Local mitochondrial physiology defined by mtDNA quality guides purifying selection
Felix Thoma, Johannes Hagen, Romina Rathberger, Francesco Padovani, David Hörl, Kurt M. Schmoller, Christof Osman
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.
Introduction
Mitochondria contain their own genome, mitochondrial DNA (mtDNA), which is present in multiple copies in each cell. mtDNA encodes a limited but critical subset of mitochondrial proteins, which are integral subunits of the respiratory chain. Mutations in these genes often result in respiratory dysfunction, contributing to the pathogenesis of a wide range of diseases [1–3]. Purifying selection, which has been shown to act in the germline of mammalian cells and Drosophila melanogaster, acts to preserve mitochondrial DNA (mtDNA) integrity, by preventing maintenance of mutant mtDNA copies [4–9].
Materials and methods
Yeast strains and plasmids
All yeast strains are derived from the W303 background. Deletion of genes was performed using homologous recombination according to protocols described in [50]. Strains generated or used for this study can be found in S1 Table. The plasmids used are listed in S2 Table.
Results
Development of a rapid pipeline for mtDNA segregation analysis
In the following, we describe the development of a novel flow cytometry–based pipeline that enables quantitative analysis of mtDNA variant segregation in cell populations derived from single heteroplasmic zygotes carrying two distinct mitochondrial genomes. This approach builds on previous microscopy-based strategies that employed heteroplasmic yeast zygotes containing mtDNA encoding the fluorescent fusion protein Atp6-mNeonGreen (mtDNAATP6-NG) alongside a non-fluorescent mtDNA (“dark” mtDNA) [12].
Discussion
In this study, we introduce FAST, a novel pipeline to analyse the segregation behaviour of mtDNA variants in heteroplasmic S. cerevisiae zygotes. Using this approach, we demonstrate purifying selection against mutant mtDNA genomes lacking components of complexes III, IV, or the ATP synthase in populations arising from heteroplasmic founder cells.
Acknowledgments
We are grateful to AG Kunz and AG Robatzek for providing access to the flow cytometers. We thank Nadja Lebedeva for her technical assistance and to Verena Peterreins for cloning the mtQUEEN-2m plasmid. We also appreciate the valuable discussions and feedback from members of the Mokranjac lab and the Mito-Club.
Citation: Thoma F, Hagen J, Rathberger R, Padovani F, Hörl D, Schmoller KM, et al. (2026) Local mitochondrial physiology defined by mtDNA quality guides purifying selection. PLoS Genet 22(1): e1011836. https://doi.org/10.1371/journal.pgen.1011836
Editor: Miguel A. Peñalva, Consejo Superior de Investigaciones Cientificas, SPAIN
Received: August 11, 2025; Accepted: December 29, 2025; Published: January 9, 2026
Copyright: © 2026 Thoma et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All cropped images used for data quantification in the figures are accessible here: https://osf.io/h63ca/overview (main figures) https://osf.io/xmh8b/overview (supplementary) The full raw microscopy data exceed 1 TB and were therefore not uploaded to an online repository; however, they can be made available upon request.
Funding: This work was supported by grants of the Deutsche Forschungsgemeinschaft (https://www.dfg.de/) awarded to CO (OS 410/3-1) and DH (HO 7333/1). The work was also supported by a grant of the Human Frontier Science Program (https://www.hfsp.org/) awarded to CO (GP021/2023). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
