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J. Biol. Chem., Vol. 279, Issue 31, 32761-32770, July 30, 2004

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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^*

Daniel F. Ortiz, James Moseley, German Calderon, Amy L. Swift, Shaohua Li, and Irwin M. Arias

From the Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111

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.

Received for publication, April 19, 2004 , and in revised form, May 24, 2004.

^* This work was supported in part by National Institutes of Health Grants RO1DK35652, T32DK07542 (to I. M. A.), and R01 DK060719 (to D. F. O.), by a pilot grant (to D. F. O.) from the Center for Gastroenterology Research on Absorptive and Secretory Processes at Tufts University, and the New England Medical Center. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 617-636-3828; Fax: 617-636-0045; E-mail: daniel.ortiz{at}tuffs.edu.

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