The proton-coupled folate transporter (PCFT; SLC46A1) is a folate-proton symporter expressed in solid tumors and is used for tumor-targeted delivery of cytotoxic antifolates. Topology modeling suggests that PCFT secondary structure includes twelve transmembrane domains (TMDs) with TMDs 6 and 7 linked by an intracellular loop (positions 236 to 265) including His247, implicated as functionally important. Single cysteine mutants were inserted from positions 241 to 251 in cysteine-less PCFT and mutant proteins were expressed in PCFT-null (R1-11) HeLa cells; none were reactive with 2-aminoethyl methanethiosulfonate biotin, suggesting that the TMD6-7 loop is intracellular. Twenty-nine single alanine mutants spanning the entire TMD6-7 loop were expressed in R1-11 cells; activity was generally preserved, with the exception of the 247, 250 and 251 mutants, partly due to decreased surface expression. Co-expression of PCFT TMD1-6 and TMD7-12 half-molecules in R1-11 cells partially restored transport activity, although removal of residues 252-265 from TMD7-12 abolished transport. Chimeric proteins, including non-homologous sequence from a thiamine transporter (ThTr1) inserted into the PCFT TMD6-7 loop (positions 236 to 250, or 251 to 265), were active, although replacement of the entire loop with ThTr1 sequence resulted in substantial loss of activity. Amino acid replacements (Ala, Arg, His, Gln, Glu) or deletions at position 247 in wild-type and PCFT-ThTr1 chimeras resulted in differential effects on transport. Collectively, our findings suggest that the PCFT TMD6-7 connecting loop confers protein stability and may serve a unique functional role that depends on secondary structure rather than particular sequence elements.
- facilitative transport
- proton-coupled folate transporter
- ©2016 The Author(s)
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