Mfn2 induces NCLX-mediated calcium release from mitochondria.
Mfn2 is a mitochondrial outer-membrane fusion protein that also functions as a tether between mitochondria and the ER. Here, we identify a previously unrecognized role for Mfn2 in promoting mitochondrial Ca2+ release via the Na+/Ca2+ exchanger NCLX. This function was uncovered through studies with the fungal toxin phomoxanthone A (PXA), which induces NCLX-dependent Ca2+ release by directly targeting Mfn2. Mfn2-dependent Ca2+ release through NCLX is similarly triggered by ROS in respiring cells treated with oligomycin or mitoPQ. ROS enhances Ca2+ release by strengthening the interaction between Mfn2 and NCLX, an interaction that also requires the mitochondrial outer-membrane protein SLC25A46. Together, these proteins coordinate mitochondrial fission and Ca2+ release to initiate mitophagy. The antioxidant N-acetylcysteine blocks ROS-induced mitochondrial fission, but inhibition of Ca2+ release with the NCLX inhibitor CGP37157 does not, indicating that ROS-driven fission is independent of Ca2+ release. In contrast, Ca2+ release is required for efficient mitophagy, as NCLX inhibition arrests this process at a later stage. We further show that Ca2+ promotes mitophagy through NEDD4-1, which is a Ca2+-responsive E3 ubiquitin ligase. Together, these findings connect mitochondrial ROS production to Ca2+ signaling, mitochondrial remodeling, and mitophagy, providing new insight into how mitochondrial dysfunction may contribute to neurodegenerative and metabolic disease.