Summary. Cholesterol is transported from the endoplasmic reticulum where it is synthesized to the plasma membrane in a fast process. Niemann-Pick disease is a fatal inherited 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 Niemann-Pick C2 (NPC2) proteins are not yet known.

This study shows the ability of bovine NPC2 (bNPC2) for intermembrane transfer of cholesterol in a novel cell-free assay-system. Our results show that NPC2 specifically extracts cholesterol from phospholipid bilayers and catalyzes the intermembrane transfer to acceptor vesicles in a dose- and time-dependent manner. This transfer activity was found to be dependent on temperature, pH, ionic strength, lipid composition of the model membranes, and the ratio of donor- to acceptor vesicles.

The presence of the lysosomal anionic phospholipids bis(monoacylglycero)phosphate-oleoyl (BMP-oleoyl) and phosphatidyl inositol significantly stimulated cholesterol transfer by NPC2, whereas bis(monoacylglycero)phosphate-myristoyl (BMP-myristoyl), phosphatidyl serine, and phosphatidic acid had no effect. When ceramide was incorporated into the vesicles, a stimulation of cholesterol transfer was observed, while the addition of sphingomyelin to vesicles inhibited cholesterol transfer. Our assay system further allowed us to identify for the first time the ability of other lysosomal proteins, most notably the GM2-activator, in transferring cholesterol from one membrane to another. This promises to be a valuable tool for further quantitative and mechanistic studies of protein-mediated lipid transfer.