Mitochondrial PEP import controls amino acid catabolism and muscle integrity through SLC25A34

Metabolic flexibility enables eukaryotic cells to adapt to stress by dynamically reallocating fuels among distinct metabolite pools. In skeletal muscle, prolonged fasting promotes a shift in substrate utilization from glucose toward amino acids, yet the molecular mechanisms governing this fuel switching remain incompletely defined. Mitochondria are central to this process by coordinating oxidative metabolism and importing/exporting metabolites through inner-membrane transporters, including members of the SLC25 carrier family. Notably, many SLC25 transporters remain “orphan carriers,” with unknown substrates and functions. Here, we identify SLC25A34 as a fasting-inducible orphan mitochondrial carrier that is highly enriched in the slow-twitch skeletal muscles. Muscle-specific Slc25a34 deficiency results in muscle hypertrophy accompanied by myopathic features, implicating SLC25A34 in muscle homeostasis. Additionally, we reconstituted SLC25A34 into proteoliposomes and directly quantified transport activity, revealing that SLC25A34 functions as a phosphoenolpyruvate (PEP) transporter. Together, these results identified SLC25A34 is a PEP transporter that regulates amino acid catabolism and muscle integrity.