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Journal of Lipid Research, Vol. 45, 287-294, February 2004
Copyright © 2004 by American Society for Biochemistry and Molecular Biology

ABCA1 and amphipathic apolipoproteins form high-affinity molecular complexes required for cholesterol efflux

M. L. Fitzgerald^*, A. L. Morris^*, A. Chroni, A. J. Mendez, V. I. Zannis and M. W. Freeman^1,*

^* Lipid Metabolism Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114
Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA 02118
Diabetes Research Institute, University of Miami School of Medicine, Miami, FL 33101

¹ To whom correspondence should be addressed. e-mail: freeman{at}molbio.mgh.harvard.edu

Apolipoproteins, such as apolipoprotein A-I (apoA-I), can stimulate cholesterol efflux from cells expressing the ATP binding cassette transporter A1 (ABCA1). The nature of the molecular interaction between these cholesterol acceptors and ABCA1 is controversial, and models suggesting a direct protein-protein interaction or indirect association have been proposed. To explore this issue, we performed competition binding and chemical cross-linking assays using six amphipathic plasma proteins and an 18 amino acid amphipathic helical peptide. All seven proteins stimulated lipid efflux and inhibited the cross-linking of apoA-I to ABCA1. Cross-linking of apoA-I to ABCA1 was saturable and occurred at high affinity (K_d of 7.0 ± 1.9 nM), as was cross-linking of apoA-II. After binding to ABCA1, apoA-I rapidly dissociated (half-life of 25 min) from the complex and was released back into the medium. A mutant form of ABCA1 (W590S) that avidly binds apoA-I but fails to promote cholesterol efflux released apoA-I with similar kinetics but without transfer of cholesterol to apoA-I.

Thus, a high-affinity, saturable, protein-protein interaction occurs between ABCA1 and all of its amphipathic protein ligands. Dissociation of the complex leads to the cellular release of cholesterol and the apolipoprotein. However, dissociation is not dependent on cholesterol transfer, which is a clearly separable event, distinguishable by ABCA1 mutants.

Abbreviations: ABCA1, ATP binding cassette transporter A1; apoA-I, apolipoprotein A-I; DSG, disuccinimidyl glutarate; DSP, dithiobis(succinimidylpropionate)

Supplementary key words ATP binding cassette transporter A1 • Tangier Disease • high density lipoprotein

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