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J. Biol. Chem., Vol. 281, Issue 42, 31594-31604, October 20, 2006

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Mechanism of Cholesterol Transfer from the Niemann-Pick Type C2 Protein to Model Membranes Supports a Role in Lysosomal Cholesterol Transport^*

Sunita R. Cheruku¹, Zhi Xu¹, Roxanne Dutia, Peter Lobel, and Judith Storch²

From the Department of Nutritional Sciences, Rutgers University, New Brunswick, New Jersey 08901 and the Center for Advanced Biotechnology and Medicine, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854

Cells acquire cholesterol either by de novo synthesis in the endoplasmic reticulum or by internalization of cholesterol-containing lipoproteins, particularly low density lipoprotein (LDL), via receptor-mediated endocytosis. The inherited disorder Niemann-Pick type C (NPC), in which abnormal LDL-cholesterol trafficking from the endo/lysosomal compartment leads to substantial cholesterol and glycolipid accumulation in lysosomes, is caused by defects in either of two genes that encode for proteins designated as NPC1 and NPC2. NPC2 is a small intralysosomal protein that has been characterized biochemically as a cholesterol binding protein. We determined the rate and mechanism by which NPC2 delivers cholesterol to model phospholipid membranes. A fluorescence dequenching assay was used to monitor the kinetics of cholesterol transfer from the protein to membranes. The endogenous tryptophan fluorescence of the NPC2 was quenched upon binding of cholesterol, and the subsequent addition of acceptor vesicles resulted in dequenching of the tryptophan signal, enabling the monitoring of cholesterol transfer to membranes. The rates of cholesterol transfer were evaluated as a function of acceptor vesicle concentration, acceptor vesicle phospholipid headgroup composition, and aqueous phase properties. The results suggest that NPC2 rapidly transports cholesterol to phospholipid vesicles via a collisional mechanism which involves a direct interaction with the acceptor membrane. Transfer of cholesterol to membranes is faster in an acidic environment and is greatly enhanced by the presence of the unique lysosomal/late endosomal phospholipid lyso-bisphosphatidic acid (LBPA) (also known as bismonoacylglycerol phosphate). Finally, we found that the rate of transfer of cholesterol from vesicles to NPC2 was dramatically increased by the presence of lyso-bisphosphatidic acid in the donor vesicles. These results support a role for the NPC2 protein in the egress of LDL derived cholesterol out of the endosomal/lysosomal compartment.

Received for publication, March 23, 2006 , and in revised form, July 10, 2006.

^* This work was supported by the Ara Parseghian Medical Research Foundation (to J. S. and P. L.). 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.

This article was selected as a Paper of the Week.

¹ These authors contributed equally to this work.

² To whom correspondence should be addressed: Dept. of Nutritional Sciences, Rutgers University, 96 Lipman Dr., New Brunswick, NJ 08901. Tel.: 732-932-1689; Fax: 732-932-6837; E-mail: storch{at}aesop.rutgers.edu.

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