Abstract

Mitochondrial dysfunction underlies a wide range of human diseases, including primary mitochondrial disorders, neurodegeneration, cancer, and ageing. To preserve cellular homeostasis, organisms have evolved adaptive mechanisms that coordinate nuclear and mitochondrial gene expression. Here, we use genome-wide CRISPR knockout screening to identify cell fitness pathways that support survival under impaired mitochondrial protein synthesis. The strongest suppressor of aberrant mitochondrial translation defects – besides a compendium of known mitochondrial translation quality control factors – is the loss of the vacuolar-type H + -ATPase (v-ATPase), a key regulator of intracellular acidification, nutrient sensing, and growth signaling. We show that partial v-ATPase loss reciprocally modulates mitochondrial membrane potential (ΔΨ m ) and cristae structure in both cancer cell lines and mitochondrial disease patient-derived models. Our findings uncover an extra-organellar buffering mechanism whereby partial v-ATPase inhibition mitigates mitochondrial dysfunction by altering pH homeostasis and driving metabolic rewiring as a protective response that promotes cell fitness.