Faculty Collaboration Database

Targeting and trafficking of the human thiamine transporter-2 in epithelial cells. J Biol Chem 2006 Feb 24;281(8):5233-45

Date

12/24/2005

Pubmed ID

16371350

DOI

10.1074/jbc.M512765200

Scopus ID

2-s2.0-33646185694   26 Citations

Abstract

Humans lack biochemical pathways for thiamine synthesis, so cellular requirements are met via specific carrier-mediated uptake pathways. Two proteins from the solute carrier SLC19A gene family have been identified as human thiamine transporters (hTHTRs), SLC19A1 (hTHTR1) and SLC19A2 (hTHTR2). Both of these transporters are co-expressed but are differentially targeted in polarized cell types that mediate vectorial thiamine transport (e.g. renal and intestinal epithelia). It is important to understand the domain structure of these proteins, namely which regions within the polypeptide sequence are important for physiological delivery to the cell surface, in order to understand the impact of clinically relevant mutations on thiamine transport. Here we have characterized the mechanisms regulating hTHTR2 distribution by using live cell imaging methods that resolve the targeting and trafficking dynamics of full-length hTHTR2, a series of hTHTR2 truncation mutants, as well as chimeras comprising the hTHTR1 and hTHTR2 sequence. We showed the following: (i) that the cytoplasmic COOH-tail of hTHTR2 is not essential for apical targeting in polarized cells; (ii) that delivery of hTHTR2 to the cell surface is critically dependent on the integrity of the transmembrane backbone of the polypeptide so that minimal truncations abrogate cell surface expression of hTHTR2; and (iii) video rate images of hTHTR2-containing intracellular vesicles displayed rapid bi-directional trafficking events to and from the cell surface impaired by microtubule-disrupting but not microfilament-disrupting agents as well as by overexpression of the dynactin subunit dynamitin (p50). Finally, we compared the behavior of hTHTR2 with that of hTHTR1 and the human reduced folate carrier (SLC19A1) to underscore commonalities in the cell surface targeting mechanisms of the entire SLC19A gene family.

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

Animals
Bacterial Proteins
Caco-2 Cells
Cell Line
Cell Line, Tumor
Cell Membrane
Cytoplasm
DNA Mutational Analysis
DNA Primers
DNA, Complementary
Dogs
Dynactin Complex
Epithelial Cells
Flow Cytometry
Green Fluorescent Proteins
Humans
Kidney
Luminescent Proteins
Membrane Transport Proteins
Microscopy, Confocal
Microscopy, Fluorescence
Microscopy, Video
Microtubule-Associated Proteins
Microtubules
Multigene Family
Mutation
Peptides
Polymerase Chain Reaction
Protein Binding
Protein Conformation
Protein Structure, Tertiary
Recombinant Fusion Proteins
Thiamine
Transfection