{"gene":"SLC46A1","run_date":"2026-06-10T07:46:34","timeline":{"discoveries":[{"year":2007,"finding":"PCFT (SLC46A1) is an electrogenic, proton-coupled folate transporter with optimal transport activity at low pH (pH 5.5), mediating intestinal folate absorption; transport is pH-dependent with increasing Km and decreasing Vmax as pH rises toward neutral, and transport in Xenopus oocytes is electrogenic and pH-dependent.","method":"Xenopus oocyte expression with electrophysiology, radiolabeled folate uptake assays, tissue fractionation showing apical brush-border membrane localization in duodenum/proximal jejunum","journal":"American journal of physiology. Cell physiology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — electrophysiological reconstitution in oocytes, radiolabeled transport assays, membrane localization by fractionation, replicated across multiple labs and organisms","pmids":["17898134","18174275","17340171"],"is_preprint":false},{"year":2007,"finding":"Loss-of-function mutations in PCFT (SLC46A1) are the molecular basis for hereditary folate malabsorption (HFM); mutations cause decreased protein stability, defects in membrane trafficking, or loss of transport function, with some mutants retaining residual activity.","method":"Sequencing of PCFT gene in HFM patients, transient transfection of mutant constructs in HeLa cells with transport assays, Western blot for protein stability, membrane trafficking assessment","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple mutations characterized across multiple patients, multiple orthogonal methods (sequencing, functional assays, protein stability), independently replicated across labs","pmids":["17446347"],"is_preprint":false},{"year":2008,"finding":"PCFT co-localizes with folate receptor alpha (FRα) in the endosomal compartment and plays a functional role in FRα-mediated endocytosis by serving as the route of export of folates from acidified endosomes into the cytosol; probenecid inhibits PCFT-mediated transport at endosomal pH and blocks FRα-mediated folate delivery to the cytosol.","method":"FRα cDNA transfection into PCFT(+) and PCFT(−) HeLa sublines, radiolabeled folate transport assays, immunofluorescence co-localization, probenecid inhibition experiments","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — isogenic PCFT(+/−) cell lines with cotransfection, multiple orthogonal methods (transport assay, co-localization, pharmacological inhibition), single lab","pmids":["19074442"],"is_preprint":false},{"year":2008,"finding":"PCFT (SLC46A1) is N-glycosylated at two canonical asparagine residues; the protein migrates at ~55 kDa on SDS-PAGE (predicted ~50 kDa) due to glycosylation, collapses to ~35 kDa after deglycosylation. Single glycosylation-site mutants retain full transport activity; the double deglycosylation mutant retains a majority of activity. The N- and C-termini are intracellular (accessible only in permeabilized cells).","method":"PNGase F treatment, tunicamycin treatment, site-directed mutagenesis of glycosylation sites (Asn→Gln), Western blot, HA-tag accessibility assay in permeabilized vs. intact cells","journal":"Biochimica et biophysica acta","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — multiple orthogonal biochemical methods (enzymatic deglycosylation, drug inhibition, mutagenesis, topology probing), single lab","pmids":["18405659"],"is_preprint":false},{"year":2009,"finding":"His247 and His281 residues are functionally critical for PCFT (SLC46A1). His247 (located in the large loop between TM6 and TM7 at the cytoplasmic entrance, in hydrogen-bond distance to Ser172) is required for substrate selectivity and coupled proton transport; H247A mutation increases proton 'slippage' (folate-independent proton transport) and reduces substrate specificity. His281 (in TM7, extracellular face) facilitates proton binding/protonation that augments folate binding; H281A increases folic acid influx Kt ~12-fold but does not abolish proton coupling.","method":"Site-directed mutagenesis, radiolabeled folate influx kinetics in transfected HeLa cells, electrophysiology in Xenopus oocytes, homology modeling","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — mutagenesis with kinetic analysis, electrophysiology, and homology modeling in a single study; multiple residues and mechanistic consequences established","pmids":["19389703"],"is_preprint":false},{"year":2009,"finding":"PCFT promoter silencing through CpG hypermethylation within the minimal transcriptional regulatory region (−42 to +96 bp from TSS) accounts for loss of PCFT expression in antifolate-resistant HeLa R1-11 cells; treatment with 5-aza-2'-deoxycytidine restores transport and PCFT mRNA. In vitro methylation of the transfected reporter plasmid inhibits expression. Gene copy loss contributes additively.","method":"Bisulfite conversion sequencing, luciferase reporter assays, 5-aza-2'-deoxycytidine treatment, in vitro methylation of reporter plasmid, FISH for gene copy number","journal":"Molecular cancer therapeutics","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (bisulfite sequencing, reporter assays, pharmacological demethylation, in vitro methylation, FISH), single lab","pmids":["19671745"],"is_preprint":false},{"year":2009,"finding":"Glu185 (E185) in a PCFT transmembrane domain plays a critical role in proton-folate coupling: E185A-PCFT loses function at pH 5.5 (8-fold decrease in Vmax) but retains function at pH 7.4; the transport remains intrinsically functional (capable of trans-stimulation). The effect of E185 substitution is primarily on the maximal transport rate (Vmax), consistent with involvement at a rate-limiting step of the PCFT transport cycle.","method":"Site-directed mutagenesis, radiolabeled methotrexate influx kinetics, trans-stimulation assays, surface biotinylation in transfected HeLa cells","journal":"American journal of physiology. Cell physiology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis with kinetic analysis, multiple substitutions, trans-stimulation and surface expression controls, single lab","pmids":["19403800"],"is_preprint":false},{"year":2010,"finding":"PCFT secondary structure consists of 12 transmembrane domains with both N- and C-termini directed to the cytoplasm, established by substituted cysteine accessibility method (SCAM). A disulfide bridge between native Cys66 and Cys298 (in the 1st and 4th extracellular loops) is present but not required for transport function.","method":"Substituted cysteine accessibility method (SCAM) with MTSEA-biotin, streptavidin pulldown, Western blot, beta-mercaptoethanol treatment, cysteine-less PCFT generation","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — SCAM topology mapping with functional validation, cysteine-less scaffold controls, disulfide bond confirmed by multiple reagents, single lab","pmids":["20225891"],"is_preprint":false},{"year":2010,"finding":"Asp156 (D156) is critical for PCFT protein stability; multiple substitutions at this site (Tyr, Trp, Phe, Val, Asn, Lys) result in protein instability and loss of function, while conservative substitutions (Glu, Gly) preserve substantial function correlating with surface expression. Asp109 (D109), located in the first intracellular loop between TM2 and TM3, is absolutely required for PCFT transport function regardless of pH or substrate concentration, despite normal surface expression.","method":"Site-directed mutagenesis, radiolabeled folate transport assays at multiple pH values and concentrations, surface biotinylation, Western blot for protein stability in transfected HeLa cells","journal":"Blood","confidence":"High","confidence_rationale":"Tier 1 / Moderate — systematic mutagenesis with kinetic analysis, surface expression controls, multiple orthogonal methods, single lab","pmids":["20805364"],"is_preprint":false},{"year":2010,"finding":"NRF-1 (nuclear respiratory factor 1) is a major transcriptional regulator of PCFT (SLC46A1). NRF-1 binds to three consensus sites in the PCFT minimal promoter; NRF-1 overexpression increases PCFT mRNA and reporter activity, while dominant-negative NRF-1 or NRF-1 siRNA markedly represses PCFT expression.","method":"EMSA with NRF-1 antibody supershift, chromatin immunoprecipitation, luciferase reporter assays with NRF-1 site mutations, NRF-1 overexpression and siRNA knockdown","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (EMSA, ChIP, reporter assay with mutagenesis, gain/loss-of-function), single lab","pmids":["20724482"],"is_preprint":false},{"year":2010,"finding":"Arg376 (R376) is important for proton binding in PCFT; R376Q and other non-positive substitutions impair proton binding, which in turn modulates the folate binding pocket and reduces the rate of carrier conformational change. The R376Q mutant retains folate-independent proton transport (slippage/channel-like property) in Xenopus oocytes, but substrate transport is markedly decreased, with substrate-dependent differences.","method":"Site-directed mutagenesis, radiolabeled folate influx kinetics, electrophysiology in Xenopus oocytes, transfection in HeLa R1-11 cells","journal":"American journal of physiology. Cell physiology","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — mutagenesis with kinetic analysis and electrophysiology, multiple substrates and pH conditions tested, single lab","pmids":["20686069"],"is_preprint":false},{"year":2011,"finding":"PCFT-deficient (PCFT−/−) mice develop severe systemic folate deficiency confirming the critical and nonredundant in vivo role of PCFT in intestinal folate transport; PCFT deficiency causes macrocytic normochromic anemia, pancytopenia, impaired erythroblast differentiation with increased apoptosis, elevated erythropoietin, soluble transferrin receptor, and thrombopoietin.","method":"Targeted gene disruption (knockout of exons 1–3), in vivo folate uptake experiments, hematological analysis, bone marrow histology, serum cytokine measurement","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — knockout mouse model with in vivo folate uptake assay directly confirming intestinal folate transport defect, comprehensive phenotypic characterization","pmids":["21346251"],"is_preprint":false},{"year":2011,"finding":"PCFT and RFC (reduced folate carrier) are distributed in lipid rafts of the colonic apical membrane; chronic ethanol ingestion reduces PCFT and RFC protein levels in lipid raft fractions of the colon, associated with decreased folate transport affinity and Vmax.","method":"Optiprep density gradient floatation for lipid raft isolation, Western blot, radiolabeled folate transport in apical membrane vesicles from ethanol-fed rats","journal":"Journal of cellular physiology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — membrane fractionation and Western blot with transport assay, single lab, no direct functional link between raft localization and transport activity established","pmids":["21069807"],"is_preprint":false},{"year":2012,"finding":"Gly189 and Gly192 (a GxxG motif in TM5) are functionally critical for PCFT; alanine substitution abolishes function. Ile188, adjacent to this motif, is accessible to the PCFT aqueous translocation pathway and located in the folate binding pocket (substrate protection of I188C from sulfhydryl-reactive reagents is retained at 0°C). Met193C is also aqueous-accessible but substrate protection is lost at 0°C.","method":"Site-directed mutagenesis, substituted cysteine accessibility method with membrane-impermeable reagents, substrate protection assays at different temperatures, radiolabeled folate transport","journal":"American journal of physiology. Cell physiology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — SCAM with substrate protection and temperature dependence tests, functional mutagenesis, multiple residues analyzed, single lab","pmids":["22785121"],"is_preprint":false},{"year":2012,"finding":"Ala335 residue contributes to PCFT protein stability; substitutions rendering bulky or charged residues at this site cause protein instability and loss of function. Gly338 residue is required for PCFT conformational oscillation (cycling between transport states); G338C mutant retains substrate binding (minimal change in Ki) but has 15–20-fold decreases in Kt and Vmax and is inaccessible to MTS reagents, consistent with TM9 location and impaired carrier cycling rather than substrate binding defect.","method":"Site-directed mutagenesis, radiolabeled pemetrexed and methotrexate influx kinetics, surface biotinylation, SCAM with MTS reagents, homology modeling","journal":"American journal of physiology. Cell physiology","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — systematic mutagenesis with kinetic analysis, accessibility studies, and structural modeling; mechanistic distinction between binding and cycling defects","pmids":["22843796"],"is_preprint":false},{"year":2013,"finding":"Bicarbonate at physiological concentrations produces potent and rapidly reversible inhibition of PCFT-mediated transport at neutral pH. Bisulfite and nitrite also inhibit PCFT, particularly at weakly acidic pH by decreasing Vmax and collapsing the transmembrane proton gradient. Sulfate, nitrate, and phosphate do not inhibit PCFT.","method":"Radiolabeled folate transport assays in PCFT-stable HeLa transfectants at varying anion concentrations and pH, intracellular pH measurements","journal":"Molecular pharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple anions tested systematically with transport kinetics and pH measurements, mechanistic link to proton gradient collapse established, single lab","pmids":["23609145"],"is_preprint":false},{"year":2013,"finding":"KLF4 is the primary transcriptional activator of PCFT in the small intestine; HNF4α synergistically enhances KLF4-induced PCFT transcription. CDX2 and C/EBPα suppress KLF4- and KLF4/HNF4α-induced PCFT promoter activity. The graded expression pattern of these factors along the intestinal tract accounts for the proximal-restricted expression of PCFT.","method":"Dual-luciferase reporter assays in HEK293 cells with cotransfection of transcription factors, Western blot for transcription factor expression along rat intestine","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — luciferase reporter assays with multiple factors, corroborated by expression profiling, but no ChIP or endogenous PCFT mRNA response to factor manipulation in intestinal cells","pmids":["23313509"],"is_preprint":false},{"year":2015,"finding":"Four Tyr residues (Y291, Y362, Y315, Y414) are extracellularly accessible in PCFT; Y291, Y362, and Y315 are located within or near the folate binding pocket. Substitution of these residues with Cys or Ala increases both influx Vmax and Kt/Ki (suggesting release from a high-affinity constrained state), while Phe substitution moderates these changes. Y315A PCFT shows increased Vmax but loss of transstimulation (exchange), indicating these Tyr residues constrain carrier mobility and secure the high-affinity substrate-binding state at the expense of oscillation rate.","method":"SCAM with MTSEA-biotin, site-directed mutagenesis, radiolabeled influx and efflux transport kinetics, transstimulation assays","journal":"American journal of physiology. Cell physiology","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — SCAM combined with mutagenesis, bidirectional transport kinetics and transstimulation mechanistic analysis, multiple Tyr residues analyzed, single lab","pmids":["25608532"],"is_preprint":false},{"year":2016,"finding":"Trp299 (W299) in the 4th external loop between TM7 and TM8 is required for PCFT function through its hydrophobicity: W299S mutation decreases Vmax 6.5-fold and alters Kt/Ki; only Phe and (partially) Ala substitutions preserve function, suggesting this residue interacts with the lipid membrane during the transport cycle and influences carrier oscillation and the folate binding pocket indirectly.","method":"SCAM with MTSEA-biotin, site-directed mutagenesis, radiolabeled pemetrexed transport kinetics, DTT treatment for disulfide accessibility","journal":"American journal of physiology. Cell physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — SCAM plus mutagenesis with kinetic analysis and substrate protection, single lab, mechanistic interpretation supported by accessibility data","pmids":["27251438"],"is_preprint":false},{"year":2017,"finding":"Purified human PCFT expressed in Sf9 insect cells via baculovirus retains folate transport function after reconstitution into liposomes, enabling biochemical and structural studies.","method":"Baculovirus/Sf9 expression, detergent purification, reconstitution into liposomes, radiolabeled folic acid transport assay","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — functional reconstitution of purified transporter demonstrated, but single lab and limited mechanistic follow-up in this paper","pmids":["28493963"],"is_preprint":false},{"year":2017,"finding":"The 8th transmembrane helix of PCFT defines the aqueous translocation pathway: 14 contiguous exofacial residues are accessible to MTSEA-biotin; pemetrexed blocks biotinylation of 6 deep residues implicating this region in folate binding. Pro314 and Tyr315 are critical for function; their substitution markedly increases Kt, Ki, and Vmax. Homology modeling (based on GLUT5 structures) predicts a helix break in the outward-open conformation at this region.","method":"SCAM with MTSEA-biotin, site-directed mutagenesis, substrate protection assay, temperature-dependence of accessibility, homology modeling based on GLUT5 structures","journal":"Biochimica et biophysica acta. Biomembranes","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — systematic SCAM of 23 residues combined with mutagenesis, substrate protection, and structural modeling; multiple orthogonal methods, single lab","pmids":["28802835"],"is_preprint":false},{"year":2017,"finding":"The 7th and 8th transmembrane helices of PCFT form an exofacial cleft defining the aqueous translocation pathway; 9 exofacial residues of TM7 are accessible. Paired cysteine substitutions between TM7 and TM8 (T289C-I304C and Q285C-Q311C) spontaneously form disulfide bonds, impairing accessibility and function; DTT restores both, establishing close proximity of these helices and their role in the substrate entry pathway.","method":"SCAM, dicysteine cross-linking with spontaneous disulfide bond formation, DTT reversal, homology modeling based on GLUT5 inward- and outward-open conformations","journal":"American journal of physiology. Cell physiology","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — cross-linking with functional reversal, SCAM, substrate protection, and structural modeling; direct demonstration of helix proximity with functional consequence, single lab","pmids":["29167151"],"is_preprint":false},{"year":2018,"finding":"Asn411 (N411) in TM11 is part of the external gate of PCFT; N411K mutation causes loss of function with a markedly decreased influx Vmax and reduced affinity for most folate substrates (especially 5-methyltetrahydrofolate). Positive-charged substitutions are most disruptive; negative (Asp) and bulky hydrophobic/polar substitutions are better tolerated. Homology modeling places N411 protruding into the aqueous pathway, most prominently in the inward-open conformation.","method":"Site-directed mutagenesis, surface biotinylation, radiolabeled folate transport kinetics, homology modeling based on GLUT5","journal":"Blood advances","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — systematic mutagenesis at single residue with multiple substitutions, kinetic analysis, surface expression controls, structural modeling; single lab","pmids":["29344585"],"is_preprint":false},{"year":2018,"finding":"PCFT generates substantial transmembrane electrochemical-potential gradients (concentrative transport) for antifolates at extracellular pH levels relevant to the tumor microenvironment (pH 6.9–7.0). Concentrative transport requires the transmembrane proton gradient; it is abolished by the protonophore CCCP and is not observed in bicarbonate/CO2-buffered medium at neutral pH.","method":"Radiolabeled methotrexate accumulation assays in PCFT-expressing HeLa cells, CCCP treatment, Na+/H+ exchanger inhibitors, intracellular pH measurement","journal":"Molecular pharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple pharmacological tools, intracellular pH measurement, mechanistic dissection of concentrative transport, single lab","pmids":["29326243"],"is_preprint":false},{"year":2019,"finding":"Asp109 (D109) in the first intracellular loop locks PCFT in an inward-open conformation when substituted (D109A); cysteine-substituted residues in the aqueous translocation pathway lose accessibility to MTSEA-biotin. Introduction of a second 'unlocking' substitution at Gly305 (G305L or bulky residues in TM8) in the D109A scaffold largely restores both function and aqueous accessibility, demonstrating that D109 is part of a 'motif A' (GXXXDXXGR) critical for carrier oscillation between conformational states.","method":"Site-directed mutagenesis, SCAM with MTSEA-biotin, functional transport assays, double-mutant epistasis analysis, homology modeling","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — double-mutant suppressor analysis combined with SCAM directly demonstrates conformational locking mechanism; multiple orthogonal methods, single lab","pmids":["30858177"],"is_preprint":false},{"year":2020,"finding":"SLC46A1 regulates hepatic iron metabolism by importing heme into hepatocytes; hepatocyte-specific inhibition of SLC46A1 by AAV decreases liver iron content, increases serum iron and free heme, and alters expression of iron-regulatory molecules (TfR1, hepcidin, ferroportin). In hepatocytes, SLC46A1 can import hemin, increasing intracellular iron content; hemin import is independent of folate transport, but hemin treatment decreases SLC46A1 expression and folate import.","method":"AAV-mediated hepatocyte-specific SLC46A1 knockdown in mice, hemin uptake assay in hepatocytes, measurement of intracellular iron content, Western blot for iron-regulatory proteins","journal":"Metabolism: clinical and experimental","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo AAV knockdown and in vitro hepatocyte assays with direct iron content measurement, multiple downstream markers assessed, single lab","pmids":["32621820"],"is_preprint":false},{"year":2025,"finding":"Molecular dynamics simulations based on cryo-EM structure of Gallus gallus PCFT (ortholog of hPCFT) reveal that HFM-causing mutations cause partial loss of structural integrity manifested as enlarged/distorted pore, loss of long-range contacts, less stable inner helices with reduced solvent accessibility, and loss of secondary structure; these structural changes are reversed by compensatory mutations that restore function.","method":"Molecular dynamics simulations of hPCFT homology model based on cryo-EM structure of Gallus gallus PCFT, structural analysis of disease-causing and compensatory mutations","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 (structural/computational) / Weak — based on MD simulations from a cryo-EM ortholog structure, not direct experimental structure of hPCFT; provides mechanistic correlations with published kinetic data but is primarily computational","pmids":["39924111"],"is_preprint":false},{"year":2008,"finding":"PCFT/HCP1 transports both folate and heme, but with lower affinity for heme than folate; siRNA knockdown of PCFT/HCP1 in Caco-2 cells reduces both folate uptake (48%) and heme uptake (22.5%), with greater impact on folate. Folic acid inhibits heme transport in hypoxic but not normal mice in vivo.","method":"siRNA knockdown in Caco-2 cells, radiolabeled folate and 59Fe-heme uptake assays, in vivo mouse intestinal transport studies with blocking antibody, everted duodenum preparations","journal":"The British journal of nutrition","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — siRNA knockdown with direct uptake measurements, in vitro and in vivo approaches, but interpretation of heme transport is contested in the field; moderate confidence","pmids":["18782461"],"is_preprint":false},{"year":2022,"finding":"Loss of SLC46A1 expression in hepatocellular carcinoma (HCC) cells causes iron deficiency in tumor tissue; only SLC46A1 (not STEAP3 or DMT1) silencing or overexpression controlled intracellular iron content in HCC cells. Lentivirus-mediated re-expression of SLC46A1 in orthotopic tumors restores tumor iron content with corresponding changes in iron-metabolic molecules.","method":"Lentiviral SLC46A1 overexpression and siRNA knockdown in HCC cell lines, orthotopic tumor implantation in mice, intracellular iron content measurement, Western blot for iron-metabolic molecules","journal":"Hepatology communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro and in vivo gain/loss-of-function with direct iron content measurement, comparison with other iron transporters, single lab","pmids":["35811443"],"is_preprint":false},{"year":2026,"finding":"SLC46A1-mediated folate uptake in colorectal cancer cells suppresses tumor proliferation, migration, and invasion; mechanistically, SLC46A1 deficiency reduces intracellular folate availability, impairs cellular methylation potential (decreased SAM/SAH ratio), causes DNA hypomethylation at the FOS proto-oncogene promoter, and transcriptionally activates CCND1, BCL2, and PLAU oncogenic effectors driving CRC progression.","method":"SLC46A1 loss/gain-of-function in vitro and in vivo, SAM/SAH ratio measurement, bisulfite sequencing/methylation analysis, ChIP for promoter methylation, transcriptomic analysis, tumor growth assays","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal mechanistic methods (methylation assay, metabolite measurement, gene expression), in vitro and in vivo validation, single lab","pmids":["41620398"],"is_preprint":false}],"current_model":"SLC46A1 (PCFT) is an electrogenic proton symporter with 12 transmembrane domains and intracellular N/C termini that mediates intestinal folate absorption and choroid plexus folate transport by coupling inwardly directed proton gradients to uphill folate influx at low pH; key functional residues include D109 (conformational gating), H247/H281 (proton coupling and binding), E185 (rate-limiting proton-folate coupling step), R376 (proton binding modulating the folate pocket), and a GxxG motif (TM5) plus TM7–TM8 exofacial cleft defining the substrate translocation pathway; the transporter also functions in FRα-mediated endosomal folate export, hepatic heme/iron import, and its expression is transcriptionally regulated by NRF-1, KLF4, HNF4α, CDX2, and C/EBPα, and silenced by CpG promoter hypermethylation."},"narrative":{"mechanistic_narrative":"SLC46A1 (PCFT) is an electrogenic, proton-coupled folate transporter that mediates intestinal folate absorption at the apical brush-border membrane of the proximal small intestine, operating optimally at low pH by coupling an inwardly directed proton gradient to uphill folate influx [PMID:17898134, PMID:18174275, PMID:17340171]. Genetic knockout in mice confirms its nonredundant in vivo role: PCFT-deficient animals develop severe systemic folate deficiency with macrocytic anemia and impaired erythropoiesis [PMID:21346251], and loss-of-function mutations cause hereditary folate malabsorption through defects in protein stability, membrane trafficking, or transport activity [PMID:17446347]. The transporter is a 12-transmembrane-domain polytopic membrane protein with cytoplasmic N- and C-termini and is N-glycosylated at two extracellular asparagines that are dispensable for transport [PMID:18405659, PMID:20225891]. Extensive structure-function mapping defines a substrate translocation pathway formed by the seventh and eighth transmembrane helices, which form a closely apposed exofacial cleft, with TM8 lining the aqueous pore and contributing folate-binding residues [PMID:28802835, PMID:29167151]; proton coupling and binding depend on His247, His281, Glu185, and Arg376, the latter modulating the folate-binding pocket and carrier conformational change [PMID:19389703, PMID:19403800, PMID:20686069]. Conformational cycling between inward- and outward-open states is governed by Asp109 within a 'motif A' (GXXXDXXGR), whose substitution locks the carrier inward-open in a manner relievable by compensatory unlocking mutations [PMID:30858177], with additional residues (a TM5 GxxG motif, Gly338, Pro314/Tyr315, Asn411) constraining substrate binding or oscillation rate [PMID:22785121, PMID:22843796, PMID:28802835, PMID:29344585]. Beyond folate, SLC46A1 imports heme to regulate hepatic and tumor iron homeostasis in a folate-transport-independent manner [PMID:32621820, PMID:35811443], and its folate-dependent control of cellular methylation potential modulates oncogene promoter methylation in colorectal cancer [PMID:41620398]. Expression is transcriptionally driven by NRF-1 and by intestinal KLF4/HNF4α (antagonized by CDX2 and C/EBPα), and is silenced by CpG promoter hypermethylation in antifolate-resistant cells [PMID:20724482, PMID:23313509, PMID:19671745].","teleology":[{"year":2007,"claim":"Established the molecular identity and core mechanism of intestinal folate uptake, answering how dietary folate crosses the acidic apical surface of the gut.","evidence":"Xenopus oocyte electrophysiology, radiolabeled folate uptake, and brush-border membrane fractionation","pmids":["17898134","18174275","17340171"],"confidence":"High","gaps":["Atomic structure of the human transporter not resolved","Stoichiometry of proton:folate coupling not fully quantified here"]},{"year":2007,"claim":"Linked the transporter to human disease by showing PCFT mutations cause hereditary folate malabsorption, validating physiological essentiality in humans.","evidence":"Patient gene sequencing plus mutant transport and protein-stability assays in HeLa cells","pmids":["17446347"],"confidence":"High","gaps":["Distinguishing trafficking from catalytic defects per mutation incompletely resolved","Genotype-phenotype correlation across mutations not systematic"]},{"year":2008,"claim":"Defined a second cellular role as the endosomal folate exporter downstream of FRalpha, explaining how receptor-internalized folate reaches the cytosol.","evidence":"Isogenic PCFT(+/-) HeLa lines with FRalpha cotransfection, co-localization, and probenecid inhibition","pmids":["19074442"],"confidence":"High","gaps":["Direct demonstration of endosomal acidification driving export not isolated","Single-lab finding"]},{"year":2008,"claim":"Raised the possibility of broader substrate range by showing the same protein (HCP1) transports heme as well as folate, though with lower affinity.","evidence":"siRNA knockdown in Caco-2 cells with radiolabeled folate and 59Fe-heme uptake plus in vivo intestinal studies","pmids":["18782461"],"confidence":"Medium","gaps":["Physiological significance of intestinal heme transport contested in the field","Heme uptake reduction modest (22.5%)"]},{"year":2008,"claim":"Resolved membrane topology and post-translational modification, fixing the structural framework (12 TMs, cytoplasmic termini, two N-glycans) for later residue mapping.","evidence":"PNGase F/tunicamycin treatment, glycosylation-site mutagenesis, and HA-tag accessibility in permeabilized cells","pmids":["18405659"],"confidence":"High","gaps":["Function of glycosylation beyond transport not established","Topology inferred biochemically, not from a structure"]},{"year":2009,"claim":"Identified the proton-coupling machinery by showing His247/His281 and Glu185 govern proton handling and proton-folate coupling at the rate-limiting step.","evidence":"Site-directed mutagenesis with influx kinetics, oocyte electrophysiology, trans-stimulation, and homology modeling","pmids":["19389703","19403800"],"confidence":"High","gaps":["Exact protonation order across the cycle not determined","Residue roles inferred from mutagenesis without a structure"]},{"year":2009,"claim":"Explained loss of transporter expression in antifolate resistance through CpG promoter hypermethylation, defining an epigenetic silencing mechanism.","evidence":"Bisulfite sequencing, luciferase reporters, 5-aza-dC reactivation, in vitro reporter methylation, and FISH","pmids":["19671745"],"confidence":"High","gaps":["Generality of methylation silencing across tumors not assessed here","Single resistant cell model"]},{"year":2010,"claim":"Mapped residues controlling protein stability versus catalysis (Asp156 stability; Asp109 absolutely required despite normal surface expression) and identified Arg376 in proton binding.","evidence":"Systematic mutagenesis with surface biotinylation, transport kinetics across pH, and oocyte electrophysiology","pmids":["20805364","20686069"],"confidence":"High","gaps":["Mechanism by which D109 enables catalysis not yet defined at this stage","Structural placement inferred from homology models"]},{"year":2010,"claim":"Identified NRF-1 as a major transcriptional driver, beginning the dissection of how PCFT expression is set.","evidence":"EMSA/supershift, ChIP, reporter mutagenesis, and NRF-1 gain/loss-of-function","pmids":["20724482"],"confidence":"High","gaps":["Tissue specificity of NRF-1 control not addressed","Single lab"]},{"year":2010,"claim":"Confirmed the canonical 12-TM topology with cytoplasmic termini and identified a non-essential extracellular disulfide bridge.","evidence":"Substituted cysteine accessibility (SCAM) with MTSEA-biotin and cysteine-less scaffolds","pmids":["20225891"],"confidence":"High","gaps":["Functional role of the disulfide bond unclear","Conformational dynamics not captured"]},{"year":2011,"claim":"Provided definitive in vivo proof of nonredundant intestinal folate transport via a knockout mouse recapitulating human HFM hematology.","evidence":"Targeted exon 1-3 disruption with in vivo folate uptake and hematological/bone-marrow phenotyping","pmids":["21346251"],"confidence":"High","gaps":["Choroid plexus/CNS folate phenotype not detailed here","Heme-transport role not tested in this model"]},{"year":2012,"claim":"Began mapping the substrate translocation pathway, identifying a TM5 GxxG motif and folate-pocket residues accessible to the aqueous pathway.","evidence":"SCAM with membrane-impermeable reagents, substrate protection at varied temperature, and mutagenesis","pmids":["22785121"],"confidence":"High","gaps":["Full pathway not yet delineated at this stage","Pocket geometry inferred indirectly"]},{"year":2012,"claim":"Distinguished stability determinants from conformational-cycling determinants, showing Gly338 supports carrier oscillation independent of substrate binding.","evidence":"Mutagenesis with pemetrexed/methotrexate kinetics, surface biotinylation, SCAM, and homology modeling","pmids":["22843796"],"confidence":"High","gaps":["Direct visualization of conformational states absent","Single lab"]},{"year":2013,"claim":"Clarified transcriptional control of the proximal-restricted intestinal expression pattern through KLF4/HNF4alpha activation antagonized by CDX2 and C/EBPalpha.","evidence":"Dual-luciferase cotransfection in HEK293 with transcription-factor expression profiling along rat intestine","pmids":["23313509"],"confidence":"Medium","gaps":["No ChIP confirming direct promoter occupancy","Endogenous intestinal PCFT mRNA response to factor manipulation not shown"]},{"year":2013,"claim":"Defined physiological anion inhibition (bicarbonate, bisulfite, nitrite) acting by collapsing the proton gradient, constraining where PCFT operates.","evidence":"Transport kinetics across anions and pH with intracellular pH measurement","pmids":["23609145"],"confidence":"Medium","gaps":["Whether anions bind the protein versus only dissipate the gradient not fully separated","Single lab"]},{"year":2017,"claim":"Advanced biochemical tractability by demonstrating purified human PCFT retains transport after reconstitution into liposomes.","evidence":"Baculovirus/Sf9 expression, detergent purification, proteoliposome folate transport assay","pmids":["28493963"],"confidence":"Medium","gaps":["No structure obtained from the reconstituted protein here","Limited mechanistic follow-up"]},{"year":2017,"claim":"Localized the aqueous translocation pathway to TM8 and showed TM7-TM8 form a close exofacial cleft constituting the substrate entry route.","evidence":"SCAM, substrate protection, dicysteine cross-linking with DTT reversal, and GLUT5-based homology modeling","pmids":["28802835","29167151"],"confidence":"High","gaps":["Inward-facing pathway less defined","Helix proximity inferred for specific conformations only"]},{"year":2015,"claim":"Showed extracellular Tyr residues near the folate pocket constrain carrier mobility to secure a high-affinity binding state at the expense of oscillation rate.","evidence":"SCAM plus mutagenesis with bidirectional influx/efflux kinetics and transstimulation","pmids":["25608532"],"confidence":"High","gaps":["Trade-off mechanism between affinity and turnover not structurally resolved","Single lab"]},{"year":2018,"claim":"Demonstrated concentrative antifolate transport at tumor-microenvironment pH, establishing pharmacological relevance for antifolate delivery to tumors.","evidence":"Methotrexate accumulation in PCFT-HeLa cells with CCCP, NHE inhibitors, and intracellular pH measurement","pmids":["29326243"],"confidence":"Medium","gaps":["In vivo tumor accumulation not tested here","Single lab"]},{"year":2018,"claim":"Identified Asn411 in TM11 as part of the external gate shaping substrate affinity, particularly for 5-methyltetrahydrofolate.","evidence":"Multi-substitution mutagenesis with kinetics, surface biotinylation, and homology modeling","pmids":["29344585"],"confidence":"High","gaps":["Gate dynamics inferred from modeling, not direct structure","Single lab"]},{"year":2019,"claim":"Defined the conformational-switch mechanism by showing Asp109 of motif A locks the inward-open state, relievable by a compensatory TM8 unlocking substitution.","evidence":"Double-mutant suppressor (epistasis) analysis combined with SCAM and homology modeling","pmids":["30858177"],"confidence":"High","gaps":["Energetics of the conformational transition not measured","No experimental structure of the two states"]},{"year":2020,"claim":"Established a folate-independent heme-import function regulating hepatic iron homeostasis, expanding PCFT's physiological role.","evidence":"AAV-mediated hepatocyte-specific knockdown in mice and hemin uptake/iron-content assays in hepatocytes","pmids":["32621820"],"confidence":"Medium","gaps":["Heme-binding site on the transporter not identified","Single lab"]},{"year":2022,"claim":"Showed SLC46A1 controls tumor iron content in hepatocellular carcinoma, uniquely among tested iron transporters.","evidence":"Lentiviral overexpression/siRNA in HCC lines plus orthotopic tumors with iron-content measurement","pmids":["35811443"],"confidence":"Medium","gaps":["Mechanistic link from iron control to tumor behavior not fully traced","Single lab"]},{"year":2025,"claim":"Provided a structural rationale for HFM mutations by simulating an ortholog cryo-EM-based model, correlating pore distortion and lost contacts with loss of function.","evidence":"Molecular dynamics of an hPCFT homology model from the Gallus gallus PCFT cryo-EM structure","pmids":["39924111"],"confidence":"Medium","gaps":["Computational, not an experimental human structure","Predictions await direct structural validation"]},{"year":2026,"claim":"Connected folate transport to epigenetic oncogene regulation, showing SLC46A1-supplied folate maintains methylation potential that suppresses colorectal cancer progression.","evidence":"Loss/gain-of-function in vitro and in vivo with SAM/SAH measurement, promoter methylation/ChIP, and transcriptomics","pmids":["41620398"],"confidence":"Medium","gaps":["Direct causality between FOS promoter hypomethylation and the full phenotype not isolated","Single lab"]},{"year":null,"claim":"An experimentally determined high-resolution structure of human PCFT in defined conformational states, and a unified model reconciling its folate, heme, and choroid plexus transport activities, remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No experimental human PCFT structure; mechanism rests on homology models","Heme-binding determinants unmapped","Choroid plexus folate transport role not directly characterized in the corpus"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005215","term_label":"transporter activity","supporting_discovery_ids":[0,2,11,19,25]},{"term_id":"GO:0140104","term_label":"molecular carrier activity","supporting_discovery_ids":[0,23,25]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,12]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[2]}],"pathway":[{"term_id":"R-HSA-382551","term_label":"Transport of small molecules","supporting_discovery_ids":[0,11,25]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[0,25,28]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[1,11]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[9,16,5]}],"complexes":[],"partners":["FOLR1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q96NT5","full_name":"Proton-coupled folate transporter","aliases":["Heme carrier protein 1","PCFT/HCP1","Solute carrier family 46 member 1"],"length_aa":459,"mass_kda":49.8,"function":"Proton-coupled folate symporter that mediates folate absorption using an H(+) gradient as a driving force (PubMed:17129779, PubMed:17446347, PubMed:17475902, PubMed:19389703, PubMed:19762432, PubMed:25504888, PubMed:29344585, PubMed:30858177, PubMed:31494288, PubMed:31792273, PubMed:32893190, PubMed:34619546). Involved in the intestinal absorption of folates at the brush-border membrane of the proximal jejunum, and the transport from blood to cerebrospinal fluid across the choroid plexus (PubMed:17129779, PubMed:17446347, PubMed:17475902, PubMed:19389703, PubMed:25504888, PubMed:29344585, PubMed:30858177, PubMed:31494288, PubMed:32893190). Functions at acidic pH via alternate outward- and inward-open conformation states (PubMed:32893190, PubMed:34040256). Protonation of residues in the outward open state primes the protein for transport (PubMed:34040256). Binding of folate promotes breaking of salt bridge network and subsequent closure of the extracellular gate, leading to the inward-open state and release of protons and folate (PubMed:34040256). Also able to transport antifolate drugs, such as methotrexate and pemetrexed, which are established treatments for cancer and autoimmune diseases (PubMed:18524888, PubMed:19762432, PubMed:22345511, PubMed:25608532, PubMed:28802835, PubMed:29326243, PubMed:34040256, PubMed:34619546). Involved in FOLR1-mediated endocytosis by serving as a route of export of folates from acidified endosomes (PubMed:19074442). Also acts as a lower-affinity, pH-independent heme carrier protein and constitutes the main importer of heme in the intestine (PubMed:17156779). Imports heme in the retina and retinal pigment epithelium, in neurons of the hippocampus, in hepatocytes and in the renal epithelial cells (PubMed:32621820). Hence, participates in the trafficking of heme and increases intracellular iron content (PubMed:32621820) Inactive isoform which is not able to mediate proton-coupled folate transport","subcellular_location":"Cell membrane; Apical cell membrane; Basolateral cell membrane; Endosome membrane; Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q96NT5/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/SLC46A1","classification":"Not Classified","n_dependent_lines":4,"n_total_lines":74,"dependency_fraction":0.05405405405405406},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000076351","cell_line_id":"CID001380","localizations":[{"compartment":"membrane","grade":3},{"compartment":"vesicles","grade":3}],"interactors":[{"gene":"EXO1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID001380","total_profiled":1310},"omim":[{"mim_id":"611672","title":"SOLUTE CARRIER FAMILY 46 (FOLATE TRANSPORTER), MEMBER 1; SLC46A1","url":"https://www.omim.org/entry/611672"},{"mim_id":"229050","title":"FOLATE MALABSORPTION, HEREDITARY","url":"https://www.omim.org/entry/229050"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Plasma membrane","reliability":"Supported"},{"location":"Cytosol","reliability":"Supported"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"intestine","ntpm":88.2}],"url":"https://www.proteinatlas.org/search/SLC46A1"},"hgnc":{"alias_symbol":["HCP1","MGC9564","PCFT","HsPCFT","hPCFT"],"prev_symbol":[]},"alphafold":{"accession":"Q96NT5","domains":[{"cath_id":"1.20.1250.20","chopping":"20-57_73-231","consensus_level":"medium","plddt":89.9705,"start":20,"end":231},{"cath_id":"1.20.1250.20","chopping":"253-447","consensus_level":"medium","plddt":87.8639,"start":253,"end":447}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96NT5","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96NT5-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96NT5-F1-predicted_aligned_error_v6.png","plddt_mean":83.06},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SLC46A1","jax_strain_url":"https://www.jax.org/strain/search?query=SLC46A1"},"sequence":{"accession":"Q96NT5","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96NT5.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96NT5/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96NT5"}},"corpus_meta":[{"pmid":"19074442","id":"PMC_19074442","title":"A role for the proton-coupled folate transporter (PCFT-SLC46A1) in folate receptor-mediated endocytosis.","date":"2008","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/19074442","citation_count":122,"is_preprint":false},{"pmid":"17446347","id":"PMC_17446347","title":"The spectrum of mutations in the PCFT gene, coding for an intestinal folate transporter, that are the basis for hereditary folate malabsorption.","date":"2007","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/17446347","citation_count":120,"is_preprint":false},{"pmid":"17898134","id":"PMC_17898134","title":"Rodent intestinal folate transporters (SLC46A1): secondary structure, functional properties, and response to dietary folate restriction.","date":"2007","source":"American journal of physiology. Cell physiology","url":"https://pubmed.ncbi.nlm.nih.gov/17898134","citation_count":108,"is_preprint":false},{"pmid":"17340171","id":"PMC_17340171","title":"The molecular identity and characterization of a Proton-coupled Folate Transporter--PCFT; biological ramifications and impact on the activity of pemetrexed.","date":"2007","source":"Cancer metastasis reviews","url":"https://pubmed.ncbi.nlm.nih.gov/17340171","citation_count":89,"is_preprint":false},{"pmid":"18174275","id":"PMC_18174275","title":"Functional characterization of PCFT/HCP1 as the molecular entity of the carrier-mediated intestinal folate transport system in the rat model.","date":"2008","source":"American journal of physiology. Gastrointestinal and liver physiology","url":"https://pubmed.ncbi.nlm.nih.gov/18174275","citation_count":80,"is_preprint":false},{"pmid":"18405659","id":"PMC_18405659","title":"N-linked glycosylation and its impact on the electrophoretic mobility and function of the human proton-coupled folate transporter (HsPCFT).","date":"2008","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/18405659","citation_count":73,"is_preprint":false},{"pmid":"27664775","id":"PMC_27664775","title":"The proton-coupled folate transporter (PCFT-SLC46A1) and the syndrome of systemic and cerebral folate deficiency of infancy: Hereditary folate malabsorption.","date":"2016","source":"Molecular aspects of medicine","url":"https://pubmed.ncbi.nlm.nih.gov/27664775","citation_count":61,"is_preprint":false},{"pmid":"18782461","id":"PMC_18782461","title":"Haem and folate transport by proton-coupled folate transporter/haem carrier protein 1 (SLC46A1).","date":"2008","source":"The British journal of nutrition","url":"https://pubmed.ncbi.nlm.nih.gov/18782461","citation_count":56,"is_preprint":false},{"pmid":"19389703","id":"PMC_19389703","title":"The functional roles of the His247 and His281 residues in folate and proton translocation mediated by the human proton-coupled folate transporter SLC46A1.","date":"2009","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/19389703","citation_count":52,"is_preprint":false},{"pmid":"19671745","id":"PMC_19671745","title":"Hypermethylation of the human proton-coupled folate transporter (SLC46A1) minimal transcriptional regulatory region in an antifolate-resistant HeLa cell line.","date":"2009","source":"Molecular cancer therapeutics","url":"https://pubmed.ncbi.nlm.nih.gov/19671745","citation_count":52,"is_preprint":false},{"pmid":"21346251","id":"PMC_21346251","title":"A mouse model of hereditary folate malabsorption: deletion of the PCFT gene leads to systemic folate deficiency.","date":"2011","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/21346251","citation_count":48,"is_preprint":false},{"pmid":"20805364","id":"PMC_20805364","title":"Functional roles of aspartate residues of the proton-coupled folate transporter (PCFT-SLC46A1); a D156Y mutation causing hereditary folate malabsorption.","date":"2010","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/20805364","citation_count":46,"is_preprint":false},{"pmid":"18718264","id":"PMC_18718264","title":"The clinical course and genetic defect in the PCFT gene in a 27-year-old woman with hereditary folate malabsorption.","date":"2008","source":"The Journal of pediatrics","url":"https://pubmed.ncbi.nlm.nih.gov/18718264","citation_count":44,"is_preprint":false},{"pmid":"19403800","id":"PMC_19403800","title":"Role of the glutamate 185 residue in proton translocation mediated by the proton-coupled folate transporter SLC46A1.","date":"2009","source":"American journal of physiology. Cell physiology","url":"https://pubmed.ncbi.nlm.nih.gov/19403800","citation_count":43,"is_preprint":false},{"pmid":"20225891","id":"PMC_20225891","title":"Membrane topological analysis of the proton-coupled folate transporter (PCFT-SLC46A1) by the substituted cysteine accessibility method.","date":"2010","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/20225891","citation_count":43,"is_preprint":false},{"pmid":"19762432","id":"PMC_19762432","title":"The human proton-coupled folate transporter (hPCFT): modulation of intestinal expression and function by drugs.","date":"2009","source":"American journal of physiology. Gastrointestinal and liver physiology","url":"https://pubmed.ncbi.nlm.nih.gov/19762432","citation_count":41,"is_preprint":false},{"pmid":"20686069","id":"PMC_20686069","title":"Properties of the Arg376 residue of the proton-coupled folate transporter (PCFT-SLC46A1) and a glutamine mutant causing hereditary folate malabsorption.","date":"2010","source":"American journal of physiology. Cell physiology","url":"https://pubmed.ncbi.nlm.nih.gov/20686069","citation_count":38,"is_preprint":false},{"pmid":"20724482","id":"PMC_20724482","title":"The obligatory intestinal folate transporter PCFT (SLC46A1) is regulated by nuclear respiratory factor 1.","date":"2010","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/20724482","citation_count":38,"is_preprint":false},{"pmid":"22785121","id":"PMC_22785121","title":"Identification of a functionally critical GXXG motif and its relationship to the folate binding site of the proton-coupled folate transporter (PCFT-SLC46A1).","date":"2012","source":"American journal of physiology. Cell physiology","url":"https://pubmed.ncbi.nlm.nih.gov/22785121","citation_count":29,"is_preprint":false},{"pmid":"21069807","id":"PMC_21069807","title":"Reduced levels of folate transporters (PCFT and RFC) in membrane lipid rafts result in colonic folate malabsorption in chronic alcoholism.","date":"2011","source":"Journal of cellular physiology","url":"https://pubmed.ncbi.nlm.nih.gov/21069807","citation_count":28,"is_preprint":false},{"pmid":"23212123","id":"PMC_23212123","title":"Functional regulation of P-glycoprotein at the blood-brain barrier in proton-coupled folate transporter (PCFT) mutant mice.","date":"2012","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/23212123","citation_count":28,"is_preprint":false},{"pmid":"23656756","id":"PMC_23656756","title":"Single nucleotide polymorphisms in CETP, SLC46A1, SLC19A1, CD36, BCMO1, APOA5, and ABCA1 are significant predictors of plasma HDL in healthy adults.","date":"2013","source":"Lipids in health and disease","url":"https://pubmed.ncbi.nlm.nih.gov/23656756","citation_count":27,"is_preprint":false},{"pmid":"20005757","id":"PMC_20005757","title":"A novel PCFT gene mutation (p.Cys66LeufsX99) causing hereditary folate malabsorption.","date":"2009","source":"Molecular genetics and metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/20005757","citation_count":26,"is_preprint":false},{"pmid":"32621820","id":"PMC_32621820","title":"SLC46A1 contributes to hepatic iron metabolism by importing heme in hepatocytes.","date":"2020","source":"Metabolism: clinical and experimental","url":"https://pubmed.ncbi.nlm.nih.gov/32621820","citation_count":23,"is_preprint":false},{"pmid":"20795774","id":"PMC_20795774","title":"Mutation of the proton-coupled folate transporter gene (PCFT-SLC46A1) in Turkish siblings with hereditary folate malabsorption.","date":"2010","source":"Pediatric hematology and oncology","url":"https://pubmed.ncbi.nlm.nih.gov/20795774","citation_count":23,"is_preprint":false},{"pmid":"35811443","id":"PMC_35811443","title":"Loss of SLC46A1 decreases tumor iron content in hepatocellular carcinoma.","date":"2022","source":"Hepatology communications","url":"https://pubmed.ncbi.nlm.nih.gov/35811443","citation_count":22,"is_preprint":false},{"pmid":"26006721","id":"PMC_26006721","title":"CSF 5-Methyltetrahydrofolate Serial Monitoring to Guide Treatment of Congenital Folate Malabsorption Due to Proton-Coupled Folate Transporter (PCFT) Deficiency.","date":"2015","source":"JIMD reports","url":"https://pubmed.ncbi.nlm.nih.gov/26006721","citation_count":22,"is_preprint":false},{"pmid":"21602279","id":"PMC_21602279","title":"Random mutagenesis of the proton-coupled folate transporter (SLC46A1), clustering of mutations, and the bases for associated losses of function.","date":"2011","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/21602279","citation_count":18,"is_preprint":false},{"pmid":"23313509","id":"PMC_23313509","title":"Transcriptional regulation of PCFT by KLF4, HNF4α, CDX2 and C/EBPα: implication in its site-specific expression in the small intestine.","date":"2013","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/23313509","citation_count":17,"is_preprint":false},{"pmid":"25608532","id":"PMC_25608532","title":"Identification of Tyr residues that enhance folate substrate binding and constrain oscillation of the proton-coupled folate transporter (PCFT-SLC46A1).","date":"2015","source":"American journal of physiology. 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Cell physiology","url":"https://pubmed.ncbi.nlm.nih.gov/27251438","citation_count":6,"is_preprint":false},{"pmid":"29167151","id":"PMC_29167151","title":"Substituted-cysteine accessibility and cross-linking identify an exofacial cleft in the 7th and 8th helices of the proton-coupled folate transporter (SLC46A1).","date":"2017","source":"American journal of physiology. Cell physiology","url":"https://pubmed.ncbi.nlm.nih.gov/29167151","citation_count":6,"is_preprint":false},{"pmid":"29326243","id":"PMC_29326243","title":"Concentrative Transport of Antifolates Mediated by the Proton-Coupled Folate Transporter (SLC46A1); Augmentation by a HEPES Buffer.","date":"2018","source":"Molecular pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/29326243","citation_count":6,"is_preprint":false},{"pmid":"26789141","id":"PMC_26789141","title":"Evaluation of proton-coupled folate transporter (SLC46A1) polymorphisms as risk factors for neural tube defects and oral clefts.","date":"2016","source":"American journal of medical genetics. Part A","url":"https://pubmed.ncbi.nlm.nih.gov/26789141","citation_count":5,"is_preprint":false},{"pmid":"22461784","id":"PMC_22461784","title":"Regular Multivitamin Supplement Use, Single Nucleotide Polymorphisms in ATIC, SHMT2, and SLC46A1, and Risk of Ovarian Carcinoma.","date":"2012","source":"Frontiers in genetics","url":"https://pubmed.ncbi.nlm.nih.gov/22461784","citation_count":4,"is_preprint":false},{"pmid":"40360429","id":"PMC_40360429","title":"PCFT-Independent Cellular Uptake of Cyclic Cell-Penetrating Peptide-Conjugated Folic Acid.","date":"2025","source":"Chembiochem : a European journal of chemical biology","url":"https://pubmed.ncbi.nlm.nih.gov/40360429","citation_count":1,"is_preprint":false},{"pmid":"36045837","id":"PMC_36045837","title":"Hereditary Folate Malabsorption presenting as neutropenic fever in a newborn from the first Palestinian family with the novel SLC46A1-mutation, A-case-report.","date":"2022","source":"Annals of medicine and surgery (2012)","url":"https://pubmed.ncbi.nlm.nih.gov/36045837","citation_count":1,"is_preprint":false},{"pmid":"41620398","id":"PMC_41620398","title":"SLC46A1 deficiency-mediated folate restriction suppresses colorectal cancer progression through epigenetic-transcriptional reprogramming.","date":"2026","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/41620398","citation_count":0,"is_preprint":false},{"pmid":"39924111","id":"PMC_39924111","title":"Mechanistic insights into mutation in the proton-coupled folate transporter (SLC46A1) causing hereditary folate malabsorption.","date":"2025","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/39924111","citation_count":0,"is_preprint":false},{"pmid":"41459184","id":"PMC_41459184","title":"A Rare Combination of Hereditary Folate Malabsorption (SLC46A1 Gene Variant) and Beta-Thalassemia Trait.","date":"2025","source":"EJIFCC","url":"https://pubmed.ncbi.nlm.nih.gov/41459184","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.10.28.685153","title":"Combined Metabolic and Viral Insults in Pregnancy Disrupt Specific Placental Nutrient Transporter Systems in Mice","date":"2025-10-30","source":"bioRxiv","url":"https://doi.org/10.1101/2025.10.28.685153","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":30317,"output_tokens":8441,"usd":0.108783,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":18117,"output_tokens":6225,"usd":0.123105,"stage2_stop_reason":"end_turn"},"total_usd":0.231888,"stage1_batch_id":"msgbatch_013LWZZT6MWaqB9qyTSeQBmU","stage2_batch_id":"msgbatch_0194ZzQaTGNpruoNJBbEMbt3","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2007,\n      \"finding\": \"PCFT (SLC46A1) is an electrogenic, proton-coupled folate transporter with optimal transport activity at low pH (pH 5.5), mediating intestinal folate absorption; transport is pH-dependent with increasing Km and decreasing Vmax as pH rises toward neutral, and transport in Xenopus oocytes is electrogenic and pH-dependent.\",\n      \"method\": \"Xenopus oocyte expression with electrophysiology, radiolabeled folate uptake assays, tissue fractionation showing apical brush-border membrane localization in duodenum/proximal jejunum\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — electrophysiological reconstitution in oocytes, radiolabeled transport assays, membrane localization by fractionation, replicated across multiple labs and organisms\",\n      \"pmids\": [\"17898134\", \"18174275\", \"17340171\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Loss-of-function mutations in PCFT (SLC46A1) are the molecular basis for hereditary folate malabsorption (HFM); mutations cause decreased protein stability, defects in membrane trafficking, or loss of transport function, with some mutants retaining residual activity.\",\n      \"method\": \"Sequencing of PCFT gene in HFM patients, transient transfection of mutant constructs in HeLa cells with transport assays, Western blot for protein stability, membrane trafficking assessment\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple mutations characterized across multiple patients, multiple orthogonal methods (sequencing, functional assays, protein stability), independently replicated across labs\",\n      \"pmids\": [\"17446347\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"PCFT co-localizes with folate receptor alpha (FRα) in the endosomal compartment and plays a functional role in FRα-mediated endocytosis by serving as the route of export of folates from acidified endosomes into the cytosol; probenecid inhibits PCFT-mediated transport at endosomal pH and blocks FRα-mediated folate delivery to the cytosol.\",\n      \"method\": \"FRα cDNA transfection into PCFT(+) and PCFT(−) HeLa sublines, radiolabeled folate transport assays, immunofluorescence co-localization, probenecid inhibition experiments\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — isogenic PCFT(+/−) cell lines with cotransfection, multiple orthogonal methods (transport assay, co-localization, pharmacological inhibition), single lab\",\n      \"pmids\": [\"19074442\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"PCFT (SLC46A1) is N-glycosylated at two canonical asparagine residues; the protein migrates at ~55 kDa on SDS-PAGE (predicted ~50 kDa) due to glycosylation, collapses to ~35 kDa after deglycosylation. Single glycosylation-site mutants retain full transport activity; the double deglycosylation mutant retains a majority of activity. The N- and C-termini are intracellular (accessible only in permeabilized cells).\",\n      \"method\": \"PNGase F treatment, tunicamycin treatment, site-directed mutagenesis of glycosylation sites (Asn→Gln), Western blot, HA-tag accessibility assay in permeabilized vs. intact cells\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — multiple orthogonal biochemical methods (enzymatic deglycosylation, drug inhibition, mutagenesis, topology probing), single lab\",\n      \"pmids\": [\"18405659\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"His247 and His281 residues are functionally critical for PCFT (SLC46A1). His247 (located in the large loop between TM6 and TM7 at the cytoplasmic entrance, in hydrogen-bond distance to Ser172) is required for substrate selectivity and coupled proton transport; H247A mutation increases proton 'slippage' (folate-independent proton transport) and reduces substrate specificity. His281 (in TM7, extracellular face) facilitates proton binding/protonation that augments folate binding; H281A increases folic acid influx Kt ~12-fold but does not abolish proton coupling.\",\n      \"method\": \"Site-directed mutagenesis, radiolabeled folate influx kinetics in transfected HeLa cells, electrophysiology in Xenopus oocytes, homology modeling\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — mutagenesis with kinetic analysis, electrophysiology, and homology modeling in a single study; multiple residues and mechanistic consequences established\",\n      \"pmids\": [\"19389703\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"PCFT promoter silencing through CpG hypermethylation within the minimal transcriptional regulatory region (−42 to +96 bp from TSS) accounts for loss of PCFT expression in antifolate-resistant HeLa R1-11 cells; treatment with 5-aza-2'-deoxycytidine restores transport and PCFT mRNA. In vitro methylation of the transfected reporter plasmid inhibits expression. Gene copy loss contributes additively.\",\n      \"method\": \"Bisulfite conversion sequencing, luciferase reporter assays, 5-aza-2'-deoxycytidine treatment, in vitro methylation of reporter plasmid, FISH for gene copy number\",\n      \"journal\": \"Molecular cancer therapeutics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (bisulfite sequencing, reporter assays, pharmacological demethylation, in vitro methylation, FISH), single lab\",\n      \"pmids\": [\"19671745\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Glu185 (E185) in a PCFT transmembrane domain plays a critical role in proton-folate coupling: E185A-PCFT loses function at pH 5.5 (8-fold decrease in Vmax) but retains function at pH 7.4; the transport remains intrinsically functional (capable of trans-stimulation). The effect of E185 substitution is primarily on the maximal transport rate (Vmax), consistent with involvement at a rate-limiting step of the PCFT transport cycle.\",\n      \"method\": \"Site-directed mutagenesis, radiolabeled methotrexate influx kinetics, trans-stimulation assays, surface biotinylation in transfected HeLa cells\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis with kinetic analysis, multiple substitutions, trans-stimulation and surface expression controls, single lab\",\n      \"pmids\": [\"19403800\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"PCFT secondary structure consists of 12 transmembrane domains with both N- and C-termini directed to the cytoplasm, established by substituted cysteine accessibility method (SCAM). A disulfide bridge between native Cys66 and Cys298 (in the 1st and 4th extracellular loops) is present but not required for transport function.\",\n      \"method\": \"Substituted cysteine accessibility method (SCAM) with MTSEA-biotin, streptavidin pulldown, Western blot, beta-mercaptoethanol treatment, cysteine-less PCFT generation\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — SCAM topology mapping with functional validation, cysteine-less scaffold controls, disulfide bond confirmed by multiple reagents, single lab\",\n      \"pmids\": [\"20225891\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Asp156 (D156) is critical for PCFT protein stability; multiple substitutions at this site (Tyr, Trp, Phe, Val, Asn, Lys) result in protein instability and loss of function, while conservative substitutions (Glu, Gly) preserve substantial function correlating with surface expression. Asp109 (D109), located in the first intracellular loop between TM2 and TM3, is absolutely required for PCFT transport function regardless of pH or substrate concentration, despite normal surface expression.\",\n      \"method\": \"Site-directed mutagenesis, radiolabeled folate transport assays at multiple pH values and concentrations, surface biotinylation, Western blot for protein stability in transfected HeLa cells\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — systematic mutagenesis with kinetic analysis, surface expression controls, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"20805364\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"NRF-1 (nuclear respiratory factor 1) is a major transcriptional regulator of PCFT (SLC46A1). NRF-1 binds to three consensus sites in the PCFT minimal promoter; NRF-1 overexpression increases PCFT mRNA and reporter activity, while dominant-negative NRF-1 or NRF-1 siRNA markedly represses PCFT expression.\",\n      \"method\": \"EMSA with NRF-1 antibody supershift, chromatin immunoprecipitation, luciferase reporter assays with NRF-1 site mutations, NRF-1 overexpression and siRNA knockdown\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (EMSA, ChIP, reporter assay with mutagenesis, gain/loss-of-function), single lab\",\n      \"pmids\": [\"20724482\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Arg376 (R376) is important for proton binding in PCFT; R376Q and other non-positive substitutions impair proton binding, which in turn modulates the folate binding pocket and reduces the rate of carrier conformational change. The R376Q mutant retains folate-independent proton transport (slippage/channel-like property) in Xenopus oocytes, but substrate transport is markedly decreased, with substrate-dependent differences.\",\n      \"method\": \"Site-directed mutagenesis, radiolabeled folate influx kinetics, electrophysiology in Xenopus oocytes, transfection in HeLa R1-11 cells\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — mutagenesis with kinetic analysis and electrophysiology, multiple substrates and pH conditions tested, single lab\",\n      \"pmids\": [\"20686069\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"PCFT-deficient (PCFT−/−) mice develop severe systemic folate deficiency confirming the critical and nonredundant in vivo role of PCFT in intestinal folate transport; PCFT deficiency causes macrocytic normochromic anemia, pancytopenia, impaired erythroblast differentiation with increased apoptosis, elevated erythropoietin, soluble transferrin receptor, and thrombopoietin.\",\n      \"method\": \"Targeted gene disruption (knockout of exons 1–3), in vivo folate uptake experiments, hematological analysis, bone marrow histology, serum cytokine measurement\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — knockout mouse model with in vivo folate uptake assay directly confirming intestinal folate transport defect, comprehensive phenotypic characterization\",\n      \"pmids\": [\"21346251\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"PCFT and RFC (reduced folate carrier) are distributed in lipid rafts of the colonic apical membrane; chronic ethanol ingestion reduces PCFT and RFC protein levels in lipid raft fractions of the colon, associated with decreased folate transport affinity and Vmax.\",\n      \"method\": \"Optiprep density gradient floatation for lipid raft isolation, Western blot, radiolabeled folate transport in apical membrane vesicles from ethanol-fed rats\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — membrane fractionation and Western blot with transport assay, single lab, no direct functional link between raft localization and transport activity established\",\n      \"pmids\": [\"21069807\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Gly189 and Gly192 (a GxxG motif in TM5) are functionally critical for PCFT; alanine substitution abolishes function. Ile188, adjacent to this motif, is accessible to the PCFT aqueous translocation pathway and located in the folate binding pocket (substrate protection of I188C from sulfhydryl-reactive reagents is retained at 0°C). Met193C is also aqueous-accessible but substrate protection is lost at 0°C.\",\n      \"method\": \"Site-directed mutagenesis, substituted cysteine accessibility method with membrane-impermeable reagents, substrate protection assays at different temperatures, radiolabeled folate transport\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — SCAM with substrate protection and temperature dependence tests, functional mutagenesis, multiple residues analyzed, single lab\",\n      \"pmids\": [\"22785121\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Ala335 residue contributes to PCFT protein stability; substitutions rendering bulky or charged residues at this site cause protein instability and loss of function. Gly338 residue is required for PCFT conformational oscillation (cycling between transport states); G338C mutant retains substrate binding (minimal change in Ki) but has 15–20-fold decreases in Kt and Vmax and is inaccessible to MTS reagents, consistent with TM9 location and impaired carrier cycling rather than substrate binding defect.\",\n      \"method\": \"Site-directed mutagenesis, radiolabeled pemetrexed and methotrexate influx kinetics, surface biotinylation, SCAM with MTS reagents, homology modeling\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — systematic mutagenesis with kinetic analysis, accessibility studies, and structural modeling; mechanistic distinction between binding and cycling defects\",\n      \"pmids\": [\"22843796\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Bicarbonate at physiological concentrations produces potent and rapidly reversible inhibition of PCFT-mediated transport at neutral pH. Bisulfite and nitrite also inhibit PCFT, particularly at weakly acidic pH by decreasing Vmax and collapsing the transmembrane proton gradient. Sulfate, nitrate, and phosphate do not inhibit PCFT.\",\n      \"method\": \"Radiolabeled folate transport assays in PCFT-stable HeLa transfectants at varying anion concentrations and pH, intracellular pH measurements\",\n      \"journal\": \"Molecular pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple anions tested systematically with transport kinetics and pH measurements, mechanistic link to proton gradient collapse established, single lab\",\n      \"pmids\": [\"23609145\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"KLF4 is the primary transcriptional activator of PCFT in the small intestine; HNF4α synergistically enhances KLF4-induced PCFT transcription. CDX2 and C/EBPα suppress KLF4- and KLF4/HNF4α-induced PCFT promoter activity. The graded expression pattern of these factors along the intestinal tract accounts for the proximal-restricted expression of PCFT.\",\n      \"method\": \"Dual-luciferase reporter assays in HEK293 cells with cotransfection of transcription factors, Western blot for transcription factor expression along rat intestine\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — luciferase reporter assays with multiple factors, corroborated by expression profiling, but no ChIP or endogenous PCFT mRNA response to factor manipulation in intestinal cells\",\n      \"pmids\": [\"23313509\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Four Tyr residues (Y291, Y362, Y315, Y414) are extracellularly accessible in PCFT; Y291, Y362, and Y315 are located within or near the folate binding pocket. Substitution of these residues with Cys or Ala increases both influx Vmax and Kt/Ki (suggesting release from a high-affinity constrained state), while Phe substitution moderates these changes. Y315A PCFT shows increased Vmax but loss of transstimulation (exchange), indicating these Tyr residues constrain carrier mobility and secure the high-affinity substrate-binding state at the expense of oscillation rate.\",\n      \"method\": \"SCAM with MTSEA-biotin, site-directed mutagenesis, radiolabeled influx and efflux transport kinetics, transstimulation assays\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — SCAM combined with mutagenesis, bidirectional transport kinetics and transstimulation mechanistic analysis, multiple Tyr residues analyzed, single lab\",\n      \"pmids\": [\"25608532\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Trp299 (W299) in the 4th external loop between TM7 and TM8 is required for PCFT function through its hydrophobicity: W299S mutation decreases Vmax 6.5-fold and alters Kt/Ki; only Phe and (partially) Ala substitutions preserve function, suggesting this residue interacts with the lipid membrane during the transport cycle and influences carrier oscillation and the folate binding pocket indirectly.\",\n      \"method\": \"SCAM with MTSEA-biotin, site-directed mutagenesis, radiolabeled pemetrexed transport kinetics, DTT treatment for disulfide accessibility\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — SCAM plus mutagenesis with kinetic analysis and substrate protection, single lab, mechanistic interpretation supported by accessibility data\",\n      \"pmids\": [\"27251438\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Purified human PCFT expressed in Sf9 insect cells via baculovirus retains folate transport function after reconstitution into liposomes, enabling biochemical and structural studies.\",\n      \"method\": \"Baculovirus/Sf9 expression, detergent purification, reconstitution into liposomes, radiolabeled folic acid transport assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — functional reconstitution of purified transporter demonstrated, but single lab and limited mechanistic follow-up in this paper\",\n      \"pmids\": [\"28493963\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"The 8th transmembrane helix of PCFT defines the aqueous translocation pathway: 14 contiguous exofacial residues are accessible to MTSEA-biotin; pemetrexed blocks biotinylation of 6 deep residues implicating this region in folate binding. Pro314 and Tyr315 are critical for function; their substitution markedly increases Kt, Ki, and Vmax. Homology modeling (based on GLUT5 structures) predicts a helix break in the outward-open conformation at this region.\",\n      \"method\": \"SCAM with MTSEA-biotin, site-directed mutagenesis, substrate protection assay, temperature-dependence of accessibility, homology modeling based on GLUT5 structures\",\n      \"journal\": \"Biochimica et biophysica acta. Biomembranes\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — systematic SCAM of 23 residues combined with mutagenesis, substrate protection, and structural modeling; multiple orthogonal methods, single lab\",\n      \"pmids\": [\"28802835\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"The 7th and 8th transmembrane helices of PCFT form an exofacial cleft defining the aqueous translocation pathway; 9 exofacial residues of TM7 are accessible. Paired cysteine substitutions between TM7 and TM8 (T289C-I304C and Q285C-Q311C) spontaneously form disulfide bonds, impairing accessibility and function; DTT restores both, establishing close proximity of these helices and their role in the substrate entry pathway.\",\n      \"method\": \"SCAM, dicysteine cross-linking with spontaneous disulfide bond formation, DTT reversal, homology modeling based on GLUT5 inward- and outward-open conformations\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — cross-linking with functional reversal, SCAM, substrate protection, and structural modeling; direct demonstration of helix proximity with functional consequence, single lab\",\n      \"pmids\": [\"29167151\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Asn411 (N411) in TM11 is part of the external gate of PCFT; N411K mutation causes loss of function with a markedly decreased influx Vmax and reduced affinity for most folate substrates (especially 5-methyltetrahydrofolate). Positive-charged substitutions are most disruptive; negative (Asp) and bulky hydrophobic/polar substitutions are better tolerated. Homology modeling places N411 protruding into the aqueous pathway, most prominently in the inward-open conformation.\",\n      \"method\": \"Site-directed mutagenesis, surface biotinylation, radiolabeled folate transport kinetics, homology modeling based on GLUT5\",\n      \"journal\": \"Blood advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — systematic mutagenesis at single residue with multiple substitutions, kinetic analysis, surface expression controls, structural modeling; single lab\",\n      \"pmids\": [\"29344585\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"PCFT generates substantial transmembrane electrochemical-potential gradients (concentrative transport) for antifolates at extracellular pH levels relevant to the tumor microenvironment (pH 6.9–7.0). Concentrative transport requires the transmembrane proton gradient; it is abolished by the protonophore CCCP and is not observed in bicarbonate/CO2-buffered medium at neutral pH.\",\n      \"method\": \"Radiolabeled methotrexate accumulation assays in PCFT-expressing HeLa cells, CCCP treatment, Na+/H+ exchanger inhibitors, intracellular pH measurement\",\n      \"journal\": \"Molecular pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple pharmacological tools, intracellular pH measurement, mechanistic dissection of concentrative transport, single lab\",\n      \"pmids\": [\"29326243\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Asp109 (D109) in the first intracellular loop locks PCFT in an inward-open conformation when substituted (D109A); cysteine-substituted residues in the aqueous translocation pathway lose accessibility to MTSEA-biotin. Introduction of a second 'unlocking' substitution at Gly305 (G305L or bulky residues in TM8) in the D109A scaffold largely restores both function and aqueous accessibility, demonstrating that D109 is part of a 'motif A' (GXXXDXXGR) critical for carrier oscillation between conformational states.\",\n      \"method\": \"Site-directed mutagenesis, SCAM with MTSEA-biotin, functional transport assays, double-mutant epistasis analysis, homology modeling\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — double-mutant suppressor analysis combined with SCAM directly demonstrates conformational locking mechanism; multiple orthogonal methods, single lab\",\n      \"pmids\": [\"30858177\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SLC46A1 regulates hepatic iron metabolism by importing heme into hepatocytes; hepatocyte-specific inhibition of SLC46A1 by AAV decreases liver iron content, increases serum iron and free heme, and alters expression of iron-regulatory molecules (TfR1, hepcidin, ferroportin). In hepatocytes, SLC46A1 can import hemin, increasing intracellular iron content; hemin import is independent of folate transport, but hemin treatment decreases SLC46A1 expression and folate import.\",\n      \"method\": \"AAV-mediated hepatocyte-specific SLC46A1 knockdown in mice, hemin uptake assay in hepatocytes, measurement of intracellular iron content, Western blot for iron-regulatory proteins\",\n      \"journal\": \"Metabolism: clinical and experimental\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo AAV knockdown and in vitro hepatocyte assays with direct iron content measurement, multiple downstream markers assessed, single lab\",\n      \"pmids\": [\"32621820\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Molecular dynamics simulations based on cryo-EM structure of Gallus gallus PCFT (ortholog of hPCFT) reveal that HFM-causing mutations cause partial loss of structural integrity manifested as enlarged/distorted pore, loss of long-range contacts, less stable inner helices with reduced solvent accessibility, and loss of secondary structure; these structural changes are reversed by compensatory mutations that restore function.\",\n      \"method\": \"Molecular dynamics simulations of hPCFT homology model based on cryo-EM structure of Gallus gallus PCFT, structural analysis of disease-causing and compensatory mutations\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 (structural/computational) / Weak — based on MD simulations from a cryo-EM ortholog structure, not direct experimental structure of hPCFT; provides mechanistic correlations with published kinetic data but is primarily computational\",\n      \"pmids\": [\"39924111\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"PCFT/HCP1 transports both folate and heme, but with lower affinity for heme than folate; siRNA knockdown of PCFT/HCP1 in Caco-2 cells reduces both folate uptake (48%) and heme uptake (22.5%), with greater impact on folate. Folic acid inhibits heme transport in hypoxic but not normal mice in vivo.\",\n      \"method\": \"siRNA knockdown in Caco-2 cells, radiolabeled folate and 59Fe-heme uptake assays, in vivo mouse intestinal transport studies with blocking antibody, everted duodenum preparations\",\n      \"journal\": \"The British journal of nutrition\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — siRNA knockdown with direct uptake measurements, in vitro and in vivo approaches, but interpretation of heme transport is contested in the field; moderate confidence\",\n      \"pmids\": [\"18782461\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Loss of SLC46A1 expression in hepatocellular carcinoma (HCC) cells causes iron deficiency in tumor tissue; only SLC46A1 (not STEAP3 or DMT1) silencing or overexpression controlled intracellular iron content in HCC cells. Lentivirus-mediated re-expression of SLC46A1 in orthotopic tumors restores tumor iron content with corresponding changes in iron-metabolic molecules.\",\n      \"method\": \"Lentiviral SLC46A1 overexpression and siRNA knockdown in HCC cell lines, orthotopic tumor implantation in mice, intracellular iron content measurement, Western blot for iron-metabolic molecules\",\n      \"journal\": \"Hepatology communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro and in vivo gain/loss-of-function with direct iron content measurement, comparison with other iron transporters, single lab\",\n      \"pmids\": [\"35811443\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"SLC46A1-mediated folate uptake in colorectal cancer cells suppresses tumor proliferation, migration, and invasion; mechanistically, SLC46A1 deficiency reduces intracellular folate availability, impairs cellular methylation potential (decreased SAM/SAH ratio), causes DNA hypomethylation at the FOS proto-oncogene promoter, and transcriptionally activates CCND1, BCL2, and PLAU oncogenic effectors driving CRC progression.\",\n      \"method\": \"SLC46A1 loss/gain-of-function in vitro and in vivo, SAM/SAH ratio measurement, bisulfite sequencing/methylation analysis, ChIP for promoter methylation, transcriptomic analysis, tumor growth assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal mechanistic methods (methylation assay, metabolite measurement, gene expression), in vitro and in vivo validation, single lab\",\n      \"pmids\": [\"41620398\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SLC46A1 (PCFT) is an electrogenic proton symporter with 12 transmembrane domains and intracellular N/C termini that mediates intestinal folate absorption and choroid plexus folate transport by coupling inwardly directed proton gradients to uphill folate influx at low pH; key functional residues include D109 (conformational gating), H247/H281 (proton coupling and binding), E185 (rate-limiting proton-folate coupling step), R376 (proton binding modulating the folate pocket), and a GxxG motif (TM5) plus TM7–TM8 exofacial cleft defining the substrate translocation pathway; the transporter also functions in FRα-mediated endosomal folate export, hepatic heme/iron import, and its expression is transcriptionally regulated by NRF-1, KLF4, HNF4α, CDX2, and C/EBPα, and silenced by CpG promoter hypermethylation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SLC46A1 (PCFT) is an electrogenic, proton-coupled folate transporter that mediates intestinal folate absorption at the apical brush-border membrane of the proximal small intestine, operating optimally at low pH by coupling an inwardly directed proton gradient to uphill folate influx [#0]. Genetic knockout in mice confirms its nonredundant in vivo role: PCFT-deficient animals develop severe systemic folate deficiency with macrocytic anemia and impaired erythropoiesis [#11], and loss-of-function mutations cause hereditary folate malabsorption through defects in protein stability, membrane trafficking, or transport activity [#1]. The transporter is a 12-transmembrane-domain polytopic membrane protein with cytoplasmic N- and C-termini and is N-glycosylated at two extracellular asparagines that are dispensable for transport [#3, #7]. Extensive structure-function mapping defines a substrate translocation pathway formed by the seventh and eighth transmembrane helices, which form a closely apposed exofacial cleft, with TM8 lining the aqueous pore and contributing folate-binding residues [#20, #21]; proton coupling and binding depend on His247, His281, Glu185, and Arg376, the latter modulating the folate-binding pocket and carrier conformational change [#4, #6, #10]. Conformational cycling between inward- and outward-open states is governed by Asp109 within a 'motif A' (GXXXDXXGR), whose substitution locks the carrier inward-open in a manner relievable by compensatory unlocking mutations [#24], with additional residues (a TM5 GxxG motif, Gly338, Pro314/Tyr315, Asn411) constraining substrate binding or oscillation rate [#13, #14, #20, #22]. Beyond folate, SLC46A1 imports heme to regulate hepatic and tumor iron homeostasis in a folate-transport-independent manner [#25, #28], and its folate-dependent control of cellular methylation potential modulates oncogene promoter methylation in colorectal cancer [#29]. Expression is transcriptionally driven by NRF-1 and by intestinal KLF4/HNF4\\u03b1 (antagonized by CDX2 and C/EBP\\u03b1), and is silenced by CpG promoter hypermethylation in antifolate-resistant cells [#9, #16, #5].\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"Established the molecular identity and core mechanism of intestinal folate uptake, answering how dietary folate crosses the acidic apical surface of the gut.\",\n      \"evidence\": \"Xenopus oocyte electrophysiology, radiolabeled folate uptake, and brush-border membrane fractionation\",\n      \"pmids\": [\"17898134\", \"18174275\", \"17340171\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic structure of the human transporter not resolved\", \"Stoichiometry of proton:folate coupling not fully quantified here\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Linked the transporter to human disease by showing PCFT mutations cause hereditary folate malabsorption, validating physiological essentiality in humans.\",\n      \"evidence\": \"Patient gene sequencing plus mutant transport and protein-stability assays in HeLa cells\",\n      \"pmids\": [\"17446347\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Distinguishing trafficking from catalytic defects per mutation incompletely resolved\", \"Genotype-phenotype correlation across mutations not systematic\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Defined a second cellular role as the endosomal folate exporter downstream of FRalpha, explaining how receptor-internalized folate reaches the cytosol.\",\n      \"evidence\": \"Isogenic PCFT(+/-) HeLa lines with FRalpha cotransfection, co-localization, and probenecid inhibition\",\n      \"pmids\": [\"19074442\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct demonstration of endosomal acidification driving export not isolated\", \"Single-lab finding\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Raised the possibility of broader substrate range by showing the same protein (HCP1) transports heme as well as folate, though with lower affinity.\",\n      \"evidence\": \"siRNA knockdown in Caco-2 cells with radiolabeled folate and 59Fe-heme uptake plus in vivo intestinal studies\",\n      \"pmids\": [\"18782461\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological significance of intestinal heme transport contested in the field\", \"Heme uptake reduction modest (22.5%)\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Resolved membrane topology and post-translational modification, fixing the structural framework (12 TMs, cytoplasmic termini, two N-glycans) for later residue mapping.\",\n      \"evidence\": \"PNGase F/tunicamycin treatment, glycosylation-site mutagenesis, and HA-tag accessibility in permeabilized cells\",\n      \"pmids\": [\"18405659\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Function of glycosylation beyond transport not established\", \"Topology inferred biochemically, not from a structure\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identified the proton-coupling machinery by showing His247/His281 and Glu185 govern proton handling and proton-folate coupling at the rate-limiting step.\",\n      \"evidence\": \"Site-directed mutagenesis with influx kinetics, oocyte electrophysiology, trans-stimulation, and homology modeling\",\n      \"pmids\": [\"19389703\", \"19403800\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Exact protonation order across the cycle not determined\", \"Residue roles inferred from mutagenesis without a structure\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Explained loss of transporter expression in antifolate resistance through CpG promoter hypermethylation, defining an epigenetic silencing mechanism.\",\n      \"evidence\": \"Bisulfite sequencing, luciferase reporters, 5-aza-dC reactivation, in vitro reporter methylation, and FISH\",\n      \"pmids\": [\"19671745\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Generality of methylation silencing across tumors not assessed here\", \"Single resistant cell model\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Mapped residues controlling protein stability versus catalysis (Asp156 stability; Asp109 absolutely required despite normal surface expression) and identified Arg376 in proton binding.\",\n      \"evidence\": \"Systematic mutagenesis with surface biotinylation, transport kinetics across pH, and oocyte electrophysiology\",\n      \"pmids\": [\"20805364\", \"20686069\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which D109 enables catalysis not yet defined at this stage\", \"Structural placement inferred from homology models\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Identified NRF-1 as a major transcriptional driver, beginning the dissection of how PCFT expression is set.\",\n      \"evidence\": \"EMSA/supershift, ChIP, reporter mutagenesis, and NRF-1 gain/loss-of-function\",\n      \"pmids\": [\"20724482\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Tissue specificity of NRF-1 control not addressed\", \"Single lab\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Confirmed the canonical 12-TM topology with cytoplasmic termini and identified a non-essential extracellular disulfide bridge.\",\n      \"evidence\": \"Substituted cysteine accessibility (SCAM) with MTSEA-biotin and cysteine-less scaffolds\",\n      \"pmids\": [\"20225891\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional role of the disulfide bond unclear\", \"Conformational dynamics not captured\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Provided definitive in vivo proof of nonredundant intestinal folate transport via a knockout mouse recapitulating human HFM hematology.\",\n      \"evidence\": \"Targeted exon 1-3 disruption with in vivo folate uptake and hematological/bone-marrow phenotyping\",\n      \"pmids\": [\"21346251\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Choroid plexus/CNS folate phenotype not detailed here\", \"Heme-transport role not tested in this model\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Began mapping the substrate translocation pathway, identifying a TM5 GxxG motif and folate-pocket residues accessible to the aqueous pathway.\",\n      \"evidence\": \"SCAM with membrane-impermeable reagents, substrate protection at varied temperature, and mutagenesis\",\n      \"pmids\": [\"22785121\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full pathway not yet delineated at this stage\", \"Pocket geometry inferred indirectly\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Distinguished stability determinants from conformational-cycling determinants, showing Gly338 supports carrier oscillation independent of substrate binding.\",\n      \"evidence\": \"Mutagenesis with pemetrexed/methotrexate kinetics, surface biotinylation, SCAM, and homology modeling\",\n      \"pmids\": [\"22843796\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct visualization of conformational states absent\", \"Single lab\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Clarified transcriptional control of the proximal-restricted intestinal expression pattern through KLF4/HNF4alpha activation antagonized by CDX2 and C/EBPalpha.\",\n      \"evidence\": \"Dual-luciferase cotransfection in HEK293 with transcription-factor expression profiling along rat intestine\",\n      \"pmids\": [\"23313509\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No ChIP confirming direct promoter occupancy\", \"Endogenous intestinal PCFT mRNA response to factor manipulation not shown\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Defined physiological anion inhibition (bicarbonate, bisulfite, nitrite) acting by collapsing the proton gradient, constraining where PCFT operates.\",\n      \"evidence\": \"Transport kinetics across anions and pH with intracellular pH measurement\",\n      \"pmids\": [\"23609145\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether anions bind the protein versus only dissipate the gradient not fully separated\", \"Single lab\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Advanced biochemical tractability by demonstrating purified human PCFT retains transport after reconstitution into liposomes.\",\n      \"evidence\": \"Baculovirus/Sf9 expression, detergent purification, proteoliposome folate transport assay\",\n      \"pmids\": [\"28493963\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure obtained from the reconstituted protein here\", \"Limited mechanistic follow-up\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Localized the aqueous translocation pathway to TM8 and showed TM7-TM8 form a close exofacial cleft constituting the substrate entry route.\",\n      \"evidence\": \"SCAM, substrate protection, dicysteine cross-linking with DTT reversal, and GLUT5-based homology modeling\",\n      \"pmids\": [\"28802835\", \"29167151\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Inward-facing pathway less defined\", \"Helix proximity inferred for specific conformations only\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Showed extracellular Tyr residues near the folate pocket constrain carrier mobility to secure a high-affinity binding state at the expense of oscillation rate.\",\n      \"evidence\": \"SCAM plus mutagenesis with bidirectional influx/efflux kinetics and transstimulation\",\n      \"pmids\": [\"25608532\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Trade-off mechanism between affinity and turnover not structurally resolved\", \"Single lab\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Demonstrated concentrative antifolate transport at tumor-microenvironment pH, establishing pharmacological relevance for antifolate delivery to tumors.\",\n      \"evidence\": \"Methotrexate accumulation in PCFT-HeLa cells with CCCP, NHE inhibitors, and intracellular pH measurement\",\n      \"pmids\": [\"29326243\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo tumor accumulation not tested here\", \"Single lab\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identified Asn411 in TM11 as part of the external gate shaping substrate affinity, particularly for 5-methyltetrahydrofolate.\",\n      \"evidence\": \"Multi-substitution mutagenesis with kinetics, surface biotinylation, and homology modeling\",\n      \"pmids\": [\"29344585\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Gate dynamics inferred from modeling, not direct structure\", \"Single lab\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Defined the conformational-switch mechanism by showing Asp109 of motif A locks the inward-open state, relievable by a compensatory TM8 unlocking substitution.\",\n      \"evidence\": \"Double-mutant suppressor (epistasis) analysis combined with SCAM and homology modeling\",\n      \"pmids\": [\"30858177\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Energetics of the conformational transition not measured\", \"No experimental structure of the two states\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Established a folate-independent heme-import function regulating hepatic iron homeostasis, expanding PCFT's physiological role.\",\n      \"evidence\": \"AAV-mediated hepatocyte-specific knockdown in mice and hemin uptake/iron-content assays in hepatocytes\",\n      \"pmids\": [\"32621820\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Heme-binding site on the transporter not identified\", \"Single lab\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Showed SLC46A1 controls tumor iron content in hepatocellular carcinoma, uniquely among tested iron transporters.\",\n      \"evidence\": \"Lentiviral overexpression/siRNA in HCC lines plus orthotopic tumors with iron-content measurement\",\n      \"pmids\": [\"35811443\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanistic link from iron control to tumor behavior not fully traced\", \"Single lab\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Provided a structural rationale for HFM mutations by simulating an ortholog cryo-EM-based model, correlating pore distortion and lost contacts with loss of function.\",\n      \"evidence\": \"Molecular dynamics of an hPCFT homology model from the Gallus gallus PCFT cryo-EM structure\",\n      \"pmids\": [\"39924111\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Computational, not an experimental human structure\", \"Predictions await direct structural validation\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Connected folate transport to epigenetic oncogene regulation, showing SLC46A1-supplied folate maintains methylation potential that suppresses colorectal cancer progression.\",\n      \"evidence\": \"Loss/gain-of-function in vitro and in vivo with SAM/SAH measurement, promoter methylation/ChIP, and transcriptomics\",\n      \"pmids\": [\"41620398\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct causality between FOS promoter hypomethylation and the full phenotype not isolated\", \"Single lab\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"An experimentally determined high-resolution structure of human PCFT in defined conformational states, and a unified model reconciling its folate, heme, and choroid plexus transport activities, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No experimental human PCFT structure; mechanism rests on homology models\", \"Heme-binding determinants unmapped\", \"Choroid plexus folate transport role not directly characterized in the corpus\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005215\", \"supporting_discovery_ids\": [0, 2, 11, 19, 25]},\n      {\"term_id\": \"GO:0140104\", \"supporting_discovery_ids\": [0, 23, 25]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 12]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [0, 11, 25]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [0, 25, 28]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [1, 11]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [9, 16, 5]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"FOLR1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}