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J. Biol. Chem., Vol. 280, Issue 47, 39653-39664, November 25, 2005

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Purification of ATP-binding Cassette Transporter A1 and Associated Binding Proteins Reveals the Importance of 1-Syntrophin in Cholesterol Efflux^*

Kei-ichiro Okuhira1, Michael L. Fitzgerald12, David A. Sarracino, Jennifer J. Manning, Susan A. Bell, Julie L. Goss, and Mason W. Freeman3

From the Lipid Metabolism Unit and the Partners Center for Genetics and Genomics, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114

ATP-binding cassette transporter A1 (ABCA1) plays a critical role in HDL cholesterol metabolism, but the mechanism by which it transports lipid across membranes is poorly understood. Because growing evidence implicates accessory proteins in this process, we developed a method by which proteins interacting with the intact transporter could be identified. cDNAs encoding wild-type ABCA1 and a mutant lacking the C-terminal PDZ binding motif of ABCA1 were transfected into 293 cells, and the expressed proteins were solubilized using detergent conditions (0.75% CHAPS, 1 mg/ml phosphatidylcholine) predicted to retain high affinity protein-protein interactions. Proteins that co-purified with ABCA1 on an antibody affinity column were identified by liquid chromatographymass spectrometric analysis. A novel interaction with the PDZ protein 1-syntrophin was identified using this approach, and this interaction was confirmed in human THP-1 macrophages and in mouse liver. Small interference RNA inhibition of 1-syntrophin expression reduced cholesterol efflux from primary skin fibroblasts by 50% while decreasing efflux 30% in bone marrow-derived macrophages. Inhibition of 1-syntrophin decreased ABCA1 protein levels, whereas overexpression of 1-syntrophin increased ABCA1 cell-surface expression and stimulated efflux to apolipoprotein A-I. These findings indicate that 1-syntrophin acts through a class-I PDZ interaction with the C terminus of ABCA1 to regulate the cellular distribution and activity of the transporter. The approach used to identify 1-syntrophin as an ABCA1-binding protein should prove useful in elucidating other protein interactions upon which ABCA1 function depends.

Received for publication, September 15, 2005

^* This work was supported by National Institutes of Health Grants HL68988, HL45098, and HL72358 (to M. W. F.) and HL074136 (to M. L. F.), as well as a fellowship grant (to K. O.) from the Uehara Memorial Foundation. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1 and S2.

¹ Both authors contributed equally to this work.

² To whom correspondence may be addressed: Lipid Metabolism Unit, Jackson 1321, Massachusetts General Hospital, Boston, MA 02114. Tel.: 617-726-1465; Fax: 617-726-2879; E-mail: mfitzger{at}molbio.mgh.harvard.edu.

³ To whom correspondence may be addressed: Lipid Metabolism Unit, Jackson 1321, Massachusetts General Hospital, Boston, MA 02114. Tel.: 617-726-5906; Fax: 617-726-2879; E-mail: freeman{at}molbio.mgh.harvard.edu.

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