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J. Biol. Chem., Vol. 283, Issue 2, 1052-1063, January 11, 2008
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Purified NPC1 Protein
I. BINDING OF CHOLESTEROL AND OXYSTEROLS TO A 1278-AMINO ACID MEMBRANE PROTEIN*
From the Department of Molecular Genetics, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390
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 for publication, September 21, 2007 , and in revised form, October 31, 2007.
* This work was supported in part by National Institutes of Health (NIH) Grant HL20948 and the Perot Family Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1.
1 Supported by a Medical Scientist Training Program Grant (5T32 GM08014).
2 Recipient of a postdoctoral fellowship from the Jane Coffin Childs Memorial Fund for Medical Research.
3 Supported by NIH T-35 Training Grant DK066141.
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.
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