Sterol-modulated glycolipid sorting occurs in Niemann-Pick C1 late endosomes

The Niemann-Pick C1 (NPC1) protein and endocytosed low density lipoprotein (LDL)-derived cholesterol were shown to enrich separate subsets of vesicles containing lysosomal associated membrane protein 2, Localization of Rab7 i n the NPC1-containing vesicles and enrichment of lysosomal hydrolases in th e cholesterol-containing vesicles confirmed that these organelles were late endosomes and lysosomes, respectively. Lyso-bisphosphatidic acid, a lipid marker of the late endosomal pathway, was found in the cholesterol-enriched lysosomes, Recruitment of NPC1 to Rab7 compartments was stimulated by cell ular uptake of cholesterol, The NPC1 compartment was shown to be enriched i ll glyco lipids, and internalization of GalNAc beta1-4[NeuAc alpha2-3]Gal b eta1-4Glc beta1-1'-ceramide (G(M2)) into endocytic vesicles depends on the presence of NPC1 protein. The glycolipid profiles of the NPC1 compartment c ould be modulated by LDL uptake and accumulation of lysosomal cholesterol, Expression in cells of biologically active NPC1 protein fused to green fluo rescent protein revealed rapidly moving and flexible tubular extensions ema nating from the NPC1-containing vesicles. We conclude that the NPC1 compart ment is a dynamic, sterol-modulated sorting organelle involved in the traff icking of plasma membrane-derived glycolipids as well as plasma membrane an d endocytosed LDL cholesterol,