A negative regulator of mitochondrial complex I assembly adapts respiration to cellular energy demand.

How mitochondrial respiration is tightly regulated by energy demand remains incompletely defined. When mammalian cells switch from glucose to galactose as a carbon source, we observed the enhanced assembly of respiratory chain complexes accompanied by a marked reduction in TMEM141, a mitochondrial inner membrane protein. Loss of TMEM141 increased mitochondrial respiration and promoted complex I assembly, whereas galactose-induced complex I assembly was markedly blunted in TMEM141-deficient cells. TMEM141 interacts with the complex I assembly factor TIMMDC1, limiting its association with complex I subunits. TMEM141 is degraded by the mitochondrial proteases AFG3L2 and YME1L1, and galactose treatment strengthens their interactions. TMEM141 deficiency increases oxidative damage and mtDNA release, leading to activation of the cGAS-STING pathway. In Drosophila, dTMEM141 localizes to mitochondria, modulates mitochondrial activity, and is required for glial cell integrity in the eye. Together, our findings reveal TMEM141 as a negative regulator of complex I assembly that adapts to oxidative phosphorylation (OXPHOS) demands.