Identification of sufficient basolateral-targeting sequence in N-terminus of human sodium-dependent vitamin C transporter 2
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Human sodium-dependent vitamin C transporters, hSVCT1 and hSVCT2 are responsible for the uptake of L-ascorbic acid in the body. Sharing 66% amino acid sequence homology, these two transporters are functionally localized in the apical and basolateral membranes in polarized epithelium cells, respectively. A necessary basolateral targeting sequence (BTS) ELMAI was found embedding within the N-terminus of hSVCT2. Deletion of this sequence disrupts basolateral targeting and results in redirection of hSVCT2 to the apical membrane. Conversely, the normally apically-localized hSVCT1 could be directed into the basolateral membrane in polarized MDCK cells if its N-terminus was replaced with the full length N-terminus of hSVCT2. However, a 15 amino acid fragment (residues 50-64 abbreviated TD) containing the necessary BTS from hSVCT2, failed to target hSVCT1 to the basolateral membrane. In this study, site-directed mutagenesis was performed to generate various mutants containing additional native and mutated hSVCT2 N-terminal sequences. In addition, the effect of a C-terminal truncation of an hSVCT1 N-terminal mutant was also studied. EGFP fluorescence quantification, live cell and DAPI-stained cell confocal imaging and transporter uptake assays were performed on stably-transfected MDCK cells to determine the functional localization of these mutants. With an additional 16 hSVCT2 N-terminal residues inserted after the 15 amino acid residues, the hSVCT1 mutant was successfully targeted to the basolateral membrane. Mutating residues within this additional 16 residues added did not change the preferentially basolateral uptake yet impaired the total uptake and resulted in an increase in cytoplasmic fluorescence. The C-terminal truncation of the basolateral targeted hSVCT1mutant resulted in decreased membrane incorporation or retention. Thus, a sufficient BTS (residues D50 to G80) was identified in the hSVCT2 N-terminus. Native hSVCT2 sequence was found to play an important role in proper basolateral-targeting and a region within the C-terminus of the protein was also needed for proper membrane incorporation and retention.