Localization of a Substrate Binding Domain of the Human Reduced Folate Carrier to Transmembrane Domain 11 by Radioaffinity Labeling and Cysteine-substituted Accessibility Methods

The human reduced folate carrier (hRFC) mediates the membrane transport of reduced folates and classical anti-folates into mammalian cells. RFC is characterized by 12 transmembrane domains (TMDs), internally oriented N and C termini, and a large central linker connecting TMDs 1–6 and 7–12. By co-expression and N-hydroxysuccinimide methotrexate (Mtx) radioaffinity labeling of hRFC TMD 1–6 and TMD 7–12 half-molecules, combined with endoproteinase GluC digestion, a substrate binding domain was previously localized to within TMDs 8–12 (Witt, T. L., Stapels, S. E., and Matherly, L. H. (2004) J. Biol. Chem. 279, 46755–46763). In this report, this region was further refined to TMDs 11–12 by digestion with 2-nitro-5-thiocyanatobenzoic acid. A transportcompetent cysteine-less hRFC was used as a template to prepare single cysteine-replacement mutant constructs in which each residue from Glu-394 to Asp-420 of TMD 11 and Tyr-435 to His-457 of TMD 12 was replaced individually by a cysteine. The mutant constructs were transfected into hRFC-null HeLa cells. Most of the 50 single cysteine-substituted constructs were expressed at high levels on Western blots. With the exception of G401C hRFC, all mutants were active for Mtx transport. Treatment with sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES) had no effect on hRFC activity for all of the cysteine mutants within TMD 12 and for the majority of the cysteine mutants within TMD 11. However, MTSES inhibited Mtx uptake by the T404C, A407C, T408C, T412C, F416C, I417C, V418C, and S419C mutants by 25–65%. Losses of activity by MTSES treatment for T404C, A407C, T412C, and I417C hRFCs were appreciably reversed in the presence of excess leucovorin, a hRFC substrate. Our results strongly suggest that residues within TMD 11 are likely critical structural and/or functional components of the putative hRFC transmembrane channel for anionic folate and anti-folate substrates.

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Faculty Opinions recommendation of Localization of a substrate binding domain of the human reduced folate carrier to transmembrane domain 11 by radioaffinity labeling and cysteine-substituted accessibility methods.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1027843.334821 ◽

2005 ◽

Author(s):

Vivian Cody

Keyword(s):

Transmembrane Domain ◽

Substrate Binding ◽

Binding Domain ◽

Reduced Folate Carrier ◽

Substrate Binding Domain

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Characterization of a cysteine-less human reduced folate carrier: localization of a substrate-binding domain by cysteine-scanning mutagenesis and cysteine accessibility methods

Biochemical Journal ◽

10.1042/bj20030301 ◽

2003 ◽

Vol 374 (1) ◽

pp. 27-36 ◽

Cited By ~ 14

Author(s):

Wei CAO ◽

Larry H. MATHERLY

Keyword(s):

Amino Acids ◽

Mammalian Cells ◽

Transmembrane Domain ◽

Chinese Hamster Ovary Cells ◽

Substrate Binding ◽

Chinese Hamster ◽

Water Soluble ◽

Cysteine Residues ◽

Reduced Folate Carrier ◽

Cysteine Scanning Mutagenesis

The human reduced folate carrier (hRFC) mediates the transport of reduced folates and classical anti-folates into mammalian cells. Whereas the functionally important domains in hRFC are poorly characterized, previous studies with anti-folate-resistant cells suggest critical roles for transmembrane domain (TMD) 1 and residues (Gly44, Glu45, Ser46 and Ile48) in or flanking this region. An hRFC mutant devoid of cysteine residues was prepared by deleting the C-terminal 56 amino acids, including four cysteine residues, and mutagenizing the remaining cysteine residues to serine residues. A fully functional cysteine-less hRFC protein was expressed in transport-impaired MtxRIIOuaR2-4 Chinese-hamster ovary cells. To explore the role of residues in or flanking TMD1 in transport, all 24 amino acids from Trp25 to Ile48 of hRFC were mutated individually to cysteine residues, and the mutant hRFCs were transfected into MtxRIIOuaR2-4 cells. All of the 24 cysteine mutants were expressed and, with the exception of R42C (Arg42→Cys), were capable of mediating methotrexate uptake above the low level in MtxRIIOuaR2-4 cells. We found that by treating the transfected cells with the small, water-soluble, thiol-reactive anionic reagent, sodium (2-sulphonatoethyl) methanethiosulphonate, methotrexate transport by several of the cysteine-substituted hRFC mutants was significantly inhibited, including Q40C, G44C, E45C and I48C. Sodium (2-sulphonatoethyl) methanethiosulphonate transport inhibition of the Q40C, G44C and I48C mutants was protected by leucovorin [(6R,S)-5-formyltetrahydrofolate], indicating that these residues lie at or near a substrate-binding site. Using surface-labelling reagents [N-biotinylaminoethyl methanethiosulphonate and 3-(N-maleimidylpropionyl)biocytin, combined with 4-acetamido-4′-maleimidylstilbene-2,2′-disulphonic acid] with cysteine mutants from positions 37–48, the extracellular TMD1 boundary was found to lie between residues 39 and 40, and amino acids 44–46 and 48 were localized to the TMD1 exofacial loop. Collectively, our results imply that amino acids 40, 44, 48 and, possibly, 42 serve important roles in hRFC transport, albeit not as structural components of the putative transmembrane channel for folate substrates.

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