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 MDH2s regulation of MT-ND3 transcription via FASTKD1 binding and CLPPs 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 C15orf61s 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. HighlightsO_LIMultiomic signatures across perturbations reveal extensive post-transcriptional regulation C_LIO_LIThe TCA cycle enzyme MDH2 binds FASTKD1 to modulate MT-ND3 transcript levels C_LIO_LIMALSU1 is a CLPP protease substrate whose deficiency causes a mitochondrial disease C_LIO_LIC15orf61 binds ATP synthase and negatively regulates its higher order assembly C_LIO_LIMMADHC inversely affects mtDNA levels potentially mediated through LONP1 C_LI