Identification of an extracellular gate for the proton-coupled folate transporter (PCFT-SLC46A1) by cysteine cross-linking

Rongbao Zhao, Mitra Najmi, Andras Fiser, I. David Goldman

Research output: Contribution to journalArticlepeer-review

13 Scopus citations


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 the Escherichia coli glycerol 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 (Gln45-TMD1, Asn90-TMD2, Leu290-TMD7, Ser407-TMD11 and Asn411-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 [3H]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 Cd2+ complex. The data were consistent with the formation of a spontaneous disulfide bond between the N90C/ S407C pair and a CuPh- and Cd2+-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 influx Vmax consistent 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.

Original languageEnglish (US)
Pages (from-to)8162-8172
Number of pages11
JournalJournal of Biological Chemistry
Issue number15
StatePublished - Apr 8 2016

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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