Purification of ABCA1 and associated binding proteins reveals the importance of β1‐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. 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.