Purification of ATP-binding cassette transporter A1 and associated binding proteins reveals the importance of beta1-syntrophin in cholesterol efflux.
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 beta1-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 beta1-syntrophin expression reduced cholesterol efflux from primary skin fibroblasts by 50% while decreasing efflux 30% in bone marrow-derived macrophages. Inhibition of beta1-syntrophin decreased ABCA1 protein levels, whereas overexpression of beta1-syntrophin increased ABCA1 cell-surface expression and stimulated efflux to apolipoprotein A-I. These findings indicate that beta1-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 beta1-syntrophin as an ABCA1-binding protein should prove useful in elucidating other protein interactions upon which ABCA1 function depends.
Okuhira
Kei-ichiro
K
Lipid Metabolism Unit, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA.
Fitzgerald
Michael L
ML
Sarracino
David A
DA
Manning
Jennifer J
JJ
Bell
Susan A
SA
Goss
Julie L
JL
Freeman
Mason W
MW
D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism*; D049029:Dystrophin-Associated Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D020868:Gene Silencing; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D008958:Models, Molecular; D046912:Multiprotein Complexes; D034741:RNA, Small Interfering / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection
C577836:ABCA1 protein, human; D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D049029:Dystrophin-Associated Proteins; D046912:Multiprotein Complexes; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; C091824:syntrophin; D002784:Cholesterol