N-linked glycosylation and its impact on the electrophoretic mobility and function of the human proton-coupled folate transporter (HsPCFT)

Ersin Selcuk Unal, Rongbao Zhao, Andong Qiu, I. David Goldman

Research output: Contribution to journalArticle

59 Scopus citations

Abstract

The human proton-coupled folate transporter (HsPCFT, SLC46A1) mediates intestinal absorption of folates and transport of folates into the liver, brain and other tissues. On Western blot, HsPCFT migrates as a broad band (~ 55 kDa), higher than predicted (~ 50 kDa) in cell lines. Western blot analysis required that membrane preparations not be incubated in the loading buffer above 50 °C to avoid aggregation of the protein. Treatment of membrane fractions from HsPCFT-transfected HeLa cells with peptidyl N-glycanase F, or cells with tunicamycin, resulted in conversion to a ~ 35 kDa species. Substitution of asparagine residues of two canonical glycosylation sites to glutamine, individually, yielded a ~ 47 kDa protein; substitution of both sites gave a smaller (~ 35 kDa) protein. Single mutants retained full transport activity; the double mutant retained a majority of activity. Transport function and molecular size were unchanged when the double mutant was hemagglutinin (HA) tagged at either the NH2 or COOH terminus and probed with an anti-HA antibody excluding degradation of the deglycosylated protein. Wild-type or deglycosylated HsPCFT HA, tagged at amino or carboxyl termini, could only be visualized on the plasma membrane when HeLa cells were first permeabilized, consistent with the intracellular location of these domains.

Original languageEnglish (US)
Pages (from-to)1407-1414
Number of pages8
JournalBiochimica et Biophysica Acta - Biomembranes
Volume1778
Issue number6
DOIs
Publication statusPublished - Jun 1 2008

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Keywords

  • Folate transport
  • HCP1
  • Hereditary folate malabsorption (HFM)
  • Intestinal folate absorption
  • PCFT glycosylation
  • PCFT secondary structure
  • PCFT, proton-coupled folate transporter
  • PCFT/HCP1
  • SLC46A1

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Cell Biology

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