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[摘要]：RFC (reduced folate carrier) is the major transporter for reduced folates and anti folates [e.g. MIX (methotrexate)]. RFC is characterized by two halves, each with six TMD (transmembrane domain) alpha helices connected by a hydrophilic loop, and cytoplasmic N- and C-termini. We previously identified TMDs 4, 5, 7, 8, 10 and 11 as forming the hydrophilic cavity for translocation of (anti)folates. The proximal end of TMD8 (positions 311-314) was implicated in substrate binding from scanning-cysteine accessibility methods; cysteine replacement of Ser(313) resulted in loss of transport. In the present study, Ser(313) was mutated to alanine, cysteine, phenylalanine and threonine. Mutant RFCs were expressed in RFC-null R5 He La cells. Replacement of Ser(313) with cysteine or phenylalanine abolished MTX transport, whereas residual activity was preserved for the alanine and threonine mutants. In stable K562 transfectants, S313A and S313T RFCs showed substantially decreased V-max, values without changes in K-t values for MTX compared with wild-type RFC. S313A and S313T RFCs differentially impacted binding of ten diverse (anti)folate substrates. Cross-linking between TMD8 and TMD5 was studied by expressing cysteine-less TMD1-6 (N-6) and TMD7-12 (C-6) half-molecules with cysteine insertions spanning these helices in R5 cells, followed by treatment with thiol-reactive homobifunctional cross-linkers. C-6-C-6 and N-6 N-6 cross-links were seen for all cysteine pairs. From the N-6 and C-6 cysteine pairs, Cys(175)/Cys(311) was cross-linked; cross-linking increased in the presence of transport substrates. The results of the present study indicate that the proximal end of TMD8 is juxtaposed to TMD5 and is conformationally active in the presence of transport substrates, and TMD8, including Ser(313), probably contributes to the RFC substrate-binding domain. |
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