Identification of HAX-1 as a protein that binds bile salt export protein and regulates its abundance in the apical membrane of Madin-Darby canine kidney cells

ATP-binding cassette (ABC)-type proteins are essential for bile formation in vertebrate liver. BSEP, MDR1, MDR2, and MRP2 ABC transporters are targeted to the apical (canalicular) membrane of hepatocytes where they execute ATP-dependent transport of bile acids, drugs, amphipathic cations, phospholipids, and conjugated organic anions, respectively. Changes in activity and abundance of transporters in the canalicular membrane regulate bile flow; however, little is known regarding cellular proteins that bind ABC transporters and regulate their trafficking. A yeast two-hybrid screen identified HAX-1 as a binding partner for BSEP, MDR1, and MDR2. The interactions were validated biochemically by glutathione S-transferase pull-down and co-immunoprecipitation assays. BSEP and HAX-1 were over-represented in rat liver subcellular fractions enriched for canalicular membrane vesicles, microsomes, and clathrin-coated vesicles. HAX-1 was bound to BSEP, MDR1, and MDR2 in canalicular membrane vesicles and co-localized with BSEP and MDR1 in the apical membrane of Madin-Darby canine kidney (MDCK) cells. RNA interference of HAX-1 increased BSEP levels in the apical membrane of MDCK cells by 71%. Pulse-chase studies indicated that HAX-1 depletion did not affect BSEP translation, post-translational modification, delivery to the plasma membrane, or half-life. HAX-1 depletion resulted in an increased peak of metabolically labeled apical membrane BSEP at 4 h and enhanced retention at 6 and 9 h. HAX-1 also interacts with cortactin. Expression of dominant negative cortactin increased steady state levels of BSEP 2-fold in the apical membrane of MDCK cells, as did expression of dominant negative EPS15. These findings suggest that HAX-1 and cortactin participate in BSEP internalization from the apical membrane.