Mitochondria have crucial roles in cellular energetics, metabolism, signalling and quality control^1–4. They contain around 1,000 different proteins that often assemble into complexes and supercomplexes such as respiratory complexes and preprotein translocases^1,3–7. The composition of the mitochondrial proteome has been characterized^1,3,5,6; however, the organization of mitochondrial proteins into stable and dynamic assemblies is poorly understood for major parts of the proteome^1,4,7. Here we report quantitative mapping of mitochondrial protein assemblies using high-resolution complexome profiling of more than 90% of the yeast mitochondrial proteome, termed MitCOM. An analysis of the MitCOM dataset resolves >5,200 protein peaks with an average of six peaks per protein and demonstrates a notable complexity of mitochondrial protein assemblies with distinct appearance for respiration, metabolism, biogenesis, dynamics, regulation and redox processes. We detect interactors of the mitochondrial receptor for cytosolic ribosomes, of prohibitin scaffolds and of respiratory complexes. The identification of quality-control factors operating at the mitochondrial protein entry gate reveals pathways for preprotein ubiquitylation, deubiquitylation and degradation. Interactions between the peptidyl-tRNA hydrolase Pth2 and the entry gate led to the elucidation of a constitutive pathway for the removal of preproteins. The MitCOM dataset—which is accessible through an interactive profile viewer—is a comprehensive resource for the identification, organization and interaction of mitochondrial machineries and pathways.

Making your way through the side of a barrel: The mechanism of membrane insertion and assembly of b-barrel proteins is a central question of outer membrane biogenesis of mitochondria, chloroplasts, and Gram-negative bacteria. Höhr et al. developed assays to address this fundamental problem. They systematically mapped precursor proteins transported by the mitochondrial Omp85 channel (Sam50) to elucidate the entire membrane insertion pathway of a precursor in the native membrane environment. Their findings directly demonstrate translocation of precursor proteins through the lumen of the mitochondrial Omp85 channel, signal recognition by β-strand exchange between channel and precursor, and exit through the lateral gate into the membrane. Science , this issue p. eaah6834

Mitochondria possess elaborate machineries for the import of proteins from the cytosol. Cytosolic factors like Hsp70 chaperones and their co-chaperones, the J-proteins, guide proteins to the mitochondrial surface. The translocase of the mitochondrial outer membrane (TOM) forms the entry gate for preproteins. How the proteins are delivered to mitochondrial preprotein receptors is poorly understood. We identify the cytosolic J-protein Xdj1 as a specific interaction partner of the central receptor Tom22. Tom22 recruits Xdj1 to the mitochondrial surface to promote import of preproteins and assembly of the TOM complex. Additionally, we find that the receptor Tom70 binds a different cytosolic J-protein, Djp1. Our findings suggest that cytosolic J-proteins target distinct TOM receptors and promote the biogenesis of mitochondrial proteins.