Association of the 16-kDa subunit c of vacuolar proton pump with the ileal Na+-dependent bile acid transporter: protein-protein interaction and intracellular trafficking.
The rat ileal apical sodium-dependent bile acid transporter (Asbt) transports conjugated bile acids in a Na+-dependent fashion and localizes specifically to the apical surface of ileal enterocytes. The mechanisms that target organic anion transporters to different domains of the ileal enterocyte plasma membrane have not been well defined. Previous studies (Sung, A.-Q., Arresa, M. A., Zeng, L., Swaby, I'K., Zhou, M. M., and Suchy, F. J. (2001) J. Biol. Chem. 276, 6825-6833) from our laboratory demonstrated that rat Asbt follows an apical sorting pathway that is brefeldin A-sensitive and insensitive to protein glycosylation, monensin treatment, and low temperature shift. Furthermore, a 14-mer signal sequence that adopts a beta-turn conformation is required for apical localization of rat Asbt. In this study, a vacuolar proton pump subunit (VPP-c, the 16-kDa subunit c of vacuolar H+-ATPase) has been identified as an interacting partner of Asbt by a bacterial two-hybrid screen. A direct protein-protein interaction between Asbt and VPP-c was confirmed in an in vitro pull-down assay and in an in vivo mammalian two-hybrid analysis. Indirect immunofluorescence confocal microscopy demonstrated that the Asbt and VPP-c colocalized in transfected COS-7 and MDCK cells. Moreover, bafilomycin A1 (a specific inhibitor of VPP) interrupted the colocalization of Asbt and VPP-c. A taurocholate influx assay and membrane biotinylation analysis showed that treatment with bafilomycin A1 resulted in a significant decrease in bile acid transport activity and the apical membrane localization of Asbt in transfected cells. Thus, these results suggest that the apical membrane localization of Asbt is mediated in part by the vacuolar proton pump associated apical sorting machinery.
Sun
An-Qiang
AQ
Department of Pediatrics, Mount Sinai School of Medicine, New York, New York 10029, USA. An-Qiang.Sun@mssm.edu
Balasubramaniyan
Natarajan
N
Liu
Chuan-Ju
CJ
Shahid
Mohammad
M
Suchy
Frederick J
FJ
D000818:Animals; D001665:Binding Sites; D007082:Ileum / Q000378:metabolism; D029363:Organic Anion Transporters, Sodium-Dependent / Q000378:metabolism*; D011485:Protein Binding; D017510:Protein Folding; D021122:Protein Subunits / Q000378:metabolism; D021381:Protein Transport; D051381:Rats; D027981:Symporters / Q000378:metabolism*; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism*
D029363:Organic Anion Transporters, Sodium-Dependent; D021122:Protein Subunits; D027981:Symporters; C071908:sodium-bile acid cotransporter; D025262:Vacuolar Proton-Translocating ATPases