Fig. 3

Figure 3. The P_D-a module of complex I is a structural component of the respirasome subcomplex

(A–I) 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 CI. Eight subunits of N (A), nine subunits of Q (C), four subunits of P_P-a (E), and five subunits of P_P-b (G) were knocked down in a total of 26 TIMMDC1^−/− cell lines. Digitonin-solubilized protein was separated by 3%–11% blue-native PAGE, and the RS was immunoblotted with anti-NDUFB6 antibody (A, C, E, and G). Quantitative analysis of RS levels was performed with KD of N (B), Q (D), P_P-a (F), and P_P-b (I) modules. P_P-b+P_D-a-containing complex in TIMMDC1^−/− cells with KD of P_P-b module (G) was quantified as in (H).

(J and K) 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 P_D-a modules. Six subunits of the P_D-a modules were knocked down independently in six TIMMDC1^−/− cells. Digitonin-solubilized protein was separated by 3%–11% blue-native PAGE, and the RS was immunoblotted with anti-NDUFB6 antibody (J). Quantitative analysis of RS levels was performed with KD of P_D-a (K).

(L and M) Blue-native PAGE and immunoblotting analysis of the RS in control (Ctrl) and TIMMDC1^−/− (KO) cells with sequence-specific KD of assembly factors. ATP5SL, FOXRED1, and TMEM70 were knocked down independently in three TIMMDC1^−/− cells. Digitonin-solubilized protein was separated by 3%–11% blue-native PAGE, and the RS was immunoblotted with anti-NDUFB6 antibody (L). Quantitative analysis of the RS levels was performed with the KD of ATP5SL, FOXRED1, and TMEM70 (M).

(N and O) 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 P_D-b modules. Six subunits of the P_D-b modules 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 (N). Quantitative analysis of RS levels was performed with KD of P_D-b (O).

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, ^∗∗p < 0.01, ^∗∗∗p < 0.001.