1. Myosin II Regulatory Light Chain Is Required for Trafficking of Bile Salt Export Protein to the Apical Membrane in Madin-Darby Canine Kidney Cells
- Author
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Wayne Chan, German Calderon, Hiroshi Hosoya, Amy L. Swift, Jamie Moseley, Irwin M. Arias, Shaohua Li, and Daniel F. Ortiz
- Subjects
Immunoprecipitation ,Recombinant Fusion Proteins ,ATP-binding cassette transporter ,Plasma protein binding ,Biology ,Kidney ,Biochemistry ,Cell Line ,Bile Acids and Salts ,Dogs ,Myosin ,Animals ,Secretion ,Phosphorylation ,Molecular Biology ,Myosin Type II ,Bile Canaliculi ,Kidney metabolism ,Cell Biology ,Apical membrane ,Molecular biology ,Transport protein ,Protein Transport ,Microscopy, Fluorescence ,Carrier Proteins ,Plasmids ,Protein Binding - Abstract
BSEP, MDR1, and MDR2 ATP binding cassette transporters are targeted to the apical (canalicular) membrane of hepatocytes, where they mediate ATP-dependent secretion of bile acids, drugs, and phospholipids, respectively. Sorting to the apical membrane is essential for transporter function; however, little is known regarding cellular proteins that bind ATP binding cassette proteins and regulate their trafficking. A yeast two-hybrid screen of a rat liver cDNA library identified the myosin II regulatory light chain, MLC2, as a binding partner for BSEP, MDR1, and MDR2. The interactions were confirmed by glutathione S-transferase pulldown and co-immunoprecipitation assays. BSEP and MLC2 were overrepresented in a rat liver subcellular fraction enriched in canalicular membrane vesicles, and MLC2 colocalized with BSEP in the apical domain of hepatocytes and polarized WifB, HepG2, and Madin-Darby canine kidney cells. Expression of a dominant negative, non-phosphorylatable MLC2 mutant reduced steady state BSEP levels in the apical domain of polarized Madin-Darby canine kidney cells. Pulse-chase studies revealed that Blebbistatin, a specific myosin II inhibitor, severely impaired delivery of newly synthesized BSEP to the apical surface. These findings indicate that myosin II is required for BSEP trafficking to the apical membrane.
- Published
- 2005