TMEM164 is an acyltransferase that forms ferroptotic polyunsaturated ether phospholipids

Ferroptosis is an iron-dependent form of cell death driven by the oxidation of polyunsaturated (PUFA) phospholipids. Large-scale genetic screens have pointed to a specialized role for PUFA ether phospholipids (ePLs) in promoting ferroptosis. Our understanding of the enzymes involved in PUFA ePL production, however, remains incomplete. Here we show using a combination of pathway mining of genetic dependency maps, AlphaFold-guided structure predictions, and targeted lipidomics that the uncharacterized transmembrane protein TMEM164 - genetic ablation of which has been shown to protect cells from ferroptosis - is a cysteine active-center enzyme that selectively transfers C20:4 acyl chains from phosphatidylcholine to lyso-ePLs to furnish PUFA-ePLs. TMEM164-null cells show substantial reductions in PUFA-ePLs, but not PUFA ester phospholipids, supporting that the selective suppression of PUFA-ePLs is sufficient to protect cells from ferroptosis and designating TMEM164 as a key enzyme specifically responsible for regulating this class of lipids.