Sterol Binding to Niemann-Pick Type C1 Disease Protein (NPC1) - Implications for its Function in Cholesterol Transport

Through studies of inborn errors of metabolism, scientists have uncovered many pathways involved in the transport of cholesterol within cells. Despite intense scientific interest, the mechanism by which cholesterol is transported between membrane compartments in animal cells remains obscure. Studies on Niemann-Pick type C disease (NPC) defined the requirement of at least two proteins involved in the transport of lipoprotein-derived cholesterol from lysosomes. Both proteins are located in the lysosomes and mutations in either NPC1, a membrane bound protein, or NPC2, a soluble protein, causes the accumulation of large amounts of cholesterol throughout the body. We encountered NPC1 in the course of isolating a cholesterol-homeostasis membrane protein that binds oxygenated metabolites of cholesterol, oxysterols. We then showed that NPC1's sterol binding domain was localized to its N-terminal luminal soluble domain (NTD) and that this domain bound cholesterol as well. We first attempted to understand the functional significance of NPC1 binding to oxysterols. We were able to demonstrate that NPC1 is not required for the regulatory actions of oxysterols since the oxysterol-mediated inhibition of the proteolytic processing of the sterol regulatory element-binding protein (SREBP) and activation of acyl-CoA:cholesterol acyltransferase (ACAT) was not defective in NPC1 mutant fibroblasts. Many studies emphasized the importance of NPC1 in the transport of LDL-derived cholesterol out of the lysosomes. However, there is contradictory evidence for the role of NPC1 in transporting plasma membrane cholesterol and endogenously synthesized cholesterol to the compartment of the endoplasmic reticulum (ER) that contains ACAT. Experiments presented in this study characterize NPC1 dependent and independent transport of cholesterol in cells. We definitively show that NPC1 is only required for the transport of cholesterol from the LDL particle that has entered the cell through receptor-mediated endocytosis. After defining that NPC1's role is only in the transport of cholesterol out of the lysosome, we began to study the mechanism involved in this transport. Inside the lysosome reside the two proteins, NPC1 and NPC2, that when mutated cause the NPC disease. Both NPC1 and NPC2 bind cholesterol with ~ 100nM affinity, but they bind to opposite ends of the cholesterol molecule. Recently an in vitro assay using purified proteins was developed to measure transfer of [3H]cholesterol between the NPC1(NTD), NPC2 and phosphatidylcholine liposomes. NPC1(NTD) donates or accepts cholesterol from liposomes very slowly, whereas NPC2 acts quickly. NPC2 stimulates the bidirectional transfer of cholesterol between NPC1(NTD) and liposomes. This evidence suggested that NPC2 could stimulate NPC1's rates of dissociation or association of cholesterol. This was the first evidence suggesting a direct interaction between both proteins. Using mutational analysis, we identified two functional subdomains of NPC1(NTD) ? one for sterol binding and the other for NPC2-mediated transfer. NPC1 with point mutations in either one of the subdomains cannot accept cholesterol from NPC2 and therefore cannot restore cholesterol exit from lysosomes in NPC1-deficient cells. These studies explain the dual requirement of these proteins in the export of cholesterol from lysosomes, defining how mutations in either protein produce similar clinical phenotypes. Based on these biochemical and tissue culture studies, in addition to the availability of the crystal structure of both proteins bound to sterols, we propose a working model: after lysosomal hydrolysis of LDL-cholesteryl esters, cholesterol binds to NPC2, which transfers it to NPC1(NTD), reversing its orientation and allowing insertion of its isooctyl s