Mammals meet their biosynthetic needs for one-carbon donors by absorption of dietary folates mediated by the protoncoupled folate transporter (PCFT) 2 (SLC46A1) (1, 2). Mutations in the PCFT gene that result in loss of the protein or its function are the basis for hereditary folate malabsorption (OMIM 229050), a rare autosomal recessive disorder (1, 3).PCFT transports protons along with folates as reflected in the current and acidification accompanying folate transport in oocytes that express this transporter (1, 4 -6). Structure-function studies, based on site-directed and random mutagenesis, have begun to define residues and domains that play a key role in PCFT function. Elucidation of the secondary structure of PCFT, along with the residues that contribute to the aqueous translocation pathway, have been documented by the substituted cysteine accessibility method (7). The protein consists of 12 transmembrane helices, divided in half by a large intracellular loop, with C and N termini oriented into cytoplasm (8 -10). Residues critical to folate/antifolate binding, proton coupling, and proton binding have been identified (5,(11)(12)(13). The roles of residues in the 2nd, 4th transmembrane domains (TMDs), and the loop connecting the 2nd and 3rd TMDs have been studied (14,15). Analyses of the functional defects that occur in subjects with hereditary folate malabsorption have provided insights into residues important to PCFT protein folding, stability, and/or trafficking to the cell membrane, substrate binding, and oscillation of the carrier between its conformational states (13, 16 -20).PCFT expression is limited to eukaryotes; there are no closely related proteins present in archaea or bacteria, so that a highresolution x-ray crystal structure with a high level of sequence identity is not available that would facilitate a structure/function analysis of the human or rodent proteins. Rather, homology modeling has identified the structure of the bacterial homolog, the glycerol-3-transporter, GlpT, as having a protein structure most like PCFT despite a low (14%) sequence identity (5,10,21). In this model, the transporter is in a conformation that is closed to the outside and open to the inside. According to the alternative access model, PCFT oscillates between this conformation and a conformation that is closed to the inside and open to the outside. This computational model has been used for correlation with functional data and for structural predictions as in the current study (5, 12-14, 21, 22).In this inward facing homology model of PCFT, TMD-1, -2, -7, and -11 are predicted to cluster together in proximity to the extracellular interface to form an extracellular gate. To confirm the presence of such a gate, Cys pairs predicted to be in proximity were introduced into these TMDs at locations near the extracellular surface. The possibility that the pairs are sufficiently close to be cross-linked and/or form a complex was evaluated by assessment of function, which should be decreased when the mobility of the gate is r...

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.

The substituted cysteine accessibility method (SCAM) is widely used to study the structure and function of channels, receptors and transporters. In its usual application, a cysteine residue is introduced into a protein which lacks native cysteines following which the accessibility of the residue to the aqueous compartment is assessed. Implicit, and generally assumed, is that if the cysteine-substituted residue is not available to react with sulfhydryl reagents it is not exposed to the extracellular compartment or within the aqueous translocation pathway. We demonstrate here, in a Hela-derived cell line, that some cysteine-substituted residues of the proton-coupled folate transporter (PCFT, SLC46A1) that are inaccessible to 2-((biotinoyl)amino)ethyl methanethiosulfonate are glutathionylated by biotinylated glutathione ethyl ester in the absence of an oxidizing agent. Intramolecular disulfide formation involving cysteine-substituted residues was also identified in some instances. These posttranslational modifications limit the accessibility of the cysteine residues to sulfhydryl-reactive reagents and can have a profound impact on the interpretation of SCAM but may not alter function. When a posttranslationally modified residue is used as a reference extracellular control, the high level of exposure required for detection on Western blot results in erroneous detection of otherwise inaccessible intracellular cysteine-substituted residues. The data indicate that in the application of SCAM, when a cysteine-substituted residue does not appear to be accessible to sulfhydryl-reactive reagents, the possibility of a posttranslational modification should be excluded. The data explain the discrepancies in the assessment, and confirm the localization, of the first intracellular loop of PCFT.

The proton-coupled folate transporter (PCFT, SLC46A1) is required for folate intestinal absorption and transport across the choroid plexus. Recent work has identified a F392V mutation causing hereditary folate malabsorption. However, the residue properties responsible for this loss of function remains unknown. Using site-directed mutagenesis, we observed complete loss-of-function with charged (Lys, Asp, Glu) and polar (Thr, Ser, Gln) Phe392 substitutions and minimal function with some neutral substitutions; however, F392M retained full function. Using the substituted-cysteine accessibility method (with MTSEA-biotin labeling), Phe392 mutations causing loss of function, while preserving membrane expression and trafficking, also resulted in loss of accessibility of the substituted cysteine in P314C-PCFT located within the aqueous translocation pathway. F392V function and accessibility of the P314C cysteine were restored by insertion of a G305L (suppressor) mutation. A S196L mutation localized in proximity to Gly305 by homology modeling was inactive. However, when inserted into the inactive F392V scaffold, function was restored (mutually compensatory mutations) as was accessibility of the P314C cysteine residue. Reduced function, documented with F392H PCFT was due to a 15-fold decrease in methotrexate influx Vmax, accompanied by a decreased influx Kt (4.5-fold) and Ki (3-fold). The data indicate that Phe392 is required for rapid oscillation of the carrier among its conformational states and suggest that this is achieved by dampening affinity of the protein for its folate substrates. F392V, and other inactivating Phe392 PCFT mutations, lock the protein in its inward-open conformation. Reach (length) and hydrophobicity of Phe392 appear to be features required for full activity.

The proton-coupled folate transporter (PCFT) mediates folate absorption across the brush-border membrane of the proximal small intestine and is required for folate transport across the choroid plexus into the cerebrospinal fluid. In this study, the functional role and accessibility of the seven PCFT Trp residues were assessed by the substituted-cysteine accessibility method. Six Trp residues at a lipid-aqueous interface tolerated Cys substitution in terms of protein stability and function. W85C, W202C, and W213C were accessible to N-biotinyl aminoethylmethanethiosulfonate; W48C and W299C were accessible only after treatment with dithiotreitol (DTT), consistent with modification of these residues by an endogenous thiol-reacting molecule and their extracellular location. Neither W107C nor W333C was accessible (even after DTT) consistent with their cytoplasmic orientation. Biotinylation was blocked by pemetrexed only for the W48C (after DTT), W85C, W202C residues. Function was impaired only for the W299C PCFT mutant located in the 4th external loop between the 7th and 8th transmembrane helices. Despite its aqueous location, function could only be fully preserved with Phe and, to a lesser extent, Ala substitutions. There was a 6.5-fold decrease in the pemetrexed influx Vmax and a 3.5- and 6-fold decrease in the influx Kt and Ki, respectively, for the W299S PCFT. The data indicate that the hydrophobicity of the W299 residue is important for function suggesting that during the transport cycle this residue interacts with the lipid membrane thereby impacting on the oscillation of the carrier and, indirectly, on the folate binding pocket.