Matrix nucleotide homeostasis couples energetic state to mitochondrial translation

Mitochondrial protein synthesis must adapt to fluctuations in organellar energetic state to sustain oxidative phosphorylation and cellular homeostasis, yet the mechanisms coupling mitochondrial bioenergetics to gene expression remain poorly understood. Here, we identify matrix nucleotide phosphorylation potential as a direct metabolic determinant of mitochondrial translation. Using ATP synthase inhibition as an experimental perturbation, we show that inhibition of the F1Fo-ATP synthase induces inner-membrane hyperpolarization that restricts ANT-mediated adenine nucleotide exchange and rapidly collapses the bioavailable matrix ATP pool. Reduced matrix ATP limits mitochondrial GTP regeneration, likely through impaired nucleoside diphosphate kinase-dependent phosphate transfer and diminished substrate-level phosphorylation, leading to global arrest of mitochondrial protein synthesis. Restoration of nucleotide exchange or selective replenishment of matrix GTP rescues translation, identifying GTP depletion as the proximal energetic constraint on mitochondrial gene expression. These findings establish an intrinsic mechanism by which energetic state directly regulates mitochondrial translational capacity through matrix nucleotide homeostasis. More broadly, our work identifies matrix GTP availability as a central energetic checkpoint coupling oxidative phosphorylation to mitochondrial gene expression, with important implications for mitochondrial stress adaptation and diseases associated with bioenergetic dysfunction.