Purified NPC1 Protein
I. BINDING OF CHOLESTEROL AND OXYSTEROLS TO A 1278-AMINO ACID MEMBRANE PROTEIN*
- Rodney E. Infante1,
- Lina Abi-Mosleh,
- Arun Radhakrishnan2,
- Jarrod D. Dale3,
- Michael S. Brown4 and
- Joseph L. Goldstein5
- Department of Molecular Genetics, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390
- ↵4 To whom correspondence may be addressed: Dept. of Molecular Genetics, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9046. Tel.: 214-648-2179; Fax: 214-648-8804; E-mail: mike.brown{at}utsouthwestern.edu.
- ↵5 To whom correspondence may be addressed: Dept. of Molecular Genetics, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9046. Tel.: 214-648-2141; Fax: 214-648-8804; E-mail: joe.goldstein{at}utsouthwestern.edu.
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 [3H]cholesterol in a reaction inhibited by Nonidet P-40 above its critical micellar concentration. Low concentrations of unlabeled 25-HC abolished binding of [3H]cholesterol, but the converse was not true, i.e. unlabeled cholesterol, even at high concentrations, did not abolish binding of [3H]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.
- Received September 21, 2007.
- Revision received October 31, 2007.
- The American Society for Biochemistry and Molecular Biology, Inc.
