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 in
cholesterol-
sphingolipid-enriched membranes, including early and recycling endosomes, but whether rafts are found in late endocytic organelles has not 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-
sphingolipid accumulation. In normal cells, the majority of [(3)H]
cholesterol released from [(3)H]
cholesterol ester-
LDL associated with
detergent-soluble membranes, was rapidly transported to the plasma membrane and became increasingly insoluble with time. In Niemann-Pick C1 (
NPC1) protein-deficient
lipidosis cells, the association of
LDL-cholesterol with DRMs was enhanced and its transport to the plasma membrane was inhibited. In addition, the
NPC1 protein was normally recovered in
detergent-soluble membranes and its association with DRMs was enhanced by lysosomal
cholesterol loading. Moreover, lysosomal
cholesterol deposition was kinetically paralleled by the sequestration of
sphingolipids and formation of multilamellar bodies in late endocytic organelles. These results suggest that late endocytic organelles are normally raft-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
sphingolipidoses, causes an overcrowding of rafts forming lamellar bodies in the degradative compartments.