Hierarchal Contribution of N- and C-Terminal Sequences to the Differential Localization of Homologous Sodium-Dependent Vitamin C Transporters, SVCT1 and SVCT2, in Epithelial Cells

Abstract

Human sodium-dependent vitamin C transporters, SVCT1 and SVCT2, share 66% sequence identity yet localize in the apical and basolateral membranes of epithelial cells, respectively. This pair thus serves as a model for studying multipass membrane protein targeting. Domain swaps, deletions, insertions, and point mutations were performed on EGFP-tagged hSVCT1 and hSVCT2 plasmids. Mutant proteins stably expressed in MDCK cells were analyzed by confocal microscopy and Transwell ascorbate transport assays. These studies identified an SVCT2 basolateral targeting sequence (BTS) in the N-terminus, which is conserved among mammalian SVCT2 forms. The less conserved N-terminus of SVCT1 is not required for apical localization. The destruction of SVCT2 BTS led to apical localization of the protein in a manner independent of the C-terminal sequence. A C-terminal sequence present in both SVCTs appears to be required for plasma membrane incorporation and retention as its deletion led to an increased level of intracellular appearance of both apically and basolaterally targeted SVCTs in the absence or presence of BTS. Nevertheless, all C-terminal deletion mutants showed preferential apical transport activity, suggesting a greater importance of this sequence for basolateral targeting. Our results collectively suggested a default apical targeting of SVCT, which is consistent with the evolution-based prediction. The SVCT sorting model with a hierarchal contribution of N- and C-terminal sequences was compared to the observations made for other multipass membrane proteins. The involvement of both intracellularly localized termini of multipass membrane proteins in the sorting pathway suggests a more complex sorting mechanism compared to that for single-pass proteins.

Molecular determinants dictating cell surface expression of the human sodium-dependent vitamin C transporter-2 in human liver cells

“…4C). Therefore, these results are consistent with the proposal that both NH 2 -and COOH-terminal sequences act in concert to regulate the cell surface delivery of hSVCT2 (36).…”

“…To investigate other molecular determinants that may function in tandem with this COOH-terminal residue, we proceeded to examine the role of the NH 2 -terminal cytoplasmic domain of hSVCT2 (36).…”

“…In contrast, a recent report (36) discovered the role of a more distal NH 2 -terminal sequence (LMAI, residues 56 -59 in hSVCT2) as a critical regulator of basolateral targeting of hSVCT2 in the polarized Madin-Darby canine kidney (MDCK) cell line. Notably, these authors demonstrated that a single point mutation L56A within the basolateral targeting sequence (albeit within a chimeric hSVCT1/hSVCT2 protein) resulted in increased intracellular retention of the chimeric transporter (36).…”

“…The relevant determinants for hepatocyte cell surface expression could be distributed within the cytoplasmic NH 2 -and/or COOH-terminal tails of hSVCT2 (10,19,21,29,30,34,36) or throughout the backbone of the polypeptide (16,31,33). Alternatively, targeting motifs may be encoded through specific protein conformations resulting from sequences throughout the entire hSVCT2 polypeptide sequence (17,32,40).…”

A fluorophore-conjugated ascorbic acid functions for the visualization of sodium vitamin C transporters in living cells

“…It must be noted that immunouorescence antibodies including anti-SVCT2 and anti-SVCT1 bind SVCTs in a quite different manner from AA or RB-A-Vc, thereby not interfering with the forming of SVCT/RB-A-Vc complexes. 27 Confocal colocalization experiments with anti-SVCT2 and anti-organelle protein markers revealed that most of the SVCT2 immunoreactivity was associated with the mitochondria, with minor colocalization at the endoplasmic reticulum and very low immunoreactivity at the plasma membrane. The transporter SVCT1 is absent from HEK-293T cells.…”