Identification of a functionally critical GXXG motif and its relationship to the folate binding site of the proton-coupled folate transporter (PCFT-SLC46A1)

Am J Physiol Cell Physiol. 2012 Sep 15;303(6):C673-81. doi: 10.1152/ajpcell.00123.2012. Epub 2012 Jul 11.


The proton-coupled folate transporter (PCFT) mediates intestinal folate absorption, and loss-of-function mutations in this gene result in the autosomal recessive disorder hereditary folate malabsorption. The current study, focused on a structure-functional analysis of this transporter, identified Gly-189 and Gly-192 (a GxxG motif) located in the fifth transmembrane domain as residues that could not be replaced with alanine without a loss of function. In contrast, function was preserved when Gly-56 and Gly-59 (the other conservative GXXG motif in human PCFT) were replaced with alanine. Similarly, Gly-93 and Gly-97, which constitute the only conserved GXXXG dimerization motif in human PCFT, tolerated alanine substitution. To explore the role of this region in folate binding, the residues around Gly-189 and Gly-192 were analyzed by the substituted cysteine accessibility method. Both I188C and M193C mutants were functional and were inhibited by membrane-impermeable sulfhydryl-reactive reagents; this could be prevented with PCFT substrate, but the protection was sustained at 0°C only for the I188C mutant, consistent with localization of Ile-188 in the PCFT folate binding pocket. The functional role of residues around Gly-189 and Gly-192 is consistent with a molecular structural model in which these two residues along with Ieu-188 are accessible to the PCFT aqueous translocation pathway.

Publication types

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

MeSH terms

  • Amino Acid Motifs / physiology
  • Amino Acid Sequence
  • Binding Sites / physiology
  • Conserved Sequence
  • Folic Acid / chemistry
  • Folic Acid / metabolism*
  • Glycine / physiology*
  • HeLa Cells
  • Humans
  • Isoleucine / genetics
  • Molecular Sequence Data
  • Protein Transport / physiology
  • Proton-Coupled Folate Transporter / chemistry*
  • Proton-Coupled Folate Transporter / physiology*
  • Protons
  • Signal Transduction / physiology


  • Proton-Coupled Folate Transporter
  • Protons
  • SLC46A1 protein, human
  • Isoleucine
  • Folic Acid
  • Glycine