title: 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.
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.
authors.affiliation: Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA. daniel.ortiz@tufts.edu
authors.forename: Daniel F
authors.initials: DF
authors.lastname: Ortiz
authors.forename: James
authors.initials: J
authors.lastname: Moseley
authors.forename: German
authors.initials: G
authors.lastname: Calderon
authors.forename: Amy L
authors.initials: AL
authors.lastname: Swift
authors.forename: Shaohua
authors.initials: S
authors.lastname: Li
authors.forename: Irwin M
authors.initials: IM
authors.lastname: Arias
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000378:metabolism*; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D000074020:ATP Binding Cassette Transporter, Subfamily B, Member 11; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001647:Bile Acids and Salts / Q000737:chemistry*; D001692:Biological Transport; D019921:Biotinylation; D002412:Cations; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D051356:Cortactin; D004285:Dogs; D004926:Escherichia coli / Q000378:metabolism; D005799:Genes, Dominant; D005982:Glutathione Transferase / Q000378:metabolism; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008099:Liver / Q000378:metabolism; D008840:Microfilament Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008969:Molecular Sequence Data; D010743:Phospholipids / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D011506:Proteins / Q000737:chemistry / Q000502:physiology*; D034622:RNA Interference; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions; D013997:Time Factors; D014162:Transfection; D020798:Two-Hybrid System Techniques
chemical_list: C093491:ABCB11 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000074020:ATP Binding Cassette Transporter, Subfamily B, Member 11; D018528:ATP-Binding Cassette Transporters; C490548:Abcb11 protein, rat; D048868:Adaptor Proteins, Signal Transducing; D001647:Bile Acids and Salts; C495868:CTTN protein, human; D002412:Cations; D051356:Cortactin; C495870:Cttn protein, rat; C104759:HAX1 protein, human; D008840:Microfilament Proteins; D010743:Phospholipids; D011506:Proteins; C095122:multidrug resistance protein 3; D005982:Glutathione Transferase