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SCAF1 drives the compositional diversity of mammalian respirasomes

Nature Structural & Molecular Biology (2024)Cite this article

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Abstract

Supercomplexes of the respiratory chain are established constituents of the oxidative phosphorylation system, but their role in mammalian metabolism has been hotly debated. Although recent studies have shown that different tissues/organs are equipped with specific sets of supercomplexes, depending on their metabolic needs, the notion that supercomplexes have a role in the regulation of metabolism has been challenged. However, irrespective of the mechanistic conclusions, the composition of various high molecular weight supercomplexes remains uncertain. Here, using cryogenic electron microscopy, we demonstrate that mammalian (mouse) tissues contain three defined types of ‘respirasome’, supercomplexes made of CI, CIII2 and CIV. The stoichiometry and position of CIV differs in the three respirasomes, of which only one contains the supercomplex-associated factor SCAF1, whose involvement in respirasome formation has long been contended. Our structures confirm that the ‘canonical’ respirasome (the C-respirasome, CICIII2CIV) does not contain SCAF1, which is instead associated to a different respirasome (the CS-respirasome), containing a second copy of CIV. We also identify an alternative respirasome (A-respirasome), with CIV bound to the ‘back’ of CI, instead of the ‘toe’. This structural characterization of mouse mitochondrial supercomplexes allows us to hypothesize a mechanistic basis for their specific role in different metabolic conditions.

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Fig. 1: SCs of mouse mitochondria.
Fig. 2: COX7A isoforms in CIV.
Fig. 3: Interfaces of CIV and position of electron carriers.
Fig. 4: Updated overview of the broad assembly pathways of mitochondrial SCs.

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Data availability

Structures of the three SCs were deposited in PDB (accessions 8PW5, 8PW6 and 8PW7) with corresponding cryo-EM density maps in EMDB (IDs 17989, 17990 and 17991). As the models were built on composite maps, the consensus and focused maps for all components of the three SCs have also been deposited on EMDB (18023, 18022, 18025, 18024, 18027, 18026, 18017, 18018, 18019, 18021, 18020, 18015, 18011, 18012, 18013 and 18014). Similarly, structures of complex I were deposited in PDB (accessions 8RGR, 8RGQ, 8RGP and 8RGT) with corresponding composite cryo-EM density maps (19147, 19146, 19145 and 19148) and consensus/focused maps (19085, 19086, 19087, 19091, 19092, 19093, 19088, 19089, 19090, 19105, 19106 and 19107) in EMDB. The following previously deposited models (PDB codes) have been used in the manuscript: 5gup, 5xth, 5iy5, 1ooc, 5z62, 3cx5, 7o3c, 5j4z, 5j7y, 7o37 and 6g2j. Uncropped gels and western blot, as well as the raw data for the activity assays summarized in Extended Data Fig. 8h, have been provided as Source data in this publication. Source data are provided with this paper.

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Acknowledgements

This research was supported by the Scientific Service Units (SSU) of IST Austria through resources provided by the Electron Microscopy Facility (EMF), the Life Science Facility (LSF), the Pre-Clinical Facility (PCF) and the IST high-performance computing cluster. The authors also acknowledge O. Petrova for her help with the complex I data acquisition. I.V. is funded by the ERC Advanced Grant 101020697 RESPICHAIN to L.S. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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  1. Irene Vercellino

    Present address: Forschungszentrum Jülich GmbH, Jülich, Germany

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  1. Institute of Science and Technology Austria, Klosterneuburg, Austria

    Irene Vercellino & Leonid A. Sazanov

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Contributions

I.V. and L.A.S. designed the project. I.V. purified the samples, prepared cryo-EM grids, acquired and processed EM data, built and analyzed the atomic models and wrote the initial draft of the manuscript. L.A.S. acquired funding, supervised the project, analyzed data and models and revised the manuscript.

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Correspondence to Leonid A. Sazanov.

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Extended data

Extended Data Fig. 1 Representative purification.

a. Ion exchange chromatography and c. gel filtration profiles with b. and d. respective BN-PAGE of the resulting fractions. (b) Coomassie stain on top and in-gel activity at the bottom, left for CIV and right for CI and (d) Coomassie stain on left, in-gel activity for CI in the middle and for CIV on the right. Peak 3 from the ion exchange was selected and subjected to gel filtration. MW markers are indicated on the left. The experiment was repeated at least three times independently with similar results.

Source data

Extended Data Fig. 2 Distribution of supercomplexes across tissues, murine strains and mammalian species.

BN-PAGE runs of CD1 mice hearts (a), brains (b), livers (c), kidneys (d), sheep heart (e) C57 mice hearts (f), brains (g), livers (h), kidneys (i), stained as indicated on top of the figure. SM is solubilised material, P3 is peak 3 of the MonoQ run, WB is Western Blot. The different species are indicated on the right for each section, and the position of 720 kDa MW marker on the left, for clarity. The experiment was repeated at least three times independently with similar results.

Source data

Extended Data Fig. 3 Processing of CD1 liver dataset.

Schematic view of the processing pipeline for the CD1 liver dataset, as explained in the Methods section (a), with angular distribution, locally filtered global and focused maps (b), final Fourier Shell Correlation (FSC) graphs for focused maps (c), global maps (d) and models (e).

Extended Data Fig. 4 Processing of CD1 brain dataset and map features.

a. Schematic view of the processing pipeline for the CD1 brain dataset, as explained in the Methods section, with b. final Fourier Shell Correlation (FSC) graphs for the global maps and c. angular distribution and locally filtered global maps. d. Respirasome models derived from the liver dataset fitted into the brain maps. Maps shown in light grey on top by themselves and on bottom with fitted models, coloured as throughout the manuscript and shown as secondary structures. e. Alphafold2 (top left, coloured by confidence values) and Alphafold multimer (top right, four subunits forming NADH-binding domain, coloured by chain) prediction for the structure of the long isoform of NDUFV3, overlaid to the full CI structure (bottom, with full CI in grey). f. Analysis of NDUFV3 long and short isoforms. Left, sequence alignment, where the first 35 residues represent the mitochondrial targeting sequence and the subsequent 25 residues are disordered in short isoform-containing samples, with only ~43 C-terminal residues ordered. Right, western blot of brain CI sample. The experiment was repeated on two independent brain preparation, plus one preparation on different tissues (ovine/murine heart and murine liver) with similar results.

Source data

Extended Data Fig. 5 Representative densities from the respirasomes models.

Representative densities for CI (a), CIII2 (b), CIVC (c) and CIVS (d): for each an alpha helix, a beta sheet, a ligand and a lipid are shown. For CI, dGTP is shown in addition to FMN.

Extended Data Fig. 6 CIV conformations and cytochrome c binding in respirasomes.

a-c. Superposition of the structures solved in this manuscript from murine liver (shown as secondary structures with piped helices and coloured as in the other figures, that is CI grey, CIII2 yellow and orange, CIVC cyan with COX7A2 light blue, CIVS ice with SCAF1 dark blue) to the other known mammalian supercomplexes featuring CIV (shown as secondary structures with piped helices and coloured in green). VS is versus. a. Superposition of the murine C-respirasome to the tight (top, PDB 5j4z) and loose (bottom, PDB 5j7y) respirasome from ovine heart, aligned on the membrane arm of CI. The circle highlights the CIV shift between tight and loose conformation. b. Superposition of the CS-respirasome to the tight (top, PDB 5j4z) and loose (bottom, PDB 5j7y) respirasome from ovine heart, aligned on the membrane arm of CI, as in a. As in a, the circles highlight the CIV shift between tight and loose conformation, outlining the region of CIVC clashing between the tight conformation of CIV in the ovine respirasome and the CIVS of the CS-respirasome (top panel). The distance between the edge helices of CI and CIV, depicted by the black lines, is measured in Å in each panel for the ovine (O.a.) and murine (M.m.) supercomplexes shown. In a, the difference in CI-to-CIV distance is 39-31=8 Å between murine C-respirasome and ovine loose respirasome and 39-18=21 Å between murine C-respirasome and ovine tight respirasome. In b, this is 35-31=4 Å between murine CS-respirasome and ovine loose and 35-18=17 Å between murine CS-respirasome and ovine tight. c. Superposition of the CS-respirasome to the unlocked mature (top, PDB 7o3c) and locked (bottom, PDB 7o37) CIII2CIV from murine heart, aligned on SCAF1-containing CIII monomer. The arrows indicate the displacement of CIVS. d-e. Cyt-c binding sites of CS-respirasome. In d, the putative contact between cyt-c bound to CIVS and Lys 46 of COX6B1 on CIVC is depicted: the insets show zoomed-in views of the binding site. In e, the binding sites of cyt-c on CIVS (left) and CIVC (right) are shown. In both panels, cyt-c is docked based on PDB 5iy5, complexes are coloured as in the rest of the manuscript, cyt-c is burgundy.

Extended Data Fig. 7 Interfaces of respirasomes.

Newly-found interaction interfaces in the CS- (a) and A- (b) respirasomes: the details are shown in the insets. CDL is cardiolipin, PC1 is phosphatidylcholine. The complexes are coloured as in the rest of the manuscript, COX6A2 is hot pink and all the lipids are grey.

Extended Data Fig. 8 Complex I structure determination and features.

a-b. Processing overview of CI particles from the liver (a) and brain (b) datasets as described in the Methods section. Representative open (light sea green) and closed (lilac) classes are overlaid in the insets, aligned on the peripheral arm, to highlight the difference in CI conformation between them, used for classification in Focus-Reverse-Classify method. c-e. Final resulting maps, including angular distribution and local resolution (c), half-map FSCs (d) and map-to-model FSCs (e). PA is peripheral arm, MA is membrane arm. f-g. Representative densities for CI from liver (f), and brain (g): as for the other supercomplexes, an alpha helix, a beta sheet, dGTP and FMN ligands and a lipid are shown for closed and open states. For the closed state of liver and brain complex I the quinone density (UQ) is also shown. h-i. CI activity, measured as reduction of A340 absorbance over time due to NADH oxidation. In h, the graph shows the result of three independent purifications from CD1 livers. In the box and whiskers representation, minimum and maximum values are indicated as top and bottom lines; the coloured squares are delimited by first and third quartiles and contain the median value as line with empty dot inside. As the representation results from three independent experiments, the minimum, median and maximum values shown correspond to the individual measurements. No error bars are shown, as no statistical analysis was performed. i shows a representative replicate with raw traces. Active (as prepared) CI is orange, deactive (heated to 37o C for 105 min without substrates, Methods) is grey.

Source data

Extended Data Table 1 Model composition (CS-respirasome used)
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Supplementary information

Source data

Source Data Fig. 1

Unprocessed western blots and gels.

Source Data Extended Data Fig. 1

Unprocessed western blots and gels.

Source Data Extended Data Fig. 2

Unprocessed western blots and gels.

Source Data Extended Data Fig. 4

Unprocessed western blots and gels.

Source Data Extended Data Fig. 8

Raw data and calculated activity for each replicate experiment.

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Vercellino, I., Sazanov, L.A. SCAF1 drives the compositional diversity of mammalian respirasomes. Nat Struct Mol Biol (2024). https://doi.org/10.1038/s41594-024-01255-0

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  • DOI: https://doi.org/10.1038/s41594-024-01255-0

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