Niemann-Pick type C disease is an inherited fatal disorder characterized by the accumulation of unesterified
cholesterol and other
lipids in the endosomal/lysosomal compartment. Two independent genes responsible for this
neurodegenerative disorder have been identified, but the precise functions of the encoded Niemann-Pick C1 (NPC1) and C2 (NPC2)
proteins are not yet known. We developed a cell-free assay for measuring intermembrane
lipid transport and examined the ability of bovine NPC2 (bNPC2) for intermembrane
cholesterol transfer. NPC2 specifically extracts
cholesterol from
phospholipid bilayers and catalyzes intermembrane transfer to acceptor vesicles in a dose- and time-dependent manner. This transfer activity is dependent on temperature, pH, ionic strength,
lipid composition of the model membranes, and the ratio of donor to acceptor vesicles. In model membranes, the presence of the lysosomal anionic
phospholipids bis(monooleoylglycero)
phosphate and
phosphatidyl inositol significantly stimulated
cholesterol transfer by NPC2, whereas bis(monomyristoylglycero)
phosphate,
phosphatidyl serine, and
phosphatidic acid had no effect. Moreover,
ceramide stimulated
cholesterol transfer slightly, whereas
sphingomyelin reduced
cholesterol transfer rates. With our assay system we identified for the first time the ability of other
lysosomal proteins, most notably the GM2-activator
protein, to mediate intermembrane
cholesterol transfer. This assay system promises to be a valuable tool for further quantitative and mechanistic studies of
protein-mediated
lipid transfer.