Johannes Hagen

The mitochondrial genome (mtDNA) encodes essential subunits of the electron transport chain and ATP synthase. Mutations in these genes impair oxidative phosphorylation, compromise mitochondrial ATP production and cellular energy supply, and can cause mitochondrial diseases. These consequences highlight the importance of mtDNA quality control (mtDNA-QC), the process by which cells selectively maintain intact mtDNA to preserve respiratory function. Here, we developed a high-throughput flow cytometry assay for Saccharomyces cerevisiae to track mtDNA segregation in cell populations derived from heteroplasmic zygotes, in which wild-type (WT) mtDNA is fluorescently labeled and mutant mtDNA remains unlabeled. Using this approach, we observe purifying selection against mtDNA lacking subunits of complex III (COB), complex IV (COX2) or the ATP synthase (ATP6), under fermentative conditions that do not require respiratory activity. By integrating cytometric data with growth assays, qPCR-based mtDNA copy-number measurements, and simulations, we find that the decline of mtDNAΔatp6 in populations derived from heteroplasmic zygotes is largely explained by the combination of its reduced mtDNA copy number—biasing zygotes toward higher contributions of intact mtDNA—and the proliferative disadvantage of cells carrying this variant. In contrast, the loss of mtDNAΔcob and mtDNAΔcox2 cannot be explained by growth defects and copy-number asymmetries alone, indicating an additional intracellular selection against these mutant genomes when intact mtDNA is present. In heteroplasmic cells containing both intact and mutant mtDNA, fluorescent reporters revealed local reductions in ATP levels and membrane potential near mutant genomes, indicating spatial heterogeneity in mitochondrial physiology that reflects local mtDNA quality. Disruption of the respiratory chain by deletion of nuclear-encoded subunits (RIP1, COX4) abolished these physiological gradients and impaired mtDNA-QC, suggesting that local bioenergetic differences are required for selective recognition. Together, our findings support a model in which yeast cells assess local respiratory function as a proxy for mtDNA integrity, enabling intracellular selection for functional

The inner membrane of mitochondria contains many membrane-embedded carrier proteins of the SLC25 family to facilitate the exchange of metabolites between the cytosol and mitochondria. These carriers use a specific import route for their biogenesis that relies on the TIM22 complex as an inner membrane translocase. The molecular details of carrier biogenesis are not well understood. Using an improved, desthiobiotin-based proximity labeling approach called Destni in living yeast cells, we identified the mitochondrial protein Aim11 as an interactor of newly imported carrier proteins. Aim11 forms a 160 kDa complex together with Iai11, Gep7 and Mtc1 in the mitochondrial inner membrane that we named the comrade-of-prohibitin (cohibitin) complex owing to its genetic interaction with prohibitins. Deletion of Aim11 impairs the import of carrier proteins into the inner membrane and renders cells hypersensitive to carrier overexpression. Our data suggest that the cohibitin complex plays a quality control function that supports the TIM22-mediated insertion of carrier proteins into the inner membrane of mitochondria.