Proteins enriched on damaged lysosomes in 293T

Lysosomal membrane permeabilization (LMP) or lysosomal membrane damage is commonly associated with aging and age-related diseases. In searching for cellular mechanisms in response to LMP, we used a proteomic approach to identify proteins enriched on damaged lysosomes. This unbiased approach identified a new phosphoinositide signaling pathway triggered by LMP to mediate rapid lysosomal repair. Specifically, LMP induces fast lysosomal recruitment of PI4K2A which generates high levels of the lipid messenger phosphatidylinositol-4-phosphate (PtdIns4P) on damaged lysosomes. Lysosomal PtdIns4P in turn recruits multiple oxysterol-binding protein (OSBP)-related protein (ORP) family members, including ORP9, ORP10, and ORP11, to orchestrate extensive membrane contact sites (MCSs) between damaged lysosomes and the endoplasmic reticulum (ER). The ORPs subsequently catalyze robust ER-to-lysosomal transport of phosphatidylserine (PS), which is critical for rapid lysosomal repair. The lipid transporter ATG2 is also recruited to damaged lysosomes, activated by PS, and is essential for rapid lysosomal repair. Our findings identify a phosphoinositide-dependent membrane tethering and lipid transport (PITT) pathway essential for the maintenance of lysosomal membrane integrity, with important implications for a wide range of diseases characterized by impaired lysosomal function.