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J. Biol. Chem., Vol. 281, Issue 38, 27894-27904, September 22, 2006

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Sterol Transfer by ABCG5 and ABCG8

IN VITRO ASSAY AND RECONSTITUTION^*

Jin Wang, Fang Sun, Da-wei Zhang, Yongming Ma, Fang Xu, Jitendra D. Belani^¶, Jonathan C. Cohen^||, Helen H. Hobbs^||**, and Xiao-Song Xie^||1

From the Eugene McDermott Center for Human Growth and Development, the Center of Human Nutrition, and the ^||Department of Internal Medicine, ^**Howard Hughes Medical Institute and Department of Molecular Genetics, the University of Texas Southwestern Medical Center, Dallas, Texas 75390-8591 and the ^¶Department of Chemistry, University of California, Irvine, California 92697-2025

ATP-binding cassette transporters G5 and G8 are half-transporters expressed on the apical membranes of enterocytes and hepatocytes that limit intestinal uptake and promote secretion of neutral sterols. Genetic defects that inactivate either half-transporter cause accumulation of cholesterol and plant sterols, resulting in premature coronary atherosclerosis. These observations suggest that G5 and G8 promote the translocation of sterols across membranes, but the primary transport substrate of the G5G8 complex has not been directly determined. Here we report the development of a sterol transfer assay using "inside-out" membrane vesicles from Sf9 cells expressing recombinant mouse G5 and G8. Radiolabeled cholesterol or sitosterol was transferred from donor liposomes to G5- and G8-containing membrane vesicles in an ATP-dependent and vanadate-sensitive manner; net transfer of cholesterol was associated with an increase in vesicular cholesterol mass. CTP, GTP, and UTP, as well as ATP, supported transfer but with lesser efficiency (ATP >> CTP > GTP > UTP). Transfer was specific for sterols and was stereoselective; minimal ATP-dependent and vanadate-sensitive transfer of cholesteryl oleate, phosphatidylcholine, or enantiomeric cholesterol was observed. These studies indicate that G5 and G8 are sufficient for reconstitution of sterol transfer activity in vitro and provide the first demonstration that sterols are direct transport substrates of the G5 and G8 heterodimer.

Received for publication, June 12, 2006 , and in revised form, July 17, 2006.

^* This work was supported by Grants HL72304 and HL20948 from the National Institutes of Health. 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.

¹ To whom correspondence should be addressed. Tel.: 214-648-7700; Fax: 214-648-7720; E-mail: xiao-song.xie{at}UTSouthwestern.edu.

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