Super-resolution complexome of human mitochondria elucidates translocase, morphology and OXPHOS networks

Mitochondria function as cellular powerhouses and central hubs in metabolism, redox and stress reactions, signaling and apoptosis ^1–5 . Defects of mitochondria lead to numerous human diseases ^1,6–8 . The integration of mitochondrial proteins into complexes and networks is crucial for their function. Whereas the composition of the human mitochondrial proteome has been studied ^8,9 , only limited information is available on the organization of the proteome into protein complexes and assemblies. Here we present a systematic mapping of the human mitochondrial complexome from HEK293T cells at super-resolution, resolving more than 7,000 abundance profile peaks of mitochondrial proteins. Proteins functioning in signaling, cell stress, protein biogenesis, turnover and membrane dynamics display particularly high complexities. High resolution and precise quantification enable discrimination between canonical constituents and non-stoichiometric regulatory interactors of the ATP synthase, major metabolite channels and import translocases. The complexome reveals membrane-spanning networks of protein insertase and morphology machinery, and co-assembly of protein import and export components at the major respiratory supercomplex, unraveling a multifunctional organization of mitochondrial machineries. This complexome represents a fully interactive resource for the systematic analysis of human mitochondrial machineries and interaction networks.