Mrx6 regulates mitochondrial DNA copy number in S. cerevisiae by engaging the evolutionarily conserved Lon protease Pim1

Mitochondrial function crucially depends on the maintenance of multiple mitochondrial DNA (mtDNA) copies. Surprisingly, the cellular mechanisms regulating mtDNA copy number remain poorly understood. Through a systematic high-throughput approach in S. cerevisiae, we determined mtDNA to nuclear DNA ratios in 5148 strains lacking non-essential genes. The screen revealed MRX6, a largely uncharacterized gene, whose deletion displayed a marked increase of mtDNA levels, while maintaining WT-like mitochondrial structure and cell size. Quantitative super-resolution imaging revealed that deletion of MRX6 alters both the size and spatial distribution of mtDNA nucleoids. We demonstrate that Mrx6 partially colocalizes with mtDNA within mitochondria and interacts with the conserved Lon protease Pim1 in a complex that also includes Mam33 and the Mrx6-related protein Pet20. Acute depletion of Pim1 phenocopied the high mtDNA levels observed in Δmrx6 cells. No further increase of mtDNA copy number was observed upon depletion of Pim1 in Δmrx6 cells, revealing an epistatic relationship between Pim1 and Mrx6. Human and bacterial Lon proteases regulate DNA replication by degrading replication initiation factors, suggesting a model in which Pim1 acts similarly with the Mrx6 complex providing a scaffold linking it to mtDNA.