Global mitochondrial connectivity map reveals the landscape of yeast functional assemblies and conserved protein...

Mitochondria are essential organelles whose functions depend on coordinated multiprotein complexes, yet their composition and organization remain incomplete. Here, we present a large-scale map of mitochondrial protein complexes by integrating affinity purification of 740 endogenously GFP-tagged mitochondrial proteins with biochemical co-fractionation of mitochondrial extracts from yeast (Saccharomyces cerevisiae) grown under respiratory conditions. Mass spectrometry identifies 13,716 high-confidence protein associations and defines 556 heteromeric complexes, many previously unknown. These assemblies reveal factors involved in coenzyme Q6 biosynthesis, membrane contact sites, phospholipid transport, and coordination with the MICOS complex during respiration. We further link 538 assemblies to 294 candidate human disease genes and construct a conservation map of 852,146 predicted mitochondrial interactions across 271 genomes, and validate key predictions in human cell lines and mouse brain tissue. Together, this work provides a comprehensive mitochondrial interactome, assigning functions to poorly characterized proteins, and offering insights into mitochondrial biology and disease-associated assemblies.