Purification of ATP-binding cassette transporter A1 and associated binding proteins reveals the importance of beta1-syntrophin in cholesterol efflux.
J Biol Chem
; 280(47): 39653-64, 2005 Nov 25.
Article
em En
| MEDLINE
| ID: mdl-16192269
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. 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.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Colesterol
/
Transportadores de Cassetes de Ligação de ATP
/
Proteínas Associadas à Distrofina
Tipo de estudo:
Prognostic_studies
/
Risk_factors_studies
Limite:
Animals
/
Humans
Idioma:
En
Revista:
J Biol Chem
Ano de publicação:
2005
Tipo de documento:
Article
País de afiliação:
Estados Unidos