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Molecular determinants dictating cell surface expression of the human sodium-dependent vitamin C transporter-2 in human liver cells. Am J Physiol Gastrointest Liver Physiol 2010 Feb;298(2):G267-74

Date

11/21/2009

Pubmed ID

19926816

Pubmed Central ID

PMC2822508

DOI

10.1152/ajpgi.00435.2009

Scopus ID

2-s2.0-76749113011   9 Citations

Abstract

The human sodium-dependent vitamin C transporter-2 (hSVCT2) plays an important role in cellular accumulation of ascorbic acid in liver cells. However, little is known about the molecular determinants that direct hSVCT2 to the cell surface in hepatocytes. We addressed this issue using live cell imaging methods to resolve the distribution and trafficking of truncated or mutated hSVCT2 constructs in a cellular model of human hepatocytes, HepG2 cells. Whereas a full-length hSVCT2-yellow fluorescent protein (YFP) fusion protein was functionally expressed at the cell surface in HepG2 cells, serial truncation and mutation analysis demonstrated an essential role for both NH(2)- and COOH-terminal sequence(s) for cell surface expression and function. Video-rate confocal imaging showed evidence of dynamic hSVCT2-YFP containing intracellular trafficking vesicles, the motility of which was impaired following disruption of microtubules using nocodazole. However, in a HepG2 cell line stably expressing hSVCT2-YFP at the cell surface, plasma membrane levels of hSVCT2 were unaffected by inhibition of microtubule-associated motor proteins; rather, surface expression of hSVCT2-YFP was increased following treatment with myosin inhibitors. Together, these results show that 1) both NH(2)- and COOH-terminal sequences are essential for proper localization of hSVCT2, 2) cell surface delivery is dependent on intact microtubules, and 3) peripheral microfilaments regulate insertion and retrieval of hSVCT2 into the plasma membrane.

Author List

Subramanian VS, Marchant JS, Said HM

Author

Jonathan S. Marchant PhD Chair, Professor in the Cell Biology, Neurobiology and Anatomy department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Ascorbic Acid
Bacterial Proteins
Carbon Radioisotopes
Carcinoma, Hepatocellular
Cell Line, Tumor
Cell Membrane
Gene Expression
Hepatocytes
Humans
Liver Neoplasms
Luminescent Proteins
Membrane Proteins
Molecular Motor Proteins
Mutagenesis
Organic Anion Transporters, Sodium-Dependent
Protein Structure, Tertiary
Protein Transport
Sodium-Coupled Vitamin C Transporters
Symporters
Transfection
jenkins-FCD Prod-482 91ad8a360b6da540234915ea01ff80e38bfdb40a