In the first part of our project, we delved into the intricate process of maintaining mitochondrial DNA (mtDNA) across cell generations, focusing on a critical but poorly understood aspect—regulating the correct number of mtDNA copies within cells. We embarked on a systematic analysis of approximately 5,000 mutant yeast strains to pinpoint the genes that are essential for maintaining normal cellular mtDNA levels. This comprehensive screen unveiled several mutants with elevated levels of mtDNA. Among them, we narrowed our focus to two enigmatic genes: MRX6 and CIM1.
Our investigation into MRX6-deficient cells unveiled clusters of mtDNA copies, indicating a deficiency in segregating replicated mtDNA copies. We found that Mrx6 interacts physically with the evolutionarily conserved protease Pim1, a central player in mitochondrial protein quality control. Depleting Pim1 mirrored the effects of MRX6 deficiency on mtDNA levels, and further depletion of MRX6 did not exacerbate the situation. These findings support a model in which MRX6 regulates mtDNA levels by modulating Pim1-mediated protein degradation.
Our attention then shifted to the CIM1 gene, identified in our systematic analysis as pivotal in regulating mtDNA copy number. Surprisingly, we discovered that Cim1 requires two HMG boxes for its function, akin to the well-characterized mammalian mtDNA packaging factor TFAM and its yeast counterpart, Abf2. Surprisingly, we found that Abf2 and Cim1 form an antagonistic relationship in which Abf2 promotes and Cim1 reduces mtDNA copy number. We observed that Pim1-mediated regulation keeps Cim1 levels low within cells, presumably to prevent toxic effects on cellular mtDNA maintenance. Taken together, our findings underscore the pivotal role of the conserved Lon protease, Pim1, in mtDNA copy number regulation.
In a second part of this project, we set out to unravel how S. cerevisiae cells maintain pristine mtDNA. We introduced innovative single-cell assays and unearthed a fascinating capability of yeast cells to detect mutant mtDNA and support the generation of progeny predominantly harboring intact mtDNA. This discovery raised the intriguing question of how cells differentiate between the quality of various mtDNA copies within the same mitochondrial network. To address this question, we engineered yeast strains expressing fluorescently-tagged mtDNA-encoded subunits and mated them with cells expressing untagged counterparts. This approach revealed the strikingly limited mobility of fluorescent mtDNA-encoded proteins within mitochondria, which depended on mitochondrial cristae, invaginations of the inner mitochondrial membrane. Based on these experimental findings, we formulated "The Sphere of Influence Model" model (Fig. 2). We propose that mtDNA-encoded proteins remain in close proximity to the copy by which they are encoded in a cristae-dependent manner, creating a spatial genotype-phenotype link. Dysfunctional mtDNA-encoded subunits will thus result in local OXPHOS complexes defects, which can be detected by cellular quality control pathways. Our study provides a foundational insight into the mechanisms facilitating the detection of low-functioning mitochondria, a compelling avenue for future research.
