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Myosin II Regulatory Light Chain Is Required for Trafficking of Bile Salt Export Protein to the Apical Membrane in Madin-Darby Canine Kidney Cells
- Source :
- Journal of Biological Chemistry. 280:23741-23747
- Publication Year :
- 2005
- Publisher :
- Elsevier BV, 2005.
-
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.
- 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
Subjects
Details
- ISSN :
- 00219258
- Volume :
- 280
- Database :
- OpenAIRE
- Journal :
- Journal of Biological Chemistry
- Accession number :
- edsair.doi.dedup.....ff9c942fc28e17b1b8c53e22dc560951