title: 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).
authors.affiliation: INRA, UMR1260 Nutrition Obesity and Risk of Thrombosis, F-13385 Marseille, France. Emmanuelle.Reboul@univ-amu.fr
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Reboul
authors.forename: Frank M
authors.initials: FM
authors.lastname: Dyka
authors.forename: Faraz
authors.initials: F
authors.lastname: Quazi
authors.forename: Robert S
authors.initials: RS
authors.lastname: Molday
mesh_terms: D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D016632:Apolipoprotein A-I / Q000378:metabolism; D001692:Biological Transport; D002784:Cholesterol / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D011485:Protein Binding
chemical_list: C577836:ABCA1 protein, human; D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters; D016632:Apolipoprotein A-I; D002784:Cholesterol