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

Mahadeo, Kris Michael; Diop-Bove, Ndeye; Shin, Daniel Sanghoon; Unal, Ersin Selcuk; Teo, Juliana; Zhao, Rongbao; Chang, Min; Fulterer, Andreas; Romero, Michael F.; Goldman, I. David

doi:10.1152/ajpcell.00113.2010

Cited by 38 publications

(57 citation statements)

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“…Given the critical roles of hPCFT in intestinal absorption of dietary folates and of mutant hPCFT in HFM (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15) and in the selective delivery of cytotoxic antifolates for targeting solid tumors (21, 24 -26), our findings of a functionally important oligomerization for hPCFT are particularly significant. For instance, in HFM, our findings may explain why all HFM patients thus far described have mutations in both pcft gene alleles because loss of a single pcft allele on hPCFT function would probably not be detected (5-15).…”

Section: Discussionmentioning

confidence: 90%

“…The role of hPCFT in intestinal folate absorption was established by demonstrating loss-of-function mutations in hPCFT in patients with the rare autosomal inherited disorder, hereditary folate malabsorption (HFM) (5). To date, 17 unique hPCFT mutations have been reported in ethnically varied kindreds (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). Although proton-coupled, this transporter is also func-tional at more physiologic pH, at which it retains appreciable affinity for pemetrexed (16), a newer antifolate currently approved for treating mesothelioma and non-squamous, nonsmall cell lung cancer (17)(18)(19).…”

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confidence: 99%

“…Based on results with substituted cysteine-scanning mutagenesis of a Cys-less hPCFT and accessibilities to thiol-reactive agents (27) and on studies of hemagglutinin (HA) epitope accessibilities to epitope-specific antibody (28,29), hPCFT has 12 TMDs with N and C termini directed to the cytosol. Structurally and/or functionally important residues have been identified in hPCFT and include (with possible roles) Glu-185 (which is required for proton coupling) (30), His-247 and Ser-172 (which modulate folate and proton access to a high affinity binding site) (31), His-281 (which is important for substrate binding) (31), Arg-376 (which impacts proton and substrate binding) (12), and Asp-156 (which contributes to PCFT protein stability) (13) (Fig. 1).…”

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confidence: 99%

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Identification and Functional Impact of Homo-oligomers of the Human Proton-coupled Folate Transporter

Hou

Desmoulin

Etnyre

et al. 2012

Journal of Biological Chemistry

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Background:The human proton-coupled folate transporter (PCFT) may exist as homo-oligomers. Results: PCFT monomers form oligomers, and wild-type and inactive mutant PCFT monomers show dominant-positive activity when co-expressed. Conclusion: Wild-type PCFT may rescue the mutant phenotype via formation of hetero-oligomers. Significance: Better understanding of PCFT oligomerization may identify therapeutic applications for hereditary folate maladsorption and delivery of PCFT-targeted chemotherapy drugs for cancer.

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Section: Discussionmentioning

confidence: 90%

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confidence: 99%

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confidence: 99%

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Identification and Functional Impact of Homo-oligomers of the Human Proton-coupled Folate Transporter

Hou

Desmoulin

Etnyre

et al. 2012

Journal of Biological Chemistry

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“…PCFT has been shown to be the major route through which folates from food sources are absorbed in the intestine, as well as the main transporter of folates across the choroid plexus barrier to the cerebrospinal fluid. These crucial physiological roles are disrupted in patients with loss of function mutations in the PCFT gene that lead to hereditary folate malabsorbtion [1][2][3][4][5][6][7]. PCFT is also involved in uptake of antifolates that are used as chemotherapeutic agents into solid tumors.…”

Section: Introductionmentioning

confidence: 99%

The monomeric state of the proton‐coupled folate transporter represents the functional unit in the plasma membrane

et al. 2013

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Folic acid is an essential vitamin required for de novo biosynthesis of nucleotides and amino acids. The proton-coupled folate transporter (PCFT; SLC46A1) has been identified as the major contributor for intestinal folate uptake. It is also involved in folate transport across the blood-brain barrier and into solid tumors. PCFT belongs to the major facilitator superfamily. Major facilitator superfamily members can exist in either monomeric or homo-oligomeric form. Here, we utilized blue native polyacrylamide gel electrophoresis (BN/PAGE) and crosslinking with bi-functional chemicals to investigate the quaternary structure of human PCFT after heterologous expression in Xenopus laevis oocytes and CHO cells. PCFT was expressed in the plasma membrane in both expression systems. The functionality of the utilized PCFT construct was confirmed in oocytes by folic acid induced currents at acidic pH. For both the oocyte and CHO expression system [ 3 H]folic acid uptake studies indicated that PCFT was functional. To analyze the oligomeric state of PCFT in the plasma membrane, plasma membranes were isolated by polymerization with colloidal silica and polyacrylic acid and subsequent centrifugation. The digitoninsolubilized non-denatured PCFT migrated during BN/PAGE as a monomer, as judged by comparison with a membrane protein (5-HT 3A receptor) of known pentameric assembly that was used to create a molecular sizing ladder. The chemical crosslinkers glutaraldehyde and dimethyl adipimidate were not able to covalently link potential higher order PCFT structures to form oligomers that were stable following SDS treatment. Together, our results demonstrate that plasma-membrane PCFT functions as a monomeric protein.

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“…[2][3][4] In only a few instances has a stable mutant protein that traffics to the cell membrane been identified, allowing characterization of the functional defect. 5,6 The topology of this transporter has been defined and is typical of the solute carrier (SLC) superfamily of transporters with 12 transmembrane helices, 6 on either side of a large internal loop, with N-and C-termini located within the cytoplasm. [7][8][9][10] The exofacial regions at the membrane-aqueous interface of transmembrane helices 1, 2, 7, and 11 were recently shown to come together to form a gate at the extracellular entrance to the aqueous translocation pathway, 11 as has been reported for other solute transporters.…”

Section: Introductionmentioning

confidence: 99%

Hereditary folate malabsorption due to a mutation in the external gate of the proton-coupled folate transporter SLC46A1

et al. 2018

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Key Points• An N411K mutation in the external gate of the proton-coupled folate transporter within the aqueous channel results in impaired function.• The N411K mutation produces a substratespecific defect in transport, resulting in hereditary folate malabsorption.Hereditary folate malabsorption (HFM) is an autosomal recessive disorder characterized by impaired intestinal folate absorption and impaired folate transport across the choroid plexus due to loss of function of the proton-coupled folate transporter (PCFT-SLC46A1). We report a novel mutation, causing HFM, affecting a residue located in the 11th transmembrane helix within the external gate. The mutant N411K-PCFT was stable, trafficked to the cell membrane, and had sufficient residual activity to characterize the transport defect and the structural requirements at this site for gate function. The influx V max of the N411K mutant was markedly decreased, as was the affinity for most, but not all, folate/antifolate substrates.The greatest loss of activity was for 5-methyltetrahydrofolate. Substitutions with positive charged residues resulted in a loss of activity (arginine . lysine . histidine). Function was retained for the negative charged aspartate, but not the larger glutamate substitutions, whereas the bulky hydrophobic (leucine), or polar (glutamine) substitutions, were tolerated.Homology models of PCFT, in the inward and outward open conformations, based upon the mammalian Glut5 fructose transporter structures, localize Asn411 protruding into the aqueous pathway. This is most prominent when the carrier is in the inward open conformation when the external gate is closed. Mutations at this site likely result in highly specific steric and electrostatic interactions between the Asn411-substituted, and other, residues in the gate region that impede carrier function. The substrate specificity of the N411K mutant may be due to alterations of substrate flows through the external gate, downstream allosteric alterations in the folate-binding pocket, or both.

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

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

Cited by 38 publications

References 31 publications

Identification and Functional Impact of Homo-oligomers of the Human Proton-coupled Folate Transporter

Identification and Functional Impact of Homo-oligomers of the Human Proton-coupled Folate Transporter

The monomeric state of the proton‐coupled folate transporter represents the functional unit in the plasma membrane

Hereditary folate malabsorption due to a mutation in the external gate of the proton-coupled folate transporter SLC46A1

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