title: Purified NPC1 protein. I. Binding of cholesterol and oxysterols to a 1278-amino acid membrane protein.
abstract: The Niemann-Pick, Type C1 protein (NPC1) is required for the transport of lipoprotein-derived cholesterol from lysosomes to endoplasmic reticulum. The 1278-amino acid, polytopic membrane protein has not been purified, and its mechanism of action is unknown. Unexpectedly, we encountered NPC1 in a search for a membrane protein that binds 25-hydroxycholesterol (25-HC) and other oxysterols. A 25-HC-binding protein was purified more than 14,000-fold from rabbit liver membranes and identified as NPC1 by mass spectroscopy. We prepared recombinant human NPC1 and confirmed its ability to bind oxysterols, including those with a hydroxyl group on the 24, 25, or 27 positions. Hydroxyl groups on the 7, 19, or 20 positions failed to confer binding. Recombinant human NPC1 also bound [(3)H]cholesterol in a reaction inhibited by Nonidet P-40 above its critical micellar concentration. Low concentrations of unlabeled 25-HC abolished binding of [(3)H]cholesterol, but the converse was not true, i.e. unlabeled cholesterol, even at high concentrations, did not abolish binding of [(3)H]25-HC. NPC1 is not required for the known regulatory actions of oxysterols. Thus, in NPC1-deficient fibroblasts 25-HC blocked the processing of sterol regulatory element-binding proteins and activated acyl-CoA:cholesterol acyltransferase in a normal fashion. The availability of assays to measure NPC1 binding in vitro may further the understanding of ways in which oxysterols regulate intracellular lipid transport.
authors.affiliation: Department of Molecular Genetics, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390, USA.
authors.forename: Rodney E
authors.initials: RE
authors.lastname: Infante
authors.forename: Lina
authors.initials: L
authors.lastname: Abi-Mosleh
authors.forename: Arun
authors.initials: A
authors.lastname: Radhakrishnan
authors.forename: Jarrod D
authors.initials: JD
authors.lastname: Dale
authors.forename: Michael S
authors.initials: MS
authors.lastname: Brown
authors.forename: Joseph L
authors.initials: JL
authors.lastname: Goldstein
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism*; D007700:Kinetics; D008055:Lipids / Q000502:physiology; D008099:Liver / Q000378:metabolism*; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D011817:Rabbits; D013261:Sterols / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D008055:Lipids; D008565:Membrane Proteins; D013261:Sterols; D002784:Cholesterol