TMEM65-dependent Ca2+ extrusion safeguards mitochondrial homeostasis

The bidirectional transport of Ca2+ into and out of mitochondria regulates metabolism, signaling, and cell fate. While influx is mediated by the Mitochondrial Calcium Uniporter (MCU) complex, efflux mechanisms are more diversified, involving Na⁺ or H⁺ exchange pathways. We here demonstrate that TMEM65 is a fundamental component of the Ca2+ efflux machinery of mitochondria. Its overexpression specifically enhances Na⁺- and Li⁺-dependent mitochondrial Ca²⁺ extrusion. This effect is inhibited by CGP-37157 and does not depends on NCLX, currently considered the bona fide mitochondrial Na+/Ca2+ exchanger. Its downregulation chronically elevates basal [Ca²⁺]mt and impairs efflux upon stimulation. In Caenorhabditis elegans, deletion of TMEM65 homologs compromises embryonic development under mild thermal stress, causing necrotic lesions that are suppressed by genetic inhibition of MCU-1. These findings highlight a molecular component that may be relevant in pathological settings in which excessive mitochondrial Ca2+ accumulation critically contribute to degenerative pathways.