Title : Substituted
cysteine accessibility reveals a novel transmembrane 2-3 reentrant loop and functional role for transmembrane
domain 2 in the human
proton-coupled folate transporter
Abstract :
- The proton-coupled folate transporter ( PCFT ) is a folate-proton symporter highly expressed in solid tumors that can selectively target cytotoxic antifolates to tumors under acidic microenvironment conditions
- Predicted topology models for PCFT suggest that the loop domain between transmembrane domains (TMDs) 2 and 3 resides in the cytosol
- Mutations involving Asp-109 or Arg-113 in the TMD2-3 loop result in loss of activity
- By structural homology to other solute carriers, TMD2 may form part of the PCFT substrate binding domain
- In this study we mutated the seven cysteine ( Cys ) residues of human PCFT to serine , creating Cys-less PCFT
- Thirty-three single- Cys mutants spanning TMD2 and the TMD2-3 loop in a Cys-less PCFT background were transfected into PCFT-null HeLa cells
- All 33 mutants were detected by Western blotting, and 28 were active for [(3)H]methotrexate uptake at pH 5.5
- For the active residues , we performed pulldown assays with membrane-impermeable 2-aminoethyl methanethiosulfonate-biotin and streptavidin beads to determine their aqueous-accessibilities
- Multiple residues in TMD2 and the TMD2-3 loop domain reacted with 2-aminoethyl methanethiosulfonate-biotin , establishing aqueous accessibilities
- Pemetrexed pretreatment inhibited biotinylation of TMD2 mutants G93C and F94C, and biotinylation of these residues inhibited methotrexate transport activity
- Our results suggest that the TMD 2-3 loop domain is aqueous-accessible and forms a novel reentrant loop structure
- Residues in TMD2 form an aqueous transmembrane pathway for folate substrates, and Gly-93 and Phe-94 may contribute to a substrate binding domain
- Characterization of PCFT structure is essential to understanding the transport mechanism including the critical determinants of substrate binding