Benedikt, A.; Job, K.; Li, X.; Sarkar, S.; Hernandez Goetz, L.; Kukhtevich, I. V.; Padovani, F.; Chadha, Y.; Paukstyte, J.; Saarikangas, J.; Skotheim, J. M.; Schneider, R.; Swaffer, M. P.; Scialdone, A.; Lanz, M. C.; Kohler, A.; Schmoller, K. M.

Eukaryotic cells maintain multiple copies of the mitochondrial genome, which is essential for cellular metabolism. Accordingly, alterations in the mitochondrial DNA (mtDNA) copy number are associated with severe human diseases and ageing. However, the mechanisms through which cells regulate mtDNA copy number and the cellular consequences of altered copy number remain poorly understood. Here, using budding yeast as a model, we show that mtDNA copy number is determined by the amount of three limiting factors, Mip1, Abf2, and Rim1. By synthetically tuning the concentrations of only these three proteins, we can modulate mtDNA dosage inside the cell. This revealed that cells are surprisingly robust to mtDNA copy number alterations, with increased copy numbers even accelerating cell growth. Our findings suggest that this robustness is due to protein dosage compensation and independent regulation of mitochondrial morphology. Mechanistically, we identified a critical role of the retrograde signalling pathway for this adaptation. We show that signalling from mitochondria to the nucleus is upregulated in cells with higher mtDNA copy number, and disruption of this regulation diminishes their faster growth. Taken together, our work reveals regulatory principles that enable cells to adapt to mtDNA copy number alterations.