This report addresses the functional role of His residues in the proton-coupled folate transporter (PCFT; SLC46A1), which mediates intestinal folate absorption. Of ten His residues, only H247A and H281A mutations altered function. The folic acid influx K t at pH 5.5 for H247A was 28.4-fold. Although wild type (WT)-PCFT K i values varied among the folates, K i values were much lower and comparable for H247-A, -R, -Q, or -E mutants. Homology modeling localized His 247 to the large loop separating transmembrane domains 6 and 7 at the cytoplasmic entrance of the translocation pathway in hydrogen-bond distance to Ser 172 . The folic acid influx K t for S172A-PCFT was decreased similar to H247A. His 281 faces the extracellular region in the seventh transmembrane domain. H281A-PCFT results in loss-of-function due to ϳ12-fold1 in the folic acid influx K t . When the pH was decreased from 5.5 to 4.5, the WT-PCFT folic acid influx K t was unchanged, but the K t decreased 4-fold for H281A. In electrophysiological studies in Xenopus oocytes, both WT-PCFT-and H281A-PCFT-mediated folic acid uptake produced current and acidification, and both exhibited a low level of folate-independent proton transport (slippage). Slippage was markedly increased for the H247A-PCFT mutant. The data suggest that disruption of the His 247 to Ser 172 interaction results in a PCFT conformational alteration causing a loss of selectivity, increased substrate access to a high affinity binding pocket, and proton transport in the absence of a folate gradient. The His 281 residue is not essential for proton coupling but plays an important role in PCFT protonation, which, in turn, augments folate binding to the carrier.This laboratory recently identified SLC46A1 as a protoncoupled folate transporter (PCFT) 2 that mediates transport of folates across the apical (brush border) membrane of enterocytes within the acidic microclimate at the absorptive surface of the proximal jejunum (1). The critical role that PCFT plays at this epithelium was confirmed with the demonstration by this laboratory that there are loss-of-function mutations in this gene in patients with the autosomal recessive disorder, hereditary folate malabsorption (Online Mendelian Inheritance in Man, 229050) (1, 2). Hereditary folate malabsorption is characterized by impaired intestinal folate absorption and impaired transport of folates into the central nervous system (3), the latter likely due to a defect in transport across the blood-choroid plexus-cerebrospinal fluid barrier. While operating most efficiently at low pH, PCFT contributes to the transport of folates and the pharmacological activity of the new generation antifolate, pemetrexed, even at neutral pH (4). PCFT may be even more important to the delivery of this, and possibly other antifolates, within the acidic interstitium of solid tumors (5, 6). PCFT may also play a role in the export of folates from acidified endosomes (7) during folate receptor-mediated endocytosis (8, 9). Although facilitative carriers in lower organisms com...

The proton-coupled folate transporter (PCFT) mediates intestinal folate absorption. Loss-of-function mutations in this gene are the molecular basis for the autosomal recessive disorder, hereditary folate malabsorption. In this study, the substituted cysteine accessibility method was utilized to localize extra- or intra-cellular loops connecting predicted PCFT transmembrane domains. Cysteine-less PCFT was generated by replacement of all seven cysteine residues with serine, and was shown to be functional, following which cysteine residues were introduced into predicted loops. HeLa cells, transiently transfected with these PCFT mutants, were then labeled with an impermeant, cysteine-specific biotinylation reagent (MTSEA-biotin) with or without permeabilization of cells. The biotinylated proteins were precipitated by streptavidin beads and assessed by Western blotting analysis. The biotinylation of PCFT was further confirmed by blocking cysteine residues with impermeant 2-sulfonatoethyl methanethiosulfonate. Two extracellular cysteine residues (66, 298) present in WT-PCFT were not biotinylated; however, in the absence of either one, biotinylation occurred. Likewise, biotinylation occurred after treatment with β-mercaptoethanol. Taken together, these analyses establish a PCFT secondary structure of twelve transmembrane domains with the N- and C- termini directed to the cytoplasm. The data indicate further that there is a disulfide bridge, which is not required for function, between the native C66 and C298 residues in the first and fourth transmembrane domains, respectively.

The proton-coupled folate transporter (PCFT) SLC46A1 mediates uphill folate transport into enterocytes in proximal small intestine coupled to the inwardly directed proton gradient. Hereditary folate malabsorption is due to loss-of-function mutations in the PCFT gene. This study addresses the functional role of conserved charged amino acid residues within PCFT transmembrane domains with a detailed analysis of the PCFT E185 residue. D156A-, E185A-, E232A-, R148A-, and R376A-PCFT mutants lost function at pH 5.5, as assessed by transient transfection in folate transport-deficient HeLa cells. At pH 7.4, function was preserved only for E185A-PCFT. Loss of function for E185A-PCFT at pH 5.5 was due to an eightfold decrease in the [(3)H]methotrexate (MTX) influx V(max); the MTX influx K(t) was identical to that of wild-type (WT)-PCFT (1.5 microM). Consistent with the intrinsic functionality of E185A-PCFT, [(3)H]MTX influx at pH 5.5 or 7.4 was trans-stimulated in cells preloaded with nonlabeled MTX or 5-formyltetrahydrofolate. Replacement of E185 with Leu, Cys, His, or Gln resulted in a phenotype similar to E185A-PCFT. However, there was greater preservation of activity (approximately 38% of WT) for the similarly charged E185D-PCFT at pH 5.5. All E185 substitution mutants were biotin accessible at the plasma membrane at a level comparable to WT-PCFT. These observations suggest that the E185 residue plays an important role in the coupled flows of protons and folate mediated by PCFT. Coupling appears to have a profound effect on the maximum rate of transport, consistent with augmentation of a rate-limiting step in the PCFT transport cycle.

. Properties of the Arg376 residue of the proton-coupled folate transporter (PCFT-SLC46A1) and a glutamine mutant causing hereditary folate malabsorption.

The human proton-coupled folate transporter (HsPCFT, SLC46A1) mediates intestinal absorption of folates and transport of folates into the liver, brain and other tissues. On Western blot, HsPCFT migrates as a broad band (~55 kDa), higher than predicted (~50 kDa) in cell lines. Western blot analysis required that membrane preparations not be incubated in the loading buffer above 50 degrees C to avoid aggregation of the protein. Treatment of membrane fractions from HsPCFT-transfected HeLa cells with peptidyl N-glycanase F, or cells with tunicamycin, resulted in conversion to a ~35 kDa species. Substitution of asparagine residues of two canonical glycosylation sites to glutamine, individually, yielded a ~47 kDa protein; substitution of both sites gave a smaller (~35 kDa) protein. Single mutants retained full transport activity; the double mutant retained a majority of activity. Transport function and molecular size were unchanged when the double mutant was hemagglutinin (HA) tagged at either the NH(2) or COOH terminus and probed with an anti-HA antibody excluding degradation of the deglycosylated protein. Wild-type or deglycosylated HsPCFT HA, tagged at amino or carboxyl termini, could only be visualized on the plasma membrane when HeLa cells were first permeabilized, consistent with the intracellular location of these domains.

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The proton-coupled folate transporter (PCFT) mediates intestinal folate absorption and transport of folates across the choroid plexus. This study focuses on the role of Tyr residues in PCFT function. The substituted Cys-accessibility method identified four Tyr residues (Y291, Y362, Y315, and Y414) that are accessible to the extracellular compartment; three of these (Y291, Y362, and Y315) are located within or near the folate binding pocket. When the Tyr residues were replaced with Cys or Ala, these mutants showed similar (up to 6-fold) increases in influx Vmax and Kt/Ki for [(3)H]methotrexate and [(3)H]pemetrexed. When the Tyr residues were replaced with Phe, these changes were moderated or absent. When Y315A PCFT was used as representative of the mutants and [(3)H]pemetrexed as the transport substrate, this substitution did not increase the efflux rate constant. Furthermore, neither influx nor efflux mediated by Y315A PCFT was transstimulated by the presence of substrate in the opposite compartment; however, substantial bidirectional transstimulation of transport was mediated by wild-type PCFT. This resulted in a threefold greater efflux rate constant for cells that express wild-type PCFT than for cells that express Y315 PCFT under exchange conditions. These data suggest that these Tyr residues, possibly through their rigid side chains, secure the carrier in a high-affinity state for its folate substrates. However, this may be achieved at the expense of constraining the carrier's mobility, thereby decreasing the rate at which the protein oscillates between its conformational states. The Vmax generated by these Tyr mutants may be so rapid that further augmentation during transstimulation may not be possible.

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