Phenotypic CRISPR screens identify NLRX1 as an essential activator of the human mitochondrial permeability transition

The mitochondrial permeability transition (mPT) is an evolutionary conserved Ca2+ overload destructive response that permeabilizes the inner mitochondrial membrane. The molecular mechanism underlying the mPT has not been established. To identify core proteins, we designed two phenotypic assays for mitochondrial Ca2+ overload and applied them to FACS-based genome-wide CRISPR screening in human cells. With secondary validation, we identified four protein encoding genes: NF2, REST, BPTF, and NRLX1. Knockout of BPTF, NF2, or REST increased mitochondrial Ca2+ retention capacity (CRC), however, Ca2+ release or sensitivity to cyclosporin A (CsA) persisted, indicative of mPT sensitizers. Knockout of the mitochondrial matrix protein NLRX1 increased CRC, abolished Ca2+ release, and was CsA-insensitive. NLRX1 was the only gene discovered as an essential mPT activator. By combining our screening results with evolutionary and functional analysis, we propose that NLRX1 stimulates peroxidized lipid pore formation, and that the mPT likely does not arise from transmembrane protein pores.