Abstract
The mitochondrial respiratory chain (MRC) complexes, crucial for aerobic energy transduction in eukaryotes, form conserved higher-order structures called supercomplexes (SCs). The elucidation of SC physiological relevance is critical for our understanding of mitochondrial function and bioenergetics but has been hindered by the limited availability of experimental models isolating SC formation as the sole variable. In baker’s yeast, SCs comprise III2IV1 and III2IV2 configurations, which enhance respiratory rates by facilitating cytochrome c diffusion along the SC surface. However, the roles of distinct SC conformations and MRC plasticity remain unclear. To address these questions, we engineered a yeast strain expressing a covalently-linked III2IV2 SC, structurally like the wild-type. Expression of this tethered SC supports robust respiratory activity but selectively impacts cytosolic NADH-driven respiration, due to distinct interactions with the NADH dehydrogenase Nde1. We propose that in yeast mitochondria, substrate-specific respirasome-like SCs contribute to the optimization of electron fluxes and support metabolic flexibility.
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Data availability
Cryo-EM maps have been deposited in the Electron Microscopy Data Bank (EMDB) under the accession codes EMD-44770 (T-SC), EMD-44774 (CIVa), and EMD-44775 (CIVb). Atomic coordinates of the T-SC, built using the overall T-SC map and the CIVa local refinement, have been deposited in the Protein Data Bank (PDB) under the accession code 9BPB. The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE81 partner repository with the dataset identifier PXD061923. Source data are provided with this paper.
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Acknowledgements
We thank Drs. Antoni Barrientos, Mario Barros, Elizabeth Craig, Martin Haslbeck, Johannes Herrmann, Nikolaus Pfanner, Rosemary Stuart, Jan-Willem Taanman, Alexander Tzagoloff, Dennis Winge, and Mikako Yagi for providing reagents. We thank Dr. Antoni Barrientos for the critical reading of the manuscript. We thank Drs. George Tsaprailis and Gogce Crynen at The Herbert Wertheim UF Scripps Institute for Biomedical Innovation & Technology for help with the mass-spectrometry analysis. We thank Drs. Iga Kucharska and Mark Yeager at the University of Miami Frost Institute for Chemistry and Molecular Science for advising on structural data analysis. Cryo-EM data was collected at the Cryo-EM Swedish National Facility, funded by the Knut and Alice Wallenberg Family, Erling Persson, and Kempe Foundations, SciLifeLab, Stockholm University, and Umeå University. The work was supported by the US Department of Defense, US Army Research Office award W911NF-21-1-0359 (to F.F.), US Department of Energy (DOE) award DE-SC0026354 (to F.F.), and University of Miami bridge award SBA2025-01 (to F.F.).
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F.F. conceived the study. F.F., M.H.E., and M.O. designed the experiments. F.F. and Z.C. generated and screened the strains expressing fused subunit pairs. F.F., Z.C., J.-N.M., G.R., and M.H.E. performed the phenotypical, biochemical and bioenergetic characterization of T-SCs. M.H.E., A.C., J.B., and M.O. performed the T-SC structural determination by cryo-EM. F.F. and M.O. provided funds. F.F. and M.H.E. prepared the figures and wrote the first draft of the paper. All authors edited the manuscript. All authors read and approved the final manuscript.
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Eldeeb, M.H., Cosner, Z., Carlström, A. et al. Mitochondrial respirasome-like supercomplexes support metabolic flexibility in yeast. Nat Commun (2026). https://doi.org/10.1038/s41467-026-72228-8
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DOI: https://doi.org/10.1038/s41467-026-72228-8
