Lipid transfer protein ORP3 mediates lysosomal repair via LC3B and ubiquitin-TAK1-p38 signaling

Lysosomal membrane damage triggers a multi-stage repair response essential for cellular homeostasis. Here we identify the oxysterol-binding protein-related protein ORP3 as a critical mediator of late-stage lysosomal membrane repair. Following lysosomal damage induced by L-leucine-leucine methyl ester (LLOME) or cationic amphiphilic drugs (CADs), ORP3 is phosphorylated and recruited to ER–lysophagosome contact sites via a signaling cascade initiated by lysosomal membrane ubiquitination, TAK1, p38 MAPK, and, to a lesser extent, IKK. p38-dependent phosphorylation promotes direct interaction between ORP3 and LC3B, which together with PI(4,5)P₂ binding, is required for autophagic lysosome recruitment. ORP3 depletion impairs late-stage lysosomal recovery, elevates lysosomal lipid peroxidation, and reduces cell survival. A lipid transfer-deficient ORP3 mutant fails to restore lysosome function despite normal recruitment, indicating that ER-to-lysophagosome transfer of phosphatidylcholine by ORP3 is functionally required. ORP3 activity is subsequently terminated by VCP/p97-mediated deubiquitination of lysosomes. These findings define ORP3 as a MAPK regulated lipid transfer protein during the late autophagic phase of the endolysosomal damage response.