# Local mitochondrial physiology defined by mtDNA quality guides purifying selection

## TL;DR

This study shows how yeast cells selectively retain healthy mitochondrial DNA by detecting local energy production differences, even when respiration isn't needed.

## Contribution

A novel high-throughput flow cytometry assay was developed to track mtDNA segregation in yeast, revealing localized physiological signals guide purifying selection.

## Key findings

- Cells preferentially retain functional mtDNA even under non-respiratory conditions.
- Local ATP levels and membrane potential near mutant mtDNA are reduced, indicating spatial physiological heterogeneity.
- Disruption of the respiratory chain abolishes physiological gradients and impairs mtDNA quality control.

## Abstract

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 (ΔΨ) 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 mitochondrial genomes.

Mitochondria are essential organelles in our cells that convert nutrients into usable cellular energy. They contain their own DNA, and mutations in this DNA can compromise mitochondrial function and contribute to disease. In this work, we asked how cells recognize and limit the transmission of defective mitochondrial DNA. Using baker’s yeast as a model system, we developed a rapid approach that allows us to track how healthy and mutant mitochondrial DNA variants are inherited when cells divide. We found that cells preferentially retain mitochondrial DNA that supports normal mitochondrial function, even under conditions where respiration is not required. By combining cell sorting, growth measurements, microscopy, and simulations, we show that this selective process is influenced not only by the initial abundance of each mitochondrial genome, but also by localized physiological differences within the mitochondrial network. Regions containing mutant mitochondrial DNA exhibit reduced membrane potential and lower ATP availability, making them distinguishable from regions harboring intact genomes. When we impaired the respiratory chain, these physiological differences disappeared and selective quality control broke down. Our findings suggest that cells rely on local mitochondrial signals to preserve functional mitochondrial DNA, offering insight into how healthy mitochondria are maintained across generations.