCR (pmol/min) measured in cells described in f. n = 3. mean ± SD, ***p < 0.001. k Graph shows basal and maximal OCR (pmol/min) measured in cells described in (g). n = 3. mean ± SD, n.s. not significant, ***p < 0.001