Mitochondria are epicentres of eukaryotic metabolism and bioenergetics. Pioneering efforts in recent decades have established the core
protein componentry of these organelles1 and have linked their dysfunction to more than 150 distinct disorders2,3. Still, hundreds of
mitochondrial proteins lack clear functions4, and the underlying genetic basis for approximately 40% of
mitochondrial disorders remains unresolved5. Here, to establish a more complete functional compendium of human
mitochondrial proteins, we profiled more than 200 CRISPR-mediated HAP1 cell knockout lines using mass spectrometry-based multiomics analyses. This effort generated approximately 8.3 million distinct biomolecule measurements, providing a deep survey of the cellular responses to mitochondrial perturbations and laying a foundation for mechanistic investigations into
protein function. Guided by these data, we discovered that PIGY upstream open reading frame (PYURF) is an
S-adenosylmethionine-dependent
methyltransferase chaperone that supports both complex I assembly and
coenzyme Q biosynthesis and is disrupted in a previously unresolved multisystemic
mitochondrial disorder. We further linked the putative
zinc transporter SLC30A9 to mitochondrial ribosomes and OxPhos integrity and established RAB5IF as the second gene harbouring pathogenic variants that cause
cerebrofaciothoracic dysplasia. Our data, which can be explored through the interactive online MITOMICS.app resource, suggest
biological roles for many other orphan
mitochondrial proteins that still lack robust functional characterization and define a rich cell signature of
mitochondrial dysfunction that can support the genetic diagnosis of
mitochondrial diseases.