The role of the N-terminus in vitamin C transport and the apical sorting of EGFP-tagged SVCT1 in MDCK cells

SVCT1 (Sodium-dependent vitamin C transporter 1) is responsible for bulk absorption of vitamin C from the diet and reabsorption in the kidney to maintain plasma levels of this nutrient. To function as such, SVCT1 localizes to the apical membrane of intestinal and renal epithelial cells. Cytoplasmic domains of membrane proteins are commonly found to contain sorting determinants, which dictate their specific membrane localization in polarized epithelial cells. SVCT1 has 12 putative transmembrane segments with both the N and C-terminals on the cytoplasmic side of the membrane. The role of the N-terminal of SVCT1 and its contribution to the apical sorting and vitamin C transport activity of this protein was previously unexamined. Deletion mutations were made in the N-terminal of human SVCT1 tagged with EGFP and exogenously expressed in MDCK (Madin-Darby canine kidney) cells. Live cell confocal imaging, functional assays, as well as EGFP quantification measurements were taken to assess the effects of the deletion mutations. The vitamin C transport activity of SVCT1 was only impaired when most of the highly conserved region before the putative TM1 was deleted. In addition, the cytoplasmic N-terminus of SVCT1 does not play a role in the apical sorting of this protein. The conclusion that SVCT1 localizes to the apical membrane by default, presents an avenue of consideration for the apical sorting of other multi-pass membrane proteins.