Human sodium dependent vitamin C transporters, hSVCT1 and hSVCT2: Role of TM1 in their activity and the identification of trafficking motifs
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In humans, ascorbate is transported by sodium dependent vitamin C transporters, SVCT1 and SVCT2 (12 TMs). SVCT1-EGFP and SVCT2-EGFP differentially localize at the apical and the basolateral surface respectively, in polarized MDCK cells. Although these transporters are very well characterized, the functional motifs and trafficking motifs are not known. This was the focus of this thesis. Site directed mutagenesis approach was used to introduce point mutations and deletions of amino acid residues into SVCT1 and SVCT2. Confocal microscopy and the 14 C vitamin C transport assays were used to investigate the subcellular localization, activity and kinetics of various mutants stably transfected in MDCK cells and the results were compared with previously published work of the SVCT1-EGFP and SVCT2-EGFP. In this thesis, the predicted TM1 of SVCTs which harbors a highly conserved motif constituted by a glutamine (Q) and histidine (H) was shown to be crucial for the transport of ascorbate and substrate selectivity but not for membrane localization. By investigating the subcellular localization of domain swaps of SVCT1 and SVCT2 a current concept of their trafficking and sorting is also proposed. It was found that amino acid residues, 55-59 in the N-terminus of SVCT2 are a part of basolateral sorting motif. It was also found that C-terminus of SVCTs do not dictate sorting but contains a region (617-634 in SVCT2), important for membrane localization/trafficking. Disruption of the basolateral signal leads to the localization of SVCT domain swaps and SVCT2-EGFP to the apical surface, suggesting the presence of apical sorting motif(s) between the TMs in both SVCT1 and SVCT2. This work makes an important contribution to understanding of the role of the QH motif in the predicted TM1 on the activity of hSVCTs, and sheds light on important regions in hSVCTs that are crucial for their trafficking and sorting in polarized MDCK cells.