Mitochondrial protein interactome elucidated by chemical cross-linking mass spectrometry

Significance Mitochondria meet the majority of living cells’ demand for ATP and, as important regulators of redox homeostasis, metabolite levels, and calcium buffering, are a critical link between cell energetics and signaling. Disruption of these processes can induce adaptive or pathological signaling responses to stress and under severe stress promote cell death. Mitochondria have a complex proteome with conformations and interactions that are not well understood. Mitochondrial dysfunction is a direct cause of rare inherited diseases and is implicated in common metabolic diseases and age-related pathology. This study illuminates protein interactions and conformational features of nearly one-third of the mitochondrial proteome. Network information on this scale will enable groundbreaking insights into mitochondrial function, dysfunction, and potential therapeutic targets for mitochondrial-based pathology. Mitochondrial protein interactions and complexes facilitate mitochondrial function. These complexes range from simple dimers to the respirasome supercomplex consisting of oxidative phosphorylation complexes I, III, and IV. To improve understanding of mitochondrial function, we used chemical cross-linking mass spectrometry to identify 2,427 cross-linked peptide pairs from 327 mitochondrial proteins in whole, respiring murine mitochondria. In situ interactions were observed in proteins throughout the electron transport chain membrane complexes, ATP synthase, and the mitochondrial contact site and cristae organizing system (MICOS) complex. Cross-linked sites showed excellent agreement with empirical protein structures and delivered complementary constraints for in silico protein docking. These data established direct physical evidence of the assembly of the complex I–III respirasome and enabled prediction of in situ interfacial regions of the complexes. Finally, we established a database and tools to harness the cross-linked interactions we observed as molecular probes, allowing quantification of conformation-dependent protein interfaces and dynamic protein complex assembly.