Figure 5. MIC26 and MIC27 are cooperatively required for the stability and assembly of the F₁F_o–ATP synthase complex.

(A) Blot showing the in-gel activity of F₁F_o–ATP synthase using isolated mitochondria of HAP1 WT, MIC26 KO, MIC27 KO, or double knockout (DKO) cells that were solubilized with increasing concentration of digitonin (g/g). The blot show oligomers, dimers, and monomers forms of F₁F_o–ATP synthase. The intensity (or activity) was reduced in DKO cells. The same mitochondrial lysate was blotted on SDS–PAGE to probe for equal loading among the samples. (B) The quantification of ratio of oligomers or dimers or monomers of F₁F_o–ATP synthase to the total intensity in the lane specific for 0.75 g/g digitonin was calculated from three independent experiments (mean ± SEM) show no significant difference among them in single knockouts or DKO cells lacking MIC26 and/or MIC27 compared with HAP1 WT. ns = P-value > 0.05 (nonsignificant). t test was used for statistical analysis. (C) Blue-native gel electrophoresis of isolated mitochondria from HAP1 WT, MIC26 KO, MIC27 KO, or DKO cells that were solubilized with increasing concentration of digitonin (g/g) is blotted and probed for F₁F_o–ATP synthase subunit, ATP5D show reduced staining in DKO cells lacking MIC26 and MIC27 with concomitant appearance of lower molecular weight complex (F₁). (D) Western blot from the lysate of HAP1 WT, MIC26 KO, MIC27 KO, or DKO cells were probed with antibodies specific to various subunits of F₁F_o–ATP synthase complex, do not show any consistent change in either of them in single knockouts or DKO cells. (E) Complexome profiling of isolated mitochondria from HAP1 WT, MIC26 KO, MIC27 KO, or DKO cells for the F₁F_o–ATP synthase complex showing the heat map of occurrence of subunits of F₁F_o–ATP synthase. F₁F_o–ATP synthase complex is reduced and subunits of the F1 part are partially dissociated from the complex in DKO cells lacking MIC26 and MIC27.