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.
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.
Varma
Saaket
S
Department of Biochemistry, University at Buffalo, Buffalo, New York 14214, USA.
Sobey
Kami
K
Campbell
Christine E
CE
Kuo
Shiu-Ming
SM
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D029363:Organic Anion Transporters, Sodium-Dependent / Q000737:chemistry* / Q000378:metabolism*; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D060347:Sodium-Coupled Vitamin C Transporters; D013329:Structure-Activity Relationship; D027981:Symporters / Q000737:chemistry* / Q000378:metabolism*
D050505:Mutant Proteins; D029363:Organic Anion Transporters, Sodium-Dependent; D021382:Protein Sorting Signals; C555162:SLC23A1 protein, human; C555163:SLC23A2 protein, human; D060347:Sodium-Coupled Vitamin C Transporters; D027981:Symporters