Figure 3. COX7A2L-KO Cells Have Enhanced Rate of De Novo CIII2 Biogenesis and Delayed Respirasomes Formation.
(A–E) HEK293T (WT) and COX7A2L-KO clone 1 (KO1) cells were cultured for 8 days in the presence of 15 mg/mL doxycycline and collected at different time points (0, 6, 15, 24, 48, 72, and 96 hr) after doxycycline removal. Mitochondria prepared from these samples were extracted with a digitonin/protein ratio of 4 g/g and analyzed by BN-PAGE in combination with (A) CI-IGA assays or (C) immunoblotting with the indicated antibodies. (B) Mean CI activity recovery after doxycycline removal quantified from (A).
(C) Mean incorporation rates of CORE2 subunit in CIII2, and of COX1 (not shown) and COX5A subunits in CIV and CIV2, or their assembly kinetics in the I+III2+IV1-n respirasomes.
(D) The signals from three independent experiments (as in C) for WT and KO cells were quantified and normalized by CII. Time point values are expressed as percentages of the untreated cells (SS) and indicated as means ± SD. *p < 0.05, **p < 0.01. I+III2+IVn, SCs containing CI, CIII2, and CIV. III2+IV, SC containing CIII2 and CIV; III2, complex III dimer (CIII2); IV, complex IV; IV2, complex IV dimer (CIV2); II, complex II.
(E) For two doxycycline experiments, samples were analyzed by SDS-PAGE for the steady-state levels of CIII subunits CORE2 and RISP. On the right panel, the signals were quantified and plotted as ratio of SDHA. The values for the two independent experiments did not differ by more than 5%.
(F) Steady-state levels of the indicated CIII subunits in HEK293T WT, KO1, and KO2 cell lines. On the right panel, the signals of CORE2 and RISP were quantified and expressed as ratio of the signal of ACTIN, used as a loading control. Error bars represent the mean ± SD of three independent experiments.
(G) Simplified current model of CIII assembly depicting the order of subunit incorporation and time of dimerization, modified from Ferna´ ndez-Vizarra and Zeviani (2015).
(H) In organello import of the indicated recombinant proteins synthesized in a reticulocyte system in the presence of 35S-methionine. The import assays were performed for 30 min in the absence or presence of the uncoupler CCCP to disrupt the mitochondrial membrane potential (J). Following import, an aliquot was treated with proteinase K to digest non-imported precursor proteins. M, mature; p, precursor.
(I and J) BN-PAGE analysis of the incorporation of the indicated radiolabeled recombinant proteins into CIII assembly intermediates, dimer, and SCs in HEK293T WT and KO1 cells during increasing times from 5 to 60 min. Import assays were performed in duplicates with similar results. Sub, subassemblies. The asterisk indicates small subassemblies that may correspond to the protein being imported bound to specific chaperones.