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

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 [H-3] 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.