In a eukaryotic cell, the ratio of mitochondrial DNA (mtDNA) to nuclear DNA (nDNA) is usually maintained within a specific range. This suggests the presence of a negative feedback loop mechanism preventing extensive mtDNA replication and depletion. However, the experimental data on this hypothetical mechanism are limited. In this study, we suggested that deletions in mtDNA, known to increase mtDNA abundance, can disrupt this mechanism, and thus, increase cell-to-cell variance in the mtDNA copy numbers. To test this, we generated Saccharomyces cerevisiae rho⁻ strains with large deletions in the mtDNA and rho⁰ strains depleted of mtDNA. Given that mtDNA contributes to the total DNA content of exponentially growing yeast cells, we showed that it can be quantified in individual cells by flow cytometry using the DNA-intercalating fluorescent dye SYTOX green. We found that the rho⁻ mutations increased both the levels and cell-to-cell heterogeneity in the total DNA content of G1 and G2/M yeast cells, with no association with the cell size. Furthermore, the depletion of mtDNA in both the rho⁺ and rho⁻ strains significantly decreased the SYTOX green signal variance. The high cell-to-cell heterogeneity of the mtDNA amount in the rho⁻ strains suggests that mtDNA copy number regulation relies on full-length mtDNA, whereas the rho⁻ mtDNAs partially escape this regulation.

During sexual reproduction, fungi usually inherit mtDNA from both parents, however, the distribution of the mtDNA in the progeny can be biased toward some mtDNA variants. For example, crossing Saccharomyces cerevisiae strain carrying wild type (rho⁺) mtDNA with the strain carrying mutant mtDNA variant with a large deletion (rho⁻) can produce up to 99–100% of rho⁻ diploid progeny. Two factors could contribute to this phenomenon. First, rho⁻ cells may accumulate more copies of mtDNA molecules per cell than wild-type cells, making rho⁻ mtDNA the prevalent mtDNA molecule in zygotes. This consequently leads to a high portion of rho⁻ diploid cells in the offspring. Second, rho⁻ mtDNA may have a competitive advantage within heteroplasmic cells, and therefore could displace rho⁺ mtDNA in a series of generations, regardless of their initial ratio. To assess the contribution of these factors, we investigated the genotypes and phenotypes of twenty two rho⁻ yeast strains. We found that indeed rho⁻ cells have a higher mtDNA copy number per cell than rho⁺ strains. Using an in silico modelling of mtDNA selection and random drift in heteroplasmic yeast cells, we assessed the intracellular fitness of mutant mtDNA variants. Our model indicates that both higher copy numbers and intracellular fitness advantage of the rho⁻ mtDNA contribute to the biased inheritance of rho⁻ mtDNA.

We searched for genes whose deletion affects mitochondrial heteroplasmy in the yeast Saccharomyces cerevisiae. In order to do this, we crossed a yeast knockout collection pool (rho⁺) with a rho^– strain containing a large deletion in its mtDNA. The resulting mixture of cells was plated onto selective media that made it possible to distinguish between diploid cells that had lost and retained wild-type mtDNA as a result of such crossing. From the resulting pools (initial one, cells that lose rho⁺ mtDNA, and those that retained it), we isolated DNA and determined the proportion of deletion strains using high-throughput sequencing. This made it possible to identify functional groups of genes that reduce the ability of yeast cells to maintain rho⁺ mtDNA in a state of heteroplasmy with rho^– mtDNA.