Abstract

Mitochondrial dysfunction is a hallmark of numerous human diseases and is often accompanied by changes in metabolic flux, mitochondrial morphology, and proteostatic signalling. In patients, such dysfunction is associated with conserved adaptive responses involving proteome remodeling, altered autophagy, and disruption of mitochondrial one-carbon metabolism. While many of these changes act as compensatory mechanisms, their chronic activation may ultimately impair cellular function. To identify modifiers of mitochondrial genome instability, we performed a genetic screen in Drosophila melanogaster expressing a proofreading-deficient mtDNA polymerase (POLγexo-). We identified critical pathways involved in nutrient sensing, insulin signalling, mitochondrial protein import, and autophagy that rescue the lethal phenotype of POL γexo- flies. Notably, hemizygosity for dilp1, atg2, tim14, or melted restored autophagic flux and proteasome activity, and supported metabolic adaptation. While mtDNA mutation frequencies remained high in most rescued lines, melted-rescued flies showed a reduction, suggesting early developmental action. Our findings further identify the nucleation step of autophagy as a key therapeutic target in mitigating mitochondrial genome instability.