Identification of an Extracellular Gate for the Proton-coupled Folate Transporter (PCFT-SLC46A1) by Cysteine Cross-linking

J Biol Chem. 2016 Apr 8;291(15):8162-72. doi: 10.1074/jbc.M115.693929. Epub 2016 Feb 16.


The proton-coupled folate transporter (PCFT, SLC46A1) is required for intestinal folate absorption and folate homeostasis in humans. A homology model of PCFT, based upon theEscherichia coliglycerol 3-phosphate transporter structure, predicted that PCFT transmembrane domains (TMDs) 1, 2, 7, and 11 form an extracellular gate in the inward-open conformation. To assess this model, five residues (Gln(45)-TMD1, Asn(90)-TMD2, Leu(290)-TMD7, Ser(407)-TMD11 and Asn(411)-TMD11) in the predicted gate were substituted with Cys to generate single and nine double mutants. Transport function of the mutants was assayed in transient transfectants by measurement of [(3)H]substrate influx as was accessibility of the Cys residues to biotinylation. Pairs of Cys residues were assessed for spontaneous formation of a disulfide bond, induction of a disulfide bond by oxidization with dichloro(1,10-phenanthroline)copper (II) (CuPh), or the formation of a Cd(2+)complex. The data were consistent with the formation of a spontaneous disulfide bond between the N90C/S407C pair and a CuPh- and Cd(2+)-induced disulfide bond and complex, respectively, for the Q45C/L290C and L290C/N411C pairs. The decrease in activity induced by cross-linkage of the Cys residue pairs was due to a decrease in the influxVmaxconsistent with restriction in the mobility of the transporter. The presence of folate substrate decreased the CuPh-induced inhibition of transport. Hence, the data support the glycerol 3-phosphate transporter-based homology model of PCFT and the presence of an extracellular gate formed by TMDs 1, 2, 7, and 11.

Keywords: cysteine-mediated cross-linking; folate; homology modeling; membrane transport; transporter.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Substitution
  • Cysteine / chemistry*
  • Cysteine / genetics
  • Cysteine / metabolism
  • Disulfides / chemistry*
  • Disulfides / metabolism
  • Folic Acid / metabolism*
  • HeLa Cells
  • Humans
  • Models, Molecular
  • Mutation
  • Oxidation-Reduction
  • Protein Conformation
  • Protein Processing, Post-Translational
  • Protein Structure, Tertiary
  • Proton-Coupled Folate Transporter / chemistry*
  • Proton-Coupled Folate Transporter / genetics
  • Proton-Coupled Folate Transporter / metabolism*


  • Disulfides
  • Proton-Coupled Folate Transporter
  • SLC46A1 protein, human
  • Folic Acid
  • Cysteine

Associated data

  • PDB/1pw54