Figure 2. COX7A2L-KO Cells Display Absence of SC III2+IV with Normal Respirasome Levels and Altered Accumulation of CIII2.

The effect of COX7A2L absence on MRC complex assembly was investigated in two COX7A2L-KO clones, clone 1 (KO1) and clone 2 (KO2), compared with the control HEK293T cells (WT).

(A) Mitochondria extracted with a digitonin/protein ratio of 4:1 (g/g) and analyzed by BN-PAGE, followed by CI- and CIV-IGA assays, or alternatively, by immunoblotting using the indicated antibodies.

(B) Subsequent 2D-BN/SDS-PAGE and immunoblot analyses were performed with antibodies against COX7A2L and the indicated OXPHOS subunits.

(C) To address the relative amount of CIII₂ in COX7A2L-KO cells, the signals from the CORE2 antibody from four BN-PAGE experiments were quantified by densitometry, normalized by CII, and indicated as mean ± SD.

(D) BN-PAGE analyses in whole-cell extracts prepared in the presence of digitonin (detergent/protein ratio, 4:1) or 1% lauryl maltoside (LM). The CIII₂ signals were quantified and normalized by CII using the histogram function of the Adobe Photoshop program on digitalized images, and the values were expressed relative to the control. Error bars represent the mean ± SD of four independent experiments.

(E) Spectrophotometric measurements of the individual activities of MRC complexes I to IV (CI–CIV) in WT and COX7A2L-KO cells. Enzyme activities are expressed as cU/U citrate synthase (CS). Error bars represent the mean ± SD of four repetitions. *p < 0.05; **p < 0.01. MegaC, megacomplexes probably containing more than one copy of CI, CIII₂, and CIV. I+III₂+IV_n, SCs containing CI, CIII₂, and CIV. I+III₂, SC containing CI and CIII₂. III₂+IV, SC containing CIII₂ and CIV. III₂, complex III dimer (CIII₂). IV, complex IV; IV₂, complex IV dimer (CIV₂). II, complex II. Subcomplexes that contain COX1 and COX4 are indicated. Apparent subcomplexes that contain CORE2 are antibody artifacts that disappear in 2D-BN/SDS-PAGE gels. See also Figure S2.