Nat Metab. 2026 Oct 27.
Mutations in mitochondrial DNA (mtDNA) impair ETC activity and oxidative phosphorylation, but their phenotypic consequences vary with heteroplasmy levels. Whether increasing mtDNA heteroplasmy creates metabolic dependencies not present in respiratory-competent cells remains unknown. Here, we show that low pyruvate carboxylase (PC) expression creates an asparagine dependency in high-mtDNA-heteroplasmy, respiration-compromised cells. In isogenic cells carrying similar mtDNA deletions, respiration declines linearly with increasing heteroplasmy, whereas redox imbalance emerges only above 50% heteroplasmy. Above 70% heteroplasmy, integrated stress-response-associated translational defects emerge, which can be reversed by either exogenous asparagine or PC overexpression. Asparagine dependence inversely correlates with PC expression across respiration-deficient cells. Consistent with this pattern, patient-derived thyroid tumour cells harbouring a high-heteroplasmy complex I mutation and low PC levels exhibit asparagine auxotrophy and sensitivity to L-asparaginase. Together, these findings demonstrate a role for mitochondrial PC in cellular asparagine synthesis under conditions of compromised respiratory activity.