Cholesterol transport via ABCA1: New insights from solid-phase binding assay.

Abstract : It is now well established that the ATP-binding cassette transporter A1 (ABCA1) plays a pivotal role in HDL metabolism, reverse cholesterol transport and net efflux of cellular cholesterol and phospholipids. We aimed to resolve some uncertainties related to the putative function of ABCA1 as a mediator of lipid transport by using a methodology developed in the laboratory to isolate a protein and study its interactions with other compounds. ABCA1 was tagged with the 1D4 peptide at the C terminus and expressed in human HEK 293 cells. Preliminary experiments showed that the tag modified neither the protein expression/localization within the cells nor the ability of ABCA1 to promote cholesterol cellular efflux to apolipoprotein A-I. ABCA1-1D4 was then purified and reconstituted in liposomes. ABCA1 displayed an ATPase activity in phospholipid liposomes that was significantly decreased by cholesterol. Finally, interactions with either cholesterol or apolipoprotein A-I were assessed by binding experiments with protein immobilized on an immunoaffinity matrix. Solid-phase binding assays showed no direct binding of cholesterol or apolipoprotein A-I to ABCA1. Overall, our data support the hypothesis that ABCA1 is able to mediate the transport of cholesterol from cells without direct interaction and that apo A-I primarily binds to membrane surface or accessory protein(s).
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Biochimie, Elsevier, 2013, 95 (4), pp.957-61. 〈10.1016/j.biochi.2012.11.009〉
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Emmanuelle Reboul, Frank Dyka, Faraz Quazi, Robert S Molday. Cholesterol transport via ABCA1: New insights from solid-phase binding assay.. Biochimie, Elsevier, 2013, 95 (4), pp.957-61. 〈10.1016/j.biochi.2012.11.009〉. 〈inserm-00765921〉



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