The human TIM22 complex: functions, substrates, and connections to disease

Mitochondria are hubs of metabolic activity in cells. In addition to supporting ATP production through nutrient catabolism, mitochondria also house many crucial biosynthetic pathways, including iron-sulphur cluster synthesis and heme synthesis. The crucial role of mitochondria in cellular function means that their dysfunction is associated with a variety of human pathologies, including cancer, neurodegenerative disease, and mitochondrial disease. The diversity of mitochondrial functions necessitates a large and versatile proteome, which in human cells contains over 1000 proteins. Targeting and import of mitochondrial proteins to the correct mitochondrial subcompartment is mediated by multi-subunit complexes called translocases. Insertion of proteins with multiple transmembrane domains into the inner mitochondrial membrane is mediated by a translocase called the Translocase of the Inner Membrane 22 (TIM22) complex. The human TIM22 complex contains two subunits, AGK and Tim29, which are not present in the more extensively characterised Saccharomyces cerevisiae TIM22 complex. AGK had been previously described as a lipid kinase, although its kinase activity is dispensable for its function at the TIM22 complex. Mutations in the AGK gene cause a mitochondrial disease called Sengers syndrome, characterised by congenital cataracts, hypertrophic cardiomyopathy, lactic acidosis, and exercise intolerance. Despite recent advances in understanding of AGK function, the extent to which the functions of AGK in lipid and protein biogenesis contribute to the pathogenesis of Sengers syndrome were unclear, and the impact of AGK mutations on protein function had not been assessed. We set out to define the impact of AGK and TIM22 complex dysfunction on mitochondrial biology to provide much needed information on how perturbations at the TIM22 complex cause mitochondrial disease. Using unbiased proteomics approaches we characterised multiple Sengers syndrome patient fibroblast and disease