Topological studies of hSVCT1, the human sodium-dependent vitamin C transporter and the influence of N-glycosylation on its intracellular targeting

Exp Cell Res. 2009 Aug 1;315(13):2312-21. doi: 10.1016/j.yexcr.2009.04.007. Epub 2009 Apr 18.


The Na(+)-dependent transporters, hSVCT1 and hSVCT2, were assessed in COS-1 cells for their membrane topology. Antibodies to N- and C-termini of hSVCT1 and C-terminus of hSVCT2 identified positive immunofluorescence only after permeabilisation, suggesting these regions are intracellular. PNGase F treatment confirmed that WT hSVCT1 (approximately 70-100 kDa) is glycosylated and site-directed mutagenesis of the three putative N-glycosylation sites, Asn138, Asn144, Asn230, demonstrated that mutants N138Q and N144Q were glycosylated (approximately 68-90 kDa) with only 31-65% of WT l-ascorbic acid (AA) uptake while the glycosylation profile of N230Q remained unaltered (approximately 98% of WT activity). However, the N138Q/N144Q double mutant displayed barely detectable membrane expression at approximately 65 kDa, no apparent glycosylation and minimal AA uptake (<10%) with no discernible improvement in expression or activity when cultured at 28 degrees C or 37 degrees C. Marker protein immunocytochemistry with N138Q/N144Q identified intracellular aggregates with hSVCT1 localised at the nuclear membrane but absent at the plasma membrane thus implicating its role as a possible intracellular transporter and suggesting N-glycosylation is required for hSVCT1 membrane targeting. Also, Lys242 on the same putative hydrophilic loop as Asn230 after biotinylation was inaccessible from the extracellular side when analysed by MALDI-TOF MS. A new hSVCT1 secondary structure model supporting these findings is proposed.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antioxidants / metabolism
  • Ascorbic Acid / metabolism
  • COS Cells
  • Chlorocebus aethiops
  • Glycosylation
  • Humans
  • Mutagenesis, Site-Directed
  • Organic Anion Transporters, Sodium-Dependent / chemistry*
  • Organic Anion Transporters, Sodium-Dependent / genetics
  • Organic Anion Transporters, Sodium-Dependent / metabolism*
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / genetics
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / metabolism
  • Peptides / genetics
  • Peptides / metabolism
  • Protein Structure, Secondary*
  • Protein Transport*
  • Sodium-Coupled Vitamin C Transporters
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Symporters / chemistry*
  • Symporters / genetics
  • Symporters / metabolism*


  • Antioxidants
  • Organic Anion Transporters, Sodium-Dependent
  • Peptides
  • Sodium-Coupled Vitamin C Transporters
  • Symporters
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
  • Ascorbic Acid