Depletion of rafts in late endocytic membranes is controlled by NPC1-dependent recycling of cholesterol to the plasma membrane

In mammalian cells, cholesterol is thought to associate with sphingolipids to form lateral membrane domains termed rafts. Increasing evidence suggests that rafts regulate protein interactions, for example, during signalling, intracellular transport and host-pathogen interactions. Rafts are present i n cholesterol-sphingolipid-enriched membranes, including early and recyclin g endosomes, but whether rafts are found in late endocytic organelles has n ot been analyzed, In this study, we analyzed the association of cholesterol and late endosomal proteins with low-density detergent-resistant membranes (DRMs) in normal cells and in cells with lysosomal cholesterol-sphingolipi d accumulation. In normal cells, the majority of [H-3]cholesterol released from [H-3]cholesterol ester-LDL associated with detergent-soluble membranes , was rapidly transported to the plasma membrane and became increasingly in soluble with time. In Niemann-Pick C1 (NPC1) protein-deficient lipidosis ce lls, the association of LDL-cholesterol with DRMs was enhanced and its tran sport to the plasma membrane was inhibited, In addition, the NPC1 protein w as normally recovered in detergent-soluble membranes and its association wi th DRMs was enhanced by lysosomal cholesterol loading, Moreover, lysosomal cholesterol deposition was kinetically paralleled by the sequestration of s phingolipids and formation of multilamellar bodies in late endocytic organe lles. These results suggest that late endocytic organelles are normally raf t-poor and that endocytosed LDL-cholesterol is efficiently recycled to the plasma membrane in an NPC1-dependent process. The cholesterol-sphingolipid accumulation characteristic to NPC disease, and potentially to other sphing olipidoses, causes an overcrowding of rafts forming lamellar bodies in the degradative compartments.