Targeting and trafficking of the human thiamine transporter-2 in epithelial cells.
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.
Subramanian
Veedamali S
VS
Department of Medicine, the University of California, Irvine, California 92697, USA.
Marchant
Jonathan S
JS
Said
Hamid M
HM
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D018938:Caco-2 Cells; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004285:Dogs; D000072159:Dynactin Complex; D004847:Epithelial Cells / Q000378:metabolism*; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006801:Humans; D007668:Kidney / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000378:metabolism*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D018715:Microscopy, Video; D008869:Microtubule-Associated Proteins / Q000737:chemistry; D008870:Microtubules / Q000737:chemistry / Q000378:metabolism; D005810:Multigene Family; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013831:Thiamine / Q000737:chemistry; D014162:Transfection
D001426:Bacterial Proteins; C000606361:DCTN2 protein, human; D017931:DNA Primers; D018076:DNA, Complementary; D000072159:Dynactin Complex; D008164:Luminescent Proteins; D026901:Membrane Transport Proteins; D008869:Microtubule-Associated Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; C492291:SLC19A3 protein, human; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D013831:Thiamine