TMEM65-dependent Ca2+ extrusion safeguards mitochondrial homeostasis
The bidirectional transport of Ca2+ into and out of mitochondria is a conserved biological process controlling multiple events, including metabolism, signaling, and cell fate. In the presence of membrane potential driving mitochondrial Ca2+ accumulation, transient changes of [Ca2+]mt in response to cytosolic [Ca2+] variations are ensured by a molecular machinery for Ca2+ influx and efflux embedded in the inner mitochondrial membrane (IMM). While it is well established that influx relies on the Mitochondrial Calcium Uniporter (MCU), efflux was expected to be molecularly diversified, given the occurrence of functionally different exchange pathways with either Na+ or H+1. Accordingly, dedicated transporters ensure proper Ca2+ homeostasis and tightly regulated mitochondrial bioenergetics, but the process is not yet fully elucidated. We here demonstrate that TMEM65, a protein with an unknown biological function, is a fundamental component of the Ca2+ efflux machinery of mitochondria. As the MCU, TMEM65 has a broad tissue expression and localizes to the IMM. Its overexpression dramatically enhances Na+- and Li+-dependent mitochondrial Ca2+ extrusion, which is abrogated by the pharmacological inhibitor CGP-37157. Conversely, its downregulation chronically elevates resting mitochondrial Ca2+ levels and inhibits efficient Ca2+ efflux upon cellular activation, culminating in mitochondrial calcium overload and causing organelle dysfunction. Since TMEM65 has been associated with a severe human mitochondrial disease2, we deleted the TMEM65 homologues in Caenorhabditis elegans (CeTMEM65). While the two CeTMEM65 orthologs are dispensable for the survival at permissive growing conditions, their loss undermines embryonic developments when eggs are exposed to mild temperature-stress. In this regard, we find that CeTMEM65 (null) alleles cause necrotic lesions that are suppressed by inhibiting the mitochondrial calcium uniporter MCU-1. Overall, these results unambiguously assign a primary role in mitochondrial Ca2+ homeostasis to the orphan protein TMEM65. More importantly, our findings describe a novel molecular component that may be relevant in pathological settings in which excessive mitochondrial Ca2+ accumulation critically contribute to degenerative pathways.