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Mechanism of super-assembly of respiratory complexes III and IV

Abstract

Respiratory chain complexes can super-assemble into quaternary structures called supercomplexes that optimize cellular metabolism1. The interaction between complexes III (CIII) and IV (CIV) is modulated by supercomplex assembly factor 1 (SCAF1, also known as COX7A2L)2. The discovery of SCAF1 represented strong genetic evidence that supercomplexes exist in vivo2,3. SCAF1 is present as a long isoform (113 amino acids) or a short isoform (111 amino acids) in different mouse strains2,4. Only the long isoform can induce the super-assembly of CIII and CIV2,3,4,5,6, but it is not clear whether SCAF1 is required for the formation of the respirasome (a supercomplex of CI, CIII2 and CIV)1,2,4,5,6. Here we show, by combining deep proteomics and immunodetection analysis, that SCAF1 is always required for the interaction between CIII and CIV and that the respirasome is absent from most tissues of animals containing the short isoform of SCAF1, with the exception of heart and skeletal muscle. We used directed mutagenesis to characterize SCAF1 regions that interact with CIII and CIV and discovered that this interaction requires the correct orientation of a histidine residue at position 73 that is altered in the short isoform of SCAF1, explaining its inability to interact with CIV. Furthermore, we find that the CIV subunit COX7A2 is replaced by SCAF1 in supercomplexes containing CIII and CIV and by COX7A1 in CIV dimers, and that dimers seem to be more stable when they include COX6A2 rather than the COX6A1 isoform.

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Figure 1: Super-assembly between CIII and CIV requires functional SCAF1.
Figure 2: Super-assembly of the different CIII and CIV structures depends on the subunit composition of CIV.
Figure 3: Functional characterization of SCAF1 domains.
Figure 4: Recovery of SCAF1 function and models of CIV super-assembly.

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Acknowledgements

This work was supported by MINECO: SAF2015-65633-R; BIO2015-67580-P; PRB2 (IPT13/0001-ISCIII-SGEFI/FEDER, ProteoRed); RD 12/0042/0054 and RD12/0042/0056)), Fundación La Marato TV3 and ERC-Starting Grant (2013 337703 zebraHeart) The CNIC is supported by MINECO and Pro-CNIC Foundation, and is a SO-MINECO (award SEV-2015-0505).

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Authors and Affiliations

Authors

Contributions

S.C. generated the B6J.129S2-Cox7a2l mice and performed the experiment shown in Figs 1a–d, i, j, 3 and 4a, b, d–f. C.G.-P. and N.M. performed the experiments shown in Fig. 1f–g and Extended Data Fig. 5a–b. R.N.-A. performed the experiments shown in Fig. 1e and cloning of mutants. A.M.G. performed the experiments shown in Extended Data Fig. 5c, d and the preparatory gels for proteomic analysis. E.C. and M.L. performed the proteomic analysis. I.E. performed structural modelling. J.V. and J.A.E. wrote the manuscript and designed the research.

Corresponding authors

Correspondence to Jesús Vázquez or José Antonio Enriquez.

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The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks S. Gygi, M. Hüttemann and D. Winge for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Specificity of tandem mass spectrometry (MS/MS) fragmentation series used to generate quantitative profiles for SCAF1-specific peptides.

a, Amino acid sequence of SCAF1113; boxes indicate structural domains. The location of the proteotypic peptides identifying the extra domain (b) and the N-terminal region of SCAF1 (c) are indicated. b, c, Quality control plots for proteotypic peptide fragmentation were generated using Vseq, a program written in R. Top, intensity versus fragment mass error plot; only the fragments with a mass error below 20 p.p.m. were assumed to be correct matches. Summation of the intensities located inside the blue dashed box represents the E-score. Lower left, representative MS/MS spectrum of the peptide indicating the matched fragments. Lower right, diagram showing the colour coded mass error of the fragments ranked according their m/z values and their correspondence with theoretical fragmentation series. Grey background colour indicates errors higher than 50 p.p.m. The b and the y fragmentation series form a V-shaped path that joins fragments with the same charge. The completeness of the ‘V’ shape assesses the quality of the identification. Note that since HCD fragmentation was used, y-type fragment ions are more intense than b-type ions.

Extended Data Figure 2 Specificity of MS/MS fragmentation series used to generate quantitative peptide profiles for COX7A2 and COX7A1.

a, b, Profiles for COX7A2 (a) and COX7A1 (b). The quality control plots are as in Extended Data Fig. 1.

Extended Data Figure 3 Specificity of MS/MS fragmentation series used to generate quantitative peptide profiles for COX6A1 and COX6A2.

a, b, Profiles for COX6A1 (a) and COX6A2 (b). The quality control plots are as in Extended Data Fig. 1.

Extended Data Figure 4 Specificity of MS/MS fragmentation series used to generate quantitative peptide profiles for COX5B and UQCRC1.

a, b, Profiles for COX5B (a) and UQCRC1 (b). The quality control plots are as in Extended Data Fig. 1.

Extended Data Figure 5 Mitochondrial import assay of CIV proteins and quantitation of CIV by mass spectrometry.

ad, Autoradiography of BNGE of CD1 mice liver digitonin-solubilized mitochondria after import of the indicated radiolabelled proteins. Labelling was fully ablated when mitochondrial import was prevented by FCCP-mediated dissipation of the membrane potential. Images represent two biological replicates. OE, overexposed. Red arrows indicate dimer CIV. e, f, Quantitative contour plots of the most abundant proteins of CV. Data-independent (DiS) mass spectrometry analysis of BNGE gel slices of digitonin-solubilized mitochondria from heart and liver from CD1 and C57BL/6J mice. e, Total spectral counts of the indicated components of ATP synthase, illustrating the reproducibility obtained upon slicing of BNGE gels. f, Total spectral counts after normalization according to ATP synthase levels.

Extended Data Figure 6 Structural models of supercomplex assembly.

ac, Structural modelling of COX6A1 and COX7A2 isoforms on the CIV dimer. The structural model for COX6A1 (in blue) is based on its homology with COX6A2 (PDB 2Y69 chain G, in red) and the structural model for COX7A2 (in orange) is based on its homology with COX7A1 (PDB 2Y69 chain J, in green). d, Tentative model showing a possible supercomplex III2+IV structure stabilized by a SCAF1 bridge. SCAF1 is proposed to substitute COX7A2 in CIV and to interact with III2 via its extra domain. The structural model for SCAF1 (red mesh) was generated based on its homology with COX7A1 (PDB 1V54, in blue) and with UQCR11 (PDB 1BGY, in green). CIV is shown in orange and the two components of CIII dimer in yellow and grey.

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Cogliati, S., Calvo, E., Loureiro, M. et al. Mechanism of super-assembly of respiratory complexes III and IV. Nature 539, 579–582 (2016). https://doi.org/10.1038/nature20157

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