Purification of ABCA1 and associated binding proteins reveals the importance of β1‐syntrophin in cholesterol efflux

Abstract

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. As 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 chromatography-mass spectrometric analysis. A novel interaction with the PDZ protein β 1-syntrophin was identified using this approach and this interaction was confirmed in human THP-1 macrophages and in mouse liver. siRNA inhibition of β 1-syntrophin expression reduced cholesterol efflux from primary skin fibroblasts by 50%, while decreasing efflux 30% in bone marrow derived macrophages. Inhibition of β 1-syntrophin decreased ABCA1 protein levels, whereas overexpression of β 1-syntrophin increased ABCA1 cell surface expression, and stimulated efflux to apoA-I. These findings indicate that β 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 β 1-syntrophin as an ABCA1 binding protein should prove useful in elucidating other protein interactions upon which ABCA1 function depends.