Abstract
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.
Highlights
ABC1 proteins in the canalicular membrane of hepatocytes generate the driving force that establishes bile flow
Mutations that impair MDR3 exit from the endoplasmic reticulum are associated with cholestasis of pregnancy [10]; mutations in BSEP that impair expression of the mutant proteins in the apical membrane of Madin-Darby canine kidney (MDCK) cells are linked to progressive familial intrahepatic cholestasis type II [11]; and deletion of two amino acids in MRP2 of a Dubin-Johnson patient resulted in intracellular retention and accelerated degradation of MRP2 [12]
We present evidence indicating that HAX-1 associates with BSEP, MDR1, and MDR2 in the canalicular membrane of rat hepatocytes and that HAX-1 and cortactin regulate BSEP abundance in the apical membrane of MDCK cells
Summary
ABC1 proteins in the canalicular membrane of hepatocytes generate the driving force that establishes bile flow. RNA interference of HAX-1 increased BSEP levels in the apical membrane of MDCK cells by 71%. We present evidence indicating that HAX-1 associates with BSEP, MDR1, and MDR2 in the canalicular membrane of rat hepatocytes and that HAX-1 and cortactin regulate BSEP abundance in the apical membrane of MDCK cells.
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