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J. Biol. Chem., Vol. 280, Issue 31, 28581-28590, August 5, 2005

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The Sterol-sensing Domain of the Niemann-Pick C1 (NPC1) Protein Regulates Trafficking of Low Density Lipoprotein Cholesterol^*

Elizabeth E. Millard, Sarah E. Gale, Nicole Dudley, Jessie Zhang, Jean E. Schaffer, and Daniel S. Ory¶||

From the Center for Cardiovascular Research, Department of Internal Medicine, Department of Molecular Biology and Pharmacology, and the ^¶Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110-1010

The Niemann-Pick C1 (NPC1) protein is a key participant in intracellular sterol trafficking and regulation of cholesterol homeostasis. NPC1 contains a pentahelical region that is evolutionarily related to sterol-sensing domains found in other polytopic proteins involved in sterol interactions or sterol metabolism, including sterol regulatory element-binding protein cleavage-activating protein and hydroxymethylglutaryl-CoA reductase. To gain insight into the role of the sterol-sensing domain of NPC1, we examined the effect of point mutations in the NPC1 sterol-sensing domain on the trafficking of low density lipoprotein-derived cholesterol and sphingolipids. We show that an NPC1 P692S loss of function mutation results in decreased cholesterol delivery to the plasma membrane and endoplasmic reticulum. By contrast, NPC1 proteins carrying a L657F or D787N point mutation, which correspond to the activating SCAP L315F and D443N mutations, respectively, exhibit a gain of function phenotype. Specifically, cell lines expressing the NPC1 L657F or D787N mutations show a nearly 2-fold increase in the rates of low density lipoprotein cholesterol trafficking to the plasma membrane and to the endoplasmic reticulum, and more rapid suppression of sterol regulatory element-binding protein-dependent gene expression. Trafficking of sphingolipids is intact in the D787N and L657F cell lines. Our finding that D787N and L657F are activating NPC1 mutations provide evidence for a conserved mechanism for the sterol-sensing domain among cholesterol homeostatic proteins.

Received for publication, December 14, 2004 , and in revised form, May 4, 2005.

^* 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.

^|| Supported by National Institutes of Health Grants HL04482 and HL67773 and the Ara Parseghian Medical Research Foundation. To whom correspondence should be addressed: Center for Cardiovascular Research, Washington University School of Medicine, Box 8086, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-8737; Fax: 314-362-0186; E-mail: dory{at}wustl.edu.

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