Cooperative Architecture of Mitochondrial Proteome Homeostasis.

Mitochondria are semi-autonomous organelles whose generation and maintenance demand precise expression, processing, and assembly of >1,000 proteins encoded across two genomes. To explore this cooperativity, we performed multiomic analyses on >200 cell lines harboring mitochondrial gene perturbations, generating >26M molecular measurements. Our data reveal that mitochondrial proteome homeostasis is heavily influenced by post-transcriptional processes. Through nearest neighbor analyses, we reveal diverse protein activities undergirding this regulation, including MDH2's regulation of MT-ND3 transcription via FASTKD1 binding and CLPP's processing of the mitoribosomal assembly factor MALSU1, which we establish as a disease gene. Through entropy analysis, we reveal unexpectedly heterogeneous protein-level variability across complexes and use complexome profiling to identify new complex-specific membership, including C15orf61's association with complex V. We further observe substantial mtDNA copy number variation, notably upon disruption of the disease-related cobalamin biosynthesis protein MMADHC. Together, we establish new protein functions and provide a multilayered view into mitochondrial proteome regulation.