Rauseo, D.; Contreras-Baeza, Y.; Faurand, H.; Carcamo, N.; Suarez, R.; von Faber-Castell, A.; Silva, F.; Mora-Gonzalez, V.; Wyss, M. T.; Baeza-Lehnert, F.; Ruminot, I.; Alvarez-Navarro, C.; San Martin, A.; Weber, B.; Sandoval, P. Y.; Barros, L. F.

We addressed the question of mitochondrial lactate metabolism using genetically-encoded sensors. The organelle was found to contain a dynamic lactate pool that leads to dose- and time-dependent protein lactylation. In neurons, mitochondrial lactate reported blood lactate levels with high fidelity. The exchange of lactate across the inner mitochondrial membrane was found to be mediated by a high affinity H+-coupled transport system involving the mitochondrial pyruvate carrier MPC. Assessment of electron transport chain activity and determination of lactate flux showed that mitochondria are tonic lactate producers, a phenomenon driven by energization and stimulated by hypoxia. We conclude that an overflow mechanism caps the redox level of mitochondria, while saving energy in the form of lactate.