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.
Highlights
The water-soluble micronutrient thiamine is essential for normal cellular functions, growth and development
Axial sections demonstrated that the plasma membrane expression of hTHTR2 was confined to the apical membrane domain, a distribution that contrasted with that of cells transfected with green fluorescent protein (GFP) alone, where fluorescence distribution was evident throughout the entire cytoplasmic volume (Fig. 1, A and B)
Nothing is known about the mechanisms that control the apical delivery of hTHTR2 in these polarized epithelia that are responsible for thiamineabsorption
Summary
Chemicals and Reagents—Custom-made [3H]thiamine (specific activity 555 GBq/mmol) was obtained from ARC Truncated constructs were generated by PCR using the primer combinations shown in Table 1 and in conditions described elsewhere [16, 19]. The PCR products and the GFP-N3 and YFP-N1 vectors were digested with the restriction enzymes XhoI and BamHI or XhoI and SacII, and the products were gel-separated and ligated together to generate in-frame fusion proteins with the fluorescent protein (GFP or YFP) fused to the COOH terminus of each hTHTR-2 construct. Cells were treated with 20 M nocodazole (30 min, on ice) and thereafter transfected at room temperature with hTHTR2GFP cDNA. Immunofluorescence—Nocodazole-treated MDCK cells (monolayers transfected with hTHTR2-GFP as well as naive controls) were fixed for 10 min in 4% paraformaldehyde solution (Electron Microscopy Sciences, Washington, PA). In all flow cytometry experiments, samples of untransfected and GFP-transfected cells were run in parallel with experimental samples to calibrate suitable parameters for identifying the intact, transfected cell population
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