Human COX7A2L Regulates Complex III Biogenesis and Promotes Supercomplex Organization Remodeling without Affecting Mitochondrial Bioenergetics

Abstract

The mitochondrial respiratory chain is organized in a dynamic set of supercomplexes (SCs). The COX7A2L protein is essential for mammalian SC III₂+IV assembly. However, its function in respirasome (SCs I+III₂+IV_n) biogenesis remains controversial. To unambiguously determine the COX7A2L role, we generated COX7A2L-knockout (COX7A2L-KO) HEK293T and U87 cells. COX7A2L-KO cells lack SC III₂+IV but have enhanced complex III steady-state levels, activity, and assembly rate, normal de novo complex IV biogenesis, and delayed respirasome formation. Nonetheless, the KOs have normal respirasome steady-state levels, and only larger structures (SCs I_1-2+III₂+IV_2-n or megacomplexes) were undetected. Functional substrate-driven competition assays showed normal mitochondrial respiration in COX7A2L-KO cells in standard and nutritional-, environmental-, and oxidative-stress-challenging conditions. We conclude that COX7A2L establishes a regulatory checkpoint for the biogenesis of CIII₂ and specific SCs, but the COX7A2L-dependent MRC remodeling is essential neither to maintain mitochondrial bioenergetics nor to cope with acute cellular stresses.

Keywords: COX7A2L; COX7RP; SCAFI; complex III; mitochondrial respiratory chain; respirasomes; supercomplexes.

Figure 1.. TALEN-Mediated Generation of COX7A2L-KO Clones in HEK293T Cells

(A) Schematic representation of the first exon of the COX7A2L locus and the sequences of recognition sites of the two TALEN pairs. (B) Immunoblot analysis of the steady-state levels of COX7A2L in HEK293T (WT) and TALEN-transfected HEK293T cell lines. VDAC was used as a loading control. (C) COX7A2L alleles in TAL-COX7A2L clones. The DNA numbering refers to the coding sequence (c.) and the protein (p.) number to the predicted full polypeptide (den Dunnen and Antonarakis, 2000). C, compound; Mut, mutant; Hetero, heterozygous; Homo, homozygous; del, deletion; -, position before starting ATG. (D) BN-PAGE analysis of whole cells extracted with digitonin (detergent/protein ratio, 4:1) separated in a 4%–8% linear gradient polyacrylamide gel, followed by CI in-gel activity (IGA) or immunoblotting with the indicated antibodies. The identity of MRC complexes and SCs is indicated in the margins. MegaC, megacomplexes probably containing more than one copy of CI, CIII₂, and CIV. See also Figure S1.

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.

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+IV_1-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+IV_n, 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.

Figure 4.. COX7A2L-KO Cells Are Capable of Normal OXPHOS Performance

Respiration of permeabilized COX7A2L KO and control HEK cells cultivated on DMEM high glucose (A–D) or DMEM high glucose supplemented with 1.2 mM piericidin A, a CI inhibitor (G). (A) Respiration of digitonin-permeabilized HEK293T cells assessed in the presence of Pyr-Glu-Mal: pyruvate (10 mM), glutamate (10 mM), and malate (5 mM) under different respiratory states (phosphorylating, resting, and uncoupled) and piericidin A-sensitive respiration (PinA sens). (B) Respiration of digitonin-permeabilized HEK293T cells assessed in the presence of Suc-G3P: succinate (10 mM) and glycerol-3-phosphate (5 mM) under different respiratory states (phosphorylating, resting, and uncoupled) and malonate-sensitive respiration (Malo sens). (C) Respiration of digitonin-permeabilized HEK cells incubated with Pyr-Glu-Mal and Suc-G3P. The Piericidin A- (PinA sens) and Malonate-sensitive (Malo sens) respiration are determined under uncoupled conditions. (D) Respective contribution of NADH and succinate dehydrogenases in providing electrons to sustain uncoupled respiration assessed with all substrates under uncoupled state. (E) OXPHOS stress paradigm based on in cello CI inhibition with rotenone or piericidin A. (F) Effect of 24-hr incubation in the presence of 50 nM rotenone in HEK293T WT and KO1 cells on SC stability, analyzed in digitonized cell extracts by BN-PAGE and immunoblotting with the indicated antibodies. (G) Respiration analysis of HEK cells cultivated during 48 hr in presence of CI inhibitor (piericidin A). The respiration of digitonin-permeabilized cells incubated with Pyr-Glu-Mal and Suc-G3P assessed under different respiratory states (phosphorylating, resting, and uncoupled). The piericidin A (PinA sens)-, malonate (Malo sens)-, and remaining antimycin A (AA)-sensitive respirations are determined under uncoupled conditions. The graph on the right side represents the respective contribution of CI and CII in providing electrons to sustain uncoupled respiration. In all the panels, error bars represent the mean ± SD of four biological repetitions. See also Figure S3.

Figure 5.. A Nutritional Challenge Induced by Switching the Carbon Source in the Media from Glucose to Galactose Does Equally Enhance Mitochondrial Bioenergetics Parameters in WT and COX7A2L-KO Cells

(A and B) Characterization of glioblastoma U87 WT and COX7A2L-KO (U-KO1) cells. The KO cells were stably transfected with an empty vector (ev) or constructs to express the long or short versions of COX7A2L. (A) and (B) show the BN-PAGE analysis of whole cells extracted with digitonin (detergent/protein ratio, 4:1) separated in a 4%–8% (A) or a 3%–12% (B) linear gradient polyacrylamide gel, followed by CI in-gel activity (IGA) (A) or immunoblotting with the indicated antibodies (B). The identity of MRC complexes and SCs is indicated in the margins. MegaC, megacomplexes probably containing more than one copy of CI, CIII₂, and CIV. (C) Scheme depicting the carbon source switch paradigm used in this study. (D) Time course quantification of COX7A2L induction by galactose in U87 WT cells. The graphs represent a quantification of the signals in three independent experiments, with error bars representing the mean ± SD. (E) Mitochondria extracted with a digitonin/protein ratio of 4:1 (g/g) from cells grown in either glucose-containing (Gluc) or galactose-containing (Gal) medium and analyzed by BN-PAGE, followed by CI-IGA assays or, alternatively, by immunoblotting using the indicated antibodies. The red arrows indicate MegaCs exclusively detected in WT cells. (F) Respiration of glioblastoma U87 cells assessed under different respiratory states (phosphorylating, resting, and uncoupled) and respective contribution of NADH and succinate dehydrogenases to the uncoupled respiration cultured in DMEM-glucose (upper panel), or DMEM-galactose (lower panel). Error bars represent the mean ± SD of four biological repetitions. See also Figure S4.

Figure 6.. The Short-COX7A2L Variant Binds to CIII2 but Does Not Rescue SC III2+IV Assembly in COX7A2L-KO Cells

Control HEK293T cells (WT) and both COX7A2L- KO (KO1 and KO2) clones carrying an empty vector (ev) or constructs to overexpress COX7A2L-Myc- DDK (long) or short-COX7A2L-Myc-DDK (short) were used in the following experiments. (A) SDS-PAGE followed by immunoblotting to estimate steady-state levels of endogenous COX7A2L (~12.6 kDa) from exogenous COX7A2L-Myc-DDK (~16.2 kDa). Membranes were also incubated with antibodies that recognize the indicated OXPHOS subunits. (B). 2D-BN/SDS-PAGE and immunoblotting using digitonin-solubilized mitochondrial extracts (detergent/protein ratio, 4:1) and the indicated antibodies. (C and D) BN-PAGE followed by immunoblotting in digitonized whole-cell extracts (C) or by CI-IGA and CIV-IGA assays and/or immunoblotting in digitonized isolated mitochondria (D). Membranes were incubated with the indicated antibodies. 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 are indicated as subCOX1. In (D), an unidentified band running a bit faster than the SC III₂+IV cross-reacting with the COX5B antibody (Ab) (but not with the CORE2 Ab) is indicated with an asterisk.

Figure 7.. Structural and Functional Rearrangements of the RC in the Absence of COX7A2L

(A) Fully assembled CIII₂ and CIV accumulate until they reach a threshold that ignites SC III₂+IV and respirasome assembly at rates arbitrarily indicated by arrows. In the absence of COX7A2L, CIII₂ levels are ~2–3-fold, SC III₂+IV is not formed, and the assembly kinetics of CIII-containing SCs is slower. (B) In physiological, nutritional, and environmental stress conditions, individual and simultaneous oxidation of NADH- and FAD-linked substrates is similar in WT and COX7A2L-KO cells. Mitochondrial CI, CIII₂, and CIV are represented in blue, green, and red, respectively. COX7A2L is represented as a black stick.