Functional role of respiratory supercomplexes in mice: SCAF1 relevance and segmentation of the Qpool

Abstract

Mitochondrial respiratory complexes assemble into supercomplexes (SC). Q-respirasome (III₂ + IV) requires the supercomplex assembly factor (SCAF1) protein. The role of this factor in the N-respirasome (I + III₂ + IV) and the physiological role of SCs are controversial. Here, we study C57BL/6J mice harboring nonfunctional SCAF1, the full knockout for SCAF1, or the wild-type version of the protein and found that exercise performance is SCAF1 dependent. By combining quantitative data-independent proteomics, 2D Blue native gel electrophoresis, and functional analysis of enriched respirasome fractions, we show that SCAF1 confers structural attachment between III₂ and IV within the N-respirasome, increases NADH-dependent respiration, and reduces reactive oxygen species (ROS). Furthermore, the expression of AOX in cells and mice confirms that CI-CIII superassembly segments the CoQ in two pools and modulates CI-NADH oxidative capacity.

Fig. 1. SCAF1-deficient phenotype and Blue-Dis characterization of SCs.

(A) BNGE followed by Western blot with anti-SCAF1 antibody showing the absence or presence of SCAF1 in the indicated tissue and mouse strain. 113: C57BL/6JOlaHsd mice with the functional version of SCAF1; 111: C57BL/6JOlaHsd mice (it harbors a nonfunctional version of SCAF1); KO: C57BL/6JOlaHsd mice without SCAF1. (B) Effect of SCAF1 on the maximum speed running in a treadmill by the indicated mouse groups. (C) Blue-DiS evidence of the formation of OXPHOS SCs. For each complex, the number of protein peptide-spectrum matches (PSMs) was plotted against normalized protein abundances (see Materials and Methods), showing that the relative proportions of proteins from CI (blue points), CIII (red points), and/or CIV (green points) are constant in specific BNGE slices from liver mitochondria indicating the presence of multimeric structures. Slices 2 to 4 correspond to a ternary structure (I + III + IV), slices 5 to 6 and 9 to 10 to binary structures (I + III and III + IV, respectively), and slices 8 and 15 to monomeric forms (I and IV, respectively). (D) Similar results are obtained in heart and brain mitochondria; for simplicity, only one slice with the tertiary structure is shown.

Fig. 2. Further characterization of SCs by Blue-DiS.

(A) Cross-correlation analysis of SC protein abundances for the indicated slices. (B) Cross-correlation analysis of SC protein abundances for the indicated mitochondrial types. (C) Normalized profiles of CI, CIII, and CIV from heart mitochondria (black lines) are accurately explained as a superimposition (red dashed lines) of six Gaussian peaks corresponding to monomeric and multimeric structures. For CIV, two additional peaks (CIV₂ and CIV_m) had to be added to the model to explain the normalized profile (arrows). (D) Gaussian components modeling of the normalized profiles for the four mitochondrial types. (E) Top: 2D-BNGE (Dig/DDM) analysis of heart mitochondria resolving complexes and supercomplexes in the first dimension and disrupting SCs into their component complexes in the second dimension. NDUFA9 in red indicates the migration of CI, COI in red indicates migration of CIV, and CORE2 in green indicates migration of CIII. (E) Bottom: Split channels from the above panel. COI immunodetection was performed first to indicate migration of CIV-containing structures, and NDUFA9 immunodetection indicates migration of CI containing structures. This gel corresponds to that shown in Fig. 5E where the immunodetection of CORE2 to localize CIII is also included. (F) Gaussian deconvolution of the heart- and liver-normalized profiles from BL6 mice, with an impaired SCAF1 protein. Asterisks indicate the position of SC III2 + IV (completely absent in both tissues) and of the respirasome, which is absent in the liver but remains detectable in the heart.

Fig. 3. Structural consequences of SCAF1 deficiency in the formation of SCs.

(A) 2D-BNGE (Dig/DDM) analysis of heart mitochondria resolving complexes and SCs in the first dimension and disrupting SCs into their component complexes in the second dimension. NDUFA9 immunodetection in red indicates the migration of CI, COI immunodetection in red indicates migration of CIV, and CORE2 immunodetection in green indicates migration of CIII. Samples from BL6, CD1, and BL6:S¹¹³ heart are compared highlighting the area of migration of the N-respirasome. Red asterisk indicates the absence of the respirasome-derived III₂ + IV only in the BL6 sample, indicating that III₂ + IV is not physically linked to SCAF1-deficient respirasomes (B) Different traces corresponding to the N-respirasome area of (A). (C) Representation of the two structurally different respirasomes. RS, SCAF1-postive N-respirasome; RnS, SCAF1-negative N-respirasome.

Fig. 4. Functional consequences of SCAF1 deficiency in the activity of the N-respirasome.

(A) Scheme representing the experimental setup to analyze the function of the respirasomes. (B) Representative oxygen consumption traces obtained with heart respirasomes excised from the BNGE and derived from either C57BL/6 or CD1 animals. The addition of different components is indicated. (C and D) NADH-dependent respiration rate normalized by TMPD respiration rate (C) or by milligram of protein (D) of heart respirasomes excised from BNGE from the indicated mouse strain and measured in a Clark oxygen electrode. (E) NADH oxidation rate by heart respirasomes eluted from BNGE excised bands of the indicated mouse strain and measured by spectrophotometry. (F and G) Representative traces (F) and quantitative data (G) of H₂O₂ production upon NADH oxidation (top) or CoQH₂ oxidation (bottom) by heart respirasomes eluted from BNGE excised bands of the indicated mouse strain and estimated by Amplex Red. When CoQH₂ oxidation was assayed, rotenone was included to prevent the interaction of CoQH₂ with CI, and the assay was performed. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 5. SCs are unstable upon mitochondrial membrane disruption.

(A) BNGE resolving complexes and SCs from CD1 liver from intact or digitonized mitochondria preincubated during the indicated time at 4°C and probed with the indicated antibody. (B) BNGE resolving complexes and SCs from CD1 liver from digitonized mitochondria preincubated during the indicated time at 4°C and probed with the indicated antibody. (C) 2D-BNGE/PAGE resolving complexes and SCs in the first dimension and protein components in the second dimension from CD1 liver digitonized preparation preincubated 72 hours at 4°C, showing that SCAF1 is processed (SCAF1*). The membrane was immunoblotted with the indicated antibodies. (D) Structure of SCAF1 sequence, mapping CIII-interacting regions (in green) and CIV-interacting regions (in yellow). The predicted calpain-1 processing site is indicated with a blue line. (E) Quantitative analysis of the SCAF1-derived tryptic (LTSSVTAYDYSGK, in blue) and calpain-1–processed (SSVTAYDYSGK, in orange) peptides. Both peptides were quantified in the BNGE slices corresponding to SCs I + III₂ + IV and III₂ + IV and to IV₂ and to CIV in fresh or in 4°C-incubated liver mitochondria-enriched fractions. The calpain-1–processed peptide was only detected in the nonfresh preparations, attached to CIV and IV₂ and also to the respirasome. A structural interpretation of these results is presented at the left; the blue and orange shadows indicate whether the SCAF1 peptide is tryptic or processed, respectively. (F) 2D-BNGE/PAGE showing that the proteolytic cleavage of SCAF1 can be prevented by inhibition of calpain-1. (G) BNGE/PAGE analysis of liver mitochondria showing that the stability of the respirasome and the SC III2 + IV is preserved after digitonization in the presence of a calpain-1 inhibitor. (H) BNGE profile for CIV (COI, red) and SCAF1 (green) in heart samples maintained at 4°C after digitonization during the indicated times.

Fig. 6. The superassembly between CI and CIII modulates the activity of CI and functionally segments the CoQ pool.

(A) Scheme representing the differential flux of electrons to AOX from the indicated mutant cell line. (B) Impact of the presence of CIII in the delivery of electrons from CI and CII to AOX. (C) Diphenyleneiodium (DPI)-sensitive NADH oxidation capacity of the mitochondrial preparation from the indicated cell line. (D) Scheme representing the simultaneous flux of electrons form either CI or CII to AOX from the indicated mutant cell line. (E) Estimation of the impact of the simultaneous addition of substrates for CII (succinate) on the CI-dependent respiration with CI substrates (glutamate and malate) in the presence or absence of CIII. (F) Impact of CIII and CIV superassembly on the maximum respiration capacity with CII (succinate) or CI (glutamate and malate) substrates versus both substrates added simultaneously. (G) Proportion of the maximum respiration achievable by CI substrates in the presence or absence of CI and CIII superassembly. (H) Analysis of the flux of electrons from NADH and CI or succinate and CII to AOX in the indicated freeze-thaw mitochondrial from wild-type (WT) cells or mutant cells lacking CIV or CIII all expressing AOX. (I) Flux of electrons from NADH and CI to AOX or CIV in intact heart mitochondria expressing AOX and monitored by oxygen consumption (left) or autofluorescence of NADH (right). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.