Fig. 2

Figure 2. Respirasome subcomplex contains an immature complex I

(A) Cartoon of complex I with different modules. The model was prepared based on modifications to the existing model (Guerrero-Castillo et al., 2017). Subunits named in the cartoon are shown in (B) and (C). Subunits of each module are shown in the figure table (bottom).

(B and C) Blue-native PAGE and immunoblotting analysis of N, Q, P_P-a, P_P-b, P_D-a, and P_D-b modules and assembly factors in the respirasome subcomplex (RS). Representative subunits corresponding to N, Q, P_P-a, P_P-b, P_D-a, and P_D-b modules and assembly factors were immunoblotted in digitonin-solubilized proteins separated by 3%–11% blue-native PAGE. N, Q, P_P-a, P_P-b, P_D-a, and P_D-b modules and assembly factors were immunoblotted with anti-NDUFS1 and NDUFS4 (B), anti-NDUFS3 and NDUFA9 (B), anti-NDUFA13 (B), anti-NDUFC2 and NDUFA11 (C), anti-NDUFB1, -NDUFB6, and -NDUFB11 (C), and anti-NDUFAF1, -FOXRED1 (B), and anti-ACAD9 (C), respectively. TOM70 was used as internal control. Ctrl, HEK293T cells; KO, TIMMDC1^−/− cells.

(D) In-gel activity assay (IGA) for CI and CIV was performed in control (Ctrl) and TIMMDC1^−/− (KO) cells. Digitonin-solubilized protein was separated by 3%–13% blue-native PAGE and incubated with substrates for the detection of CI, CIV, and CI+CIV. CI was immunoblotted with anti-NDUFB6 antibody. Coomassie-blue-stained blue-native gel (blue) was used as a loading control.