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Papers In Press, published online ahead of print June 1, 2008
J. Lipid Res., doi:10.1194/jlr.M800048-JLR200

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Journal of Lipid Research, Vol. 49, 1322-1332, June 2008
Copyright © 2008 by American Society for Biochemistry and Molecular Biology

An analysis of the role of a retroendocytosis pathway in ABCA1-mediated cholesterol efflux from macrophages

Loren E. Faulkner^1,*, Stacey E. Panagotopulos^1,*, Jacob D. Johnson, Laura A. Woollett^*, David Y. Hui^*, Scott R. Witting, J. Nicholas Maiorano^* and W. Sean Davidson^2,*

^* Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati OH, 45237
Department of Parasitology, Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Silver Spring, MD 20910
Department of Medical and Molecular Genetics, Indiana University and Purdue University at Indianapolis, Indianapolis IN, 46202

This work was supported by RO1 Grants HL-62542 and HL-67093 from the National Heart Lung and Blood Institute (W.S.D.), a summer graduate student fellowship from the University of Cincinnati Research Counsel (L.E.F), and a University of Cincinnati Dean's Distinguished Dissertation Fellowship (S.E.P.).

The online version of this article (available at http://www.jlr.org) contains supplementary data in the form of two figures.

Published, JLR Papers in Press, March 22, 2008.

¹ L. Faulkner and S. Panagotopulos contributed equally to this work.

² To whom correspondence should be addressed. e-mail: Sean.Davidson{at}UC.edu

The ATP binding cassette transporter A-1 (ABCA1) is critical for apolipoprotein-mediated cholesterol efflux, an important mechanism employed by macrophages to avoid becoming lipid-laden foam cells, the hallmark of early atherosclerotic lesions. It has been proposed that lipid-free apolipoprotein A-I (apoA-I) enters the cell and is resecreted as a lipidated particle via a retroendocytosis pathway during ABCA1-mediated cholesterol efflux from macrophages. To determine the functional importance of such a pathway, confocal microscopy was used to characterize the internalization of a fully functional apoA-I cysteine mutant containing a thiol-reactive fluorescent probe in cultured macrophages. ApoA-I was also endogenously labeled with ³⁵S-methionine to quantify cellular uptake and to determine the metabolic fate of the internalized protein. It was found that apoA-I was specifically taken inside macrophages and that a small amount of intact apoA-I was resecreted from the cells. However, a majority of the label that reappeared in the media was degraded. We estimate that the mass of apoA-I retroendocytosed is not sufficient to account for the HDL produced by the cholesterol efflux reaction. Furthermore, we have demonstrated that lipid-free apoA-I-mediated cholesterol efflux from macrophages can be pharmacologically uncoupled from apoA-I internalization into cells. On the basis these findings, we present a model in which the ABCA1-mediated lipid transfer process occurs primarily at the membrane surface in macrophages, but still accounts for the observed specific internalization of apoA-I.

Supplementary key words ATP binding cassette transporter A-1 • apolipoprotein A-I • cell biology • endocytosis • macrophage • confocal microscopy • protein expression

Abbreviations: ABCA1, ATP binding cassette transporter A-1; apoA-I, apolipoprotein A-I; CHO, Chinese hamster ovary; CVD, cardiovascular disease; GFP, green fluorescent protein; IPTG, isopropyl-β-D-thiogalactoside; RCT, reverse cholesterol transport; STB, standard Tris buffer; TCEP, Tris-(2-carboxyethyl) phosphine

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