Functional and mechanistic roles of the human proton-coupled folate transporter transmembrane domain 6–7 linker

Abstract

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 the PCFT secondary structure includes 12 transmembrane domains (TMDs) with TMDs 6 and 7 linked by an intracellular loop (positions 236–265) including His247, implicated as functionally important. Single-cysteine (Cys) mutants were inserted from positions 241 to 251 in Cys-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. Coexpression 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 a nonhomologous sequence from a thiamine transporter (ThTr1) inserted into the PCFT TMD6–7 loop (positions 236–250 or 251–265), were active, although replacement of the entire loop with the ThTr1 sequence resulted in substantial loss of activity. Amino acid replacements (Ala, Arg, His, Gln, and 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.

Identifying the novel key genes in renal cell carcinoma by bioinformatics analysis and cell experiments

“…Furthermore, it has been found that extracellular exosome, vesicle and organelle promote the tumor [ 24 ]. Moreover, studies have shown that cofactor binding, coenzyme binding, and secondary active transmembrane transporter activity often play a major role in the progression of cancer [ 25 – 27 ]. In conclusion, all these theories are consistent with our results.…”

The evolving biology of the proton‐coupled folate transporter: New insights into regulation, structure, and mechanism

“…Indeed, the role of the TMD6‐7 loop domain in hPCFT function appears to be largely unrelated to specific primary sequence elements, as long as sufficient secondary structure is preserved and proper spacing between the TMD1‐6 and TMD7‐12 segments is ensured to maintain protein stability and to facilitate optimal membrane translocation. Consistent with this notion, hPCFT could be coexpressed as TMD1–6 and TMD7‐12 half‐molecules to restore transport activity, albeit in low levels 77 …”

“…Loss‐of‐function mutations in hPCFT from patients with HFM syndrome have been used to identify amino acids that may be structurally or functionally important 1,9,10,12,20,21,51–65 . Further interrogation of individual amino acids used systematic site‐directed mutagenesis based on sequence conservation and predictions of membrane topology 44,54,65–77 . The computation of three‐dimensional comparative protein structure models is feasible for MFS members with unknown structures based on previously solved MFS structures.…”

“…The functional and mechanistic importance of the hPCFT TMD6‐7 connecting loop was further studied by site‐directed, deletion, and insertion mutagenesis 77 . Whereas removal of residues 252‐265 from TMD7‐12 abolished transport, chimeric proteins including non‐homologous sequence replacements from a thiamine transporter (ThTr1) inserted into the hPCFT TMD6‐7 loop region (positions 236 to 250, or 251 to 265; designated tp ‐ and pt ‐hPCFT, respectively) were active; however, replacement of the entire TMD6‐7 loop with ThTr1 sequence resulted in substantial loss of transport activity 77 . His247 replacements with Ala, Arg, Gln, and Glu, or His247 deletion, in wild‐type hPCFT significantly suppressed transport, 44 yet the same His247 replacements in pt ‐hPCFT showed no obvious effect on transport activity 77 .…”

“…Particularly notable is the extent to which characterization of the causal basis for HFM 1,9,12,20,21,51–64 has shed light on PCFT structure and mechanism, including the identification of mechanistically important residues and regions involved in substrate binding or proton coupling. Of course, the availability of cryo‐EM structures of cPCFT in its apo form and in complex with the pemetrexed 13 provides exciting new opportunities to interpret the extensive mutant studies on hPCFT 44,54,65–77 in light of critical determinants of (anti)folate binding and the mechanism of (anti)folate transport mediated by PCFT. The structure‐based pharmacophore for PCFT 13 establishes an important new framework for further rational design of PCFT‐selective antifolates for tumor targeting and decreased toxicity to normal tissues which express substantial RFC without PCFT.…”