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Fig. 4
Figure 4. Respirasome subcomplex contains CIII/CIV for TIMMDC1-mediated respirasome assembly (A) Blue-native PAGE and fluorescent immunoblotting analysis of respiratory supercomplexes in control (Ctrl) and TIMMDC1−/− (KO) cells. Digitonin-solubilized protein was separated by 3%–11% blue-native PAGE and immunoblotting with anti-NDUFC2 (CI), -UQCRC2 (CIII), -MT-CO1 (CIV), and fluorophore-conjugated secondary antibodies. (B and C) Blue-native PAGE and immunoblotting analysis of the RS in control (Ctrl) and TIMMDC1−/− (KO) cells with sequence-specific KD of nuclear-encoded constitutive subunits of CIII. Nine nuclear-encoded subunits for CIII were knocked down independently in TIMMDC1−/− cells. Digitonin-solubilized protein was separated by 3%–11% blue-native PAGE, and the RS was immunoblotted with anti-NDUFB6 antibody (B). Quantitative analysis of RS levels was performed with KD of CIII (C). (D and E) Blue-native PAGE and immunoblotting analysis of the RS in control (Ctrl) and TIMMDC1−/− (KO) cells with small interfering (siRNA)-mediated KD of four assembly factors (COX14, COA1, COA3, and SURF1) of CIV. TIMMDC1−/− cells were transfected with four siRNA sequences simultaneously. Digitonin-solubilized protein was separated by 3%–11% blue-native PAGE, and the RS was immunoblotted with anti-NDUFB6 antibody (D). Quantitative analysis of RS levels was performed (E). (F) Protein interactions with exogenous TIMMDC1 were detected by co-immunoprecipitation/liquid chromatography-mass spectrometry (co-IP/LC-MS) in HEK293T cells. Proteins were sorted by fold-change and p value (<0.05). Proteins that were not found in control cells but were abundant in TIMMDC1 OE cells and, therefore, have no fold-change or p value, are included in the figure table (right panel). (G) Protein interactions with exogenous TIMMDC1 were detected by coIP/WB in HEK293T cells. The exogenous TIMMDC1 was tagged with FLAG, and anti-FLAG antibody was used to immunoprecipitate the protein complex. Antibodies against CI (NDUFB6 and NDUFS3), CIII (UQCRC2), and CIV (MTCO1, MTCO2, and COX4I1) were used to detect respiratory complexes co-immunoprecipitating with TIMMDC1. (H and I) Assembly dynamics of respirasome and the RS in TIMMDC1−/− cells and paired control (Ctrl) cells. Cells were pretreated with chloramphenicol (CAP, 40 μg/mL) for 7 days. Assembly of respirasome, RS, and TIMMDC1 in digitonin-solubilized cells was examined by CAP removal, followed by blue-native PAGE (3%–11%). Respirasome and the RS were identified by antibodies against CI (NDUFB6) and CIII (UQCRC2) (H) or CI (NDUFB6), CIV (MT-CO1), and TIMMDC1 (I). TOM70 was used as an internal control. Quantitative data were generated from three independent experiments. Data are presented as the means ± SEM. ∗p < 0.05.