Reduced Folate Transport Research Articles

Host-dependent resistance of Group A Streptococcus to sulfamethoxazole mediated by a horizontally-acquired reduced folate transporter

Described antimicrobial resistance mechanisms enable bacteria to avoid the direct effects of antibiotics and can be monitored by in vitro susceptibility testing and genetic methods. Here we describe a mechanism of sulfamethoxazole resistance that requires a host metabolite for activity. Using a combination of in vitro evolution and metabolic rescue experiments, we identify an energy-coupling factor (ECF) transporter S component gene (thfT) that enables Group A Streptococcus to acquire extracellular reduced folate compounds. ThfT likely expands the substrate specificity of an endogenous ECF transporter to acquire reduced folate compounds directly from the host, thereby bypassing the inhibition of folate biosynthesis by sulfamethoxazole. As such, ThfT is a functional equivalent of eukaryotic folate uptake pathways that confers very high levels of resistance to sulfamethoxazole, yet remains undetectable when Group A Streptococcus is grown in the absence of reduced folates. Our study highlights the need to understand how antibiotic susceptibility of pathogens might function during infections to identify additional mechanisms of resistance and reduce ineffective antibiotic use and treatment failures, which in turn further contribute to the spread of antimicrobial resistance genes amongst bacterial pathogens.

Antibody Characterization Report for Reduced folate transporter SLC19A1 (FOLT)

Antibody Characterization Report for Reduced folate transporter SLC19A1 (FOLT)

Folic acid deficiency and vision: a review.

Folic acid (FA), also termed folate, is an essential vitamin for health at all ages since it participates in the biosynthesis of nucleotides, amino acids, neurotransmitters, and certain vitamins. It is therefore crucial for rapidly growing tissues such as those of the fetus. It is becoming clear that FA deficiency and impaired folate pathways are implicated in many diseases of both early life and old age. FA can be transported into the cell by the folate receptor, the reduced folate transporter, and proton-coupled folate transporter. Folate transport proteins are present in certain eye tissues, which explains why FA plays an important role in eye development. The purpose of this literature review is to investigate the evidence relating FA deficiency to eye diseases.

High-fat diet consumption reduces hepatic folate transporter expression via nuclear respiratory factor-1.

Folate is an essential micronutrient for biological function. The liver, a primary organ for folate metabolism and storage, plays an important role in folate homeostasis. Proton-coupled folate transporter (PCFT) and reduced folate carrier (RFC) are the major folate transporters responsible for folate uptake at basolateral membrane of hepatocytes. Low serum folate levels are frequently associated with obesity. We investigated the mechanism that regulated folate status in a mouse model with diet-induced obesity. Mice (C57BL/6J) were fed a high-fat diet (60% kcal fat) for 8weeks. Mice displayed increased hepatic lipid accumulation and decreased folate levels in the liver and serum compared to mice fed a normal chow diet (10% kcal fat). High-fat diet-fed mice had low expression of PCFT and RFC and decreased nuclear respiratory factor-1 (NRF-1)/DNA-binding activity. Treatment with NRF-1 siRNA or palmitic acid reduced folate transporter expression in hepatocytes. Inhibition of NRF-1 mediated folate transporter expression significantly reduced intracellular folate levels. These results suggest that chronic consumption of high-fat diets impairs folate transporter expression via NRF-1-dependent mechanism, leading to reduced hepatic folate storage. Understanding the regulation of folate homeostasis in obesity may have an important implication in current guideline of folate intake. KEY MESSAGES: Serum and liver folate levels are decreased in diet-induced obese mice. Chronic high-fat diet consumption impairs expression of hepatic PCFT and RFC. NRF-1 regulates hepatic folate transporters expression and folate levels.

Inhibition of Methotrexate Uptake via Organic Anion Transporters OAT1 and OAT3 by Glucuronides of Nonsteroidal Anti-inflammatory Drugs.

Combination therapy of non-steroidal anti-inflammatory drugs (NSAIDs) and methotrexate (MTX) sometimes triggers adverse effects, such as liver injury, renal failure, gastrointestinal disorders, and myelosuppression, owing to the reduction of MTX clearance. Previous reports have suggested that NSAIDs inhibit renal MTX uptake via organic anion transporters (OATs) and reduced folate transporter (RFC)-1 and efflux via multidrug resistance-associated proteins (MRPs). Recently, our laboratory found inhibitory effects of NSAIDs-glucuronide (NSAIDs-Glu), a major metabolite of NSAIDs, on MRP-mediated MTX transport as a new site of interaction between MTX and NSAIDs. However, it remains unclear that whether NSAIDs-Glu inhibit renal uptake of MTX. Therefore, the present study aimed to evaluate inhibitory effects of several NSAIDs-Glu (diclofenac, R- and S-ibuprofen, R- and S-flurbiprofen, and R- and S-naproxen) on human OAT1 and OAT3-mediated MTX transport. In this study, [3H]MTX uptake was observed by using human OAT1 and OAT3-overexpressing HEK293 cells in the presence or absence of NSAIDs-Glu. All examined NSAIDs-Glu exhibited concentration-dependent inhibitory effects on MTX uptake via OAT1 and OAT3. Our results indicated that NSAIDs-Glu are more potent (5- to 15-fold) inhibitors of OAT3 than OAT1. Moreover, stereoselective inhibitory effects of NSAIDs-Glu on OATs-mediated MTX uptake were not observed, unlike on MRPs-mediated transport. These findings suggest that inhibition of OAT1 and OAT3-mediated renal uptake of MTX by plasma NSAIDs-Glu may be one of the competitive sites underlying complex drug interaction between MTX and NSAIDs.

The Role of Folate Transport in Antifolate Drug Action in Trypanosoma brucei

The aim of this study was to identify and characterize mechanisms of resistance to antifolate drugs in African trypanosomes. Genome-wide RNAi library screens were undertaken in bloodstream form Trypanosoma brucei exposed to the antifolates methotrexate and raltitrexed. In conjunction with drug susceptibility and folate transport studies, RNAi knockdown was used to validate the functions of the putative folate transporters. The transport kinetics of folate and methotrexate were further characterized in whole cells. RNA interference target sequencing experiments identified a tandem array of genes encoding a folate transporter family, TbFT1–3, as major contributors to antifolate drug uptake. RNAi knockdown of TbFT1–3 substantially reduced folate transport into trypanosomes and reduced the parasite's susceptibly to the classical antifolates methotrexate and raltitrexed. In contrast, knockdown of TbFT1–3 increased susceptibly to the non-classical antifolates pyrimethamine and nolatrexed. Both folate and methotrexate transport were inhibited by classical antifolates but not by non-classical antifolates or biopterin. Thus, TbFT1–3 mediates the uptake of folate and classical antifolates in trypanosomes, and TbFT1–3 loss-of-function is a mechanism of antifolate drug resistance.

Decreased activity of folate transporters in lipid rafts resulted in reduced hepatic folate uptake in chronic alcoholism in rats

Folic acid is an essential nutrient that is required for one-carbon biosynthetic processes and for methylation of biomolecules. Deficiency of this micronutrient leads to disturbances in normal physiology of cell. Chronic alcoholism is well known to be associated with folate deficiency, which is due in part to folate malabsorption. The present study deals with the regulatory mechanisms of folate uptake in liver during chronic alcoholism. Male Wistar rats were fed 1g/kg body weight/day ethanol (20% solution) orally for 3months, and the molecular mechanisms of folate uptake were studied in liver. The characterization of the folate transport system in liver basolateral membrane (BLM) suggested it to be a carrier mediated and acidic pH dependent, with the major involvement of proton coupled folate transporter and folate binding protein in the uptake. The folate transporters were found to be associated with lipid raft microdomain of liver BLM. Moreover, ethanol ingestion decreased the folate transport by altering the Vmax of folate transport process and downregulated the expression of folate transporters in lipid rafts. The decreased transporter levels were associated with reduced protein and mRNA levels of these transporters in liver. The deranged folate uptake together with reduced folate transporter levels in lipid rafts resulted in reduced folate levels in liver and thereby to its reduced levels in serum of ethanol-fed rats. The chronic ethanol ingestion led to decreased folate uptake in liver, which was associated with the decreased number of transporter molecules in the lipid rafts that can be ascribed to the reduced synthesis of these transporters.

Characterization of uptake of folates by rat and human blood–brain barrier endothelial cells

This study aimed to characterize (3)H-folic acid ((3)H-FA) and (14)C-methyltetrahydrofolic acid ((14)C-MTHF) uptake by rat (RBE4) and human (hCMEC/D3) blood-brain barrier (BBB) endothelial cell lines. Uptake of (3)H-FA and (14)C-MTHF by RBE4 cells was time dependent and linear for the first 2 min of incubation; uptake by hCMEC/D3 cells showed a less marked time-dependency and a greater experimental variability. So, further experiments were performed in RBE4 cells only. Uptake of (3)H-FA was stimulated at acidic and alkaline pH, Na(+) dependent, stimulated when F(-) substituted for Cl(-), energy independent, inhibited by premetrexed, stimulated by cytochalasin D, and unaffected by MTHF, DIDS, SITS, methotrexate, monensin, and FA. Uptake of (14)C-MTHF was found to be pH-, Na(+)-, Cl(-)- and energy independent, inhibited by premetrexed and methotrexate, stimulated by cytochalasin D, and unaffected by FA, DIDS, SITS, monensin, and MTHF. RT-PCR analysis showed mRNA expression of reduced folate transporter (RFC), but neither of FRalpha nor of proton-coupled folate transporter (PCFT) in RBE4 cells, and mRNA expression of RFC and PCFT, but not of FRalpha, in hCMEC/D3 cells. In conclusion, both human and rat BBB endothelial cells show little capacity for (3)H-FA and (14)C-MTHF apical uptake. Hence, these cell lines do not appear to be a good model to study the transport of folates at the BBB.

Modulation of folic acid uptake into BeWo cells by cannabinoids

The aim of this study was to investigate the influence of cannabinoids (CB) on the uptake of folic acid (FA) by the choriocarcinoma cell line (BeWo cells). Acutely, anandamide caused a 15% decrease in 3H‐FA uptake (pH 7.5). Moreover, tetrahydrocannabinol (THC) caused a 30% increase, and AM630 a 15% decrease (pH 6.5). Neither the inhibitory effect of anandamide nor the stimulatory effect of THC were changed by the CB receptor type 1 or type 2 antagonists (AM251 and AM630, respectively). Chronically (48 h), THC and AM251 decreased by 20% the uptake of 3H‐FA, and anandamide and AM630 increased it by 30% (pH 7.5). Moreover, CP 55,940 increased uptake of 3H‐FA by 30% (pH 6.5). RT‐PCR analysis showed that the mRNA levels of the Reduced Folate Transporter 1 increased by 9% after chronic treatment with AM630, and the mRNA levels of the Proton‐coupled Folate Transporter decreased by 17% and increased by 18% after chronic treatment with THC and AM251, respectively. In conclusion, uptake of 3H‐FA by BeWo cells is significantly, although not very markedly, changed by several distinct CB receptor agonists and antagonists, either after acute and after chronic exposure of the cells. The acute effects of CB receptor agonists do not seem to be CB receptor‐mediated, and with a few exceptions the chronic effects do not seem to be related to changes in the expression levels of FA transporters.Supported by FCT (PTDC/SAU‐FCF/67805/2006)

Effect of Cannabinoids upon the Uptake of Folic Acid by BeWo Cells

Folic acid (FA) is an essential micronutrient that is particularly important during pregnancy for normal placental and fetal development and growth. The placenta and the fetus become frequently exposed to drugs of abuse such as cannabinoids because of maternal use of these substances. The aim of this study was to investigate the influence of cannabinoids on the uptake of FA by BeWo cells. Acute treatment with anandamide (1–10 μmol/l) caused a 15% decrease in ³H-FA uptake at pH 7.5. Moreover, tetrahydrocannabinol (THC) (1–10 μmol/l) caused a 30% increase, and AM630 (1 μmol/l) a 15% decrease in this parameter at pH 6.5. Neither the inhibitory effect of anandamide nor the stimulatory effect of THC were changed in the presence of cannabi- noid receptor type 1 or type 2 antagonists (AM251 and AM630, respectively). Chronic treatment (48 h) with THC (100 nmol/l) and AM251 (100 nmol/l) decreased the uptake of ³H-FA by 20% at pH 7.5, and anandamide (1 μmol/l) and AM630 (10–500 nmol/l) increased it by 30%. Moreover, CP55,940 (10 nmol/l) increased the uptake of ³H-FA by 30% at pH 6.5. RT-PCR analysis showed that the mRNA levels of the reduced folate transporter 1 increased by 9% after chronic treatment with AM630 (500 nmol/l). The mRNA levels of the proton-coupled folate transporter decreased by 17% and increased by 18% after chronic treatment with THC (0.1 μmol/l) and AM251 (100 nmol/l), respectively. In conclusion, ³H-FA uptake by BeWo cells is significantly, although not very markedly, changed by several distinct CB receptor agonists and antagonists, both after acute and chronic exposure of the cells. The acute effects of cannabinoid receptor agonists do not seem to be mediated by the cannabinoid receptor, and with a few exceptions the chronic effects do not seem to be related to changes in the expression levels of FA transporters.

Folate Receptor Autoimmunity and Cerebral Folate Deficiency in Low-Functioning Autism with Neurological Deficits

Reduced folate transport to the CNS was identified in two autism spectrum disorders, i.e., Rett syndrome and infantile low-functioning autism with neurological abnormalities. Twenty-five patients with early-onset low-functioning autism with or without neurological deficits, were evaluated for serum folate, cerebrospinal fluid (CSF) 5-methyltetrahydrofolate (5MTHF), and serum FR autoantibodies of the blocking type to determine the significance of folate receptor (FR) autoantibodies with respect to folate transport across the blood-CSF barrier. In spite of normal serum folate, CSF 5MTHF was low in 23 of 25 patients. The reduced CSF folate in 19 of these 23 patients could be explained by serum FR autoantibodies blocking the folate binding site of the membrane-attached FR on the choroid epithelial cells. Oral folinic acid supplements led to normal CSF 5MTHF and partial or complete clinical recovery after 12 months. Serum FR autoimmunity appears to represent an important factor in the pathogenesis of reduced folate transport to the nervous system among children with early-onset low-functioning autism associated with or without neurological deficits. Early detection of FR autoantibodies may be a key factor in the prevention and therapeutic intervention among this subgroup of patients with autism.

Decreased Expression of Transporters Reduces Folate Uptake across Renal Absorptive Surfaces in Experimental Alcoholism

In this study, we examined the mechanistic insights of folate reabsorption during alcoholism, considering enhanced renal excretion as one of the major contributing factors to alcohol-induced folate deficiency. Male Wistar rats were fed 1g/kg body weight/day ethanol (20% solution) orally for 3 months. The results on characterization of the folate transport system in renal basolateral membrane (BLM) suggested it to be a carrier-mediated, acidic pH-dependent and saturable one. Chronic ethanol feeding decreased the uptake mainly by increasing the K (m) and decreasing the V (max) of the transport process at the BLM surface. At the molecular level, reduced folate transport activity in renal tissue during chronic ethanol ingestion was attributable to decreased expression of reduced folate carrier (RFC) and folate binding protein (FBP). Antibodies against RFC protein revealed a parallel change in RFC expression in both brush border and BLM surfaces during chronic alcoholism. Such findings highlight the role of downregulation of RFC and FBP expression and provide mechanistic insight into the observed reduced folate transport efficiency at renal absorptive surfaces in alcoholism, which may result in low blood folate levels commonly observed in alcoholics.

Folate Receptor Autoantibodies and Spinal Fluid 5-Methyltetrahydrofolate Deficiency in Rett Syndrome

Rett syndrome was associated with low cerebrospinal fluid (CSF) 5-methyltetrahydrofolate (5MTHF) in 42-50% of European patients whereas approximately 93% of the patients from North-America had a normal CSF 5MTHF status. We determined the CSF folate status in Rett patients living in North- and South-Western Europe and measured serum folate receptor (FR) autoantibodies of the blocking type to explain the reduced folate transport across the choroid plexus. Irrespective of their MECP2 genotype and despite normal plasma folate values, 14 of 33 Rett patients (42%) had low CSF folate levels. Blocking FR autoantibodies were found in 8 of the Rett patients (24%), 6 of whom had low CSF folate levels. FR autoimmunity was primarily found within the group of Rett patients with low CSF folate status with a higher incidence in North-Western Europe. In Rett patients from North-America 74 of 76 girls had higher folate values in both serum and CSF than European patients. The food folate fortification in North-America may account for the higher folate levels and may prevent CFD in these Rett patients. FR autoimmunity occurred predominantly in Rett patients from North-Western Europe and may contribute to cerebral folate deficiency (CFD).

Mitochondrial Complex I Encephalomyopathy and Cerebral 5-Methyltetrahydrofolate Deficiency

Folate transport to the brain depends on ATP-driven folate receptor-mediated transport across choroid plexus epithelial cells. Failure of ATP production in Kearns-Sayre syndrome syndrome provides one explanation for the finding of low spinal fluid (CSF) 5-methyltetrahydrofolate (5MTHF) levels in this condition. Therefore, we suspect the presence of reduced folate transport across the blood-spinal fluid barrier in other mitochondrial encephalopathies. In the present patient with mitochondrial complex I encephalomyopathy a low 5-methyltetrahydrofolate level was found in the CSF. Serum folate receptor autoantibodies were negative and could not explain the low spinal fluid folate levels. The epileptic seizures did not respond to primidone monotherapy, but addition of ubiquinone-10 and radical scavengers reduced seizure frequency. Add-on treatment with folinic acid led to partial clinical improvement including full control of epilepsy, followed by marked recovery from demyelination of the brainstem, thalamus, basal ganglia and white matter. Cerebral folate deficiency is not only present in Kearns-Sayre syndrome but may also be secondary to the failure of mitochondrial ATP production in other mitochondrial encephalopathies. Treatment with folinic acid in addition to supplementation with radical scavengers and cofactors of deficient respiratory enzymes can result in partial clinical improvement and reversal of abnormal myelination patterns on neuro-imaging.

Absorption of folate by Caco-2 cells is not affected by high glucose concentration

The aim of this work was to investigate the effect of high glucose exposure on the absorption of folate by Caco-2 cells. We verified that apical high glucose did not affect the apical uptake of [ 3H]folate. Both different concentrations of glucose (10–45 mM) and different exposure times (10 min–24 h) were tested. Furthermore, apical high glucose (30 mM) did not affect the intracellular steady-state levels of [ 3H]folate, and simultaneous apical and basolateral high glucose (30 mM) did not change the apical-to-basolateral apparent permeability ( P app) to [ 3H]folate. Both the apical uptake and the steady-state intracellular levels of [ 3H]folate were strongly reduced by 5-methyltetrahydrofolate, methotrexate, SITS (4-acetamido-4′-isothiocyanato-2,2′-stilbenedisulfonic acid), DIDS (4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid) and indomethacin, but were not affected or only hardly affected by p-aminohippuric acid and fumitremorgin C. Moreover, DIDS and indomethacin significantly reduced (by 50–60%) the apical-to-basolateral P app to [ 3H]folate, but [ 3H]folate present in the cells at the end of the experiment was higher in the case of indomethacin. Fumitremorgin C had no effect. The effect of the drugs tested was not changed or only hardly changed by high glucose. In conclusion, absorption of [ 3H]folate is not modulated by either apical or basolateral high glucose exposure in Caco-2 cells. Moreover, our results suggest that the apical uptake of [ 3H]folate by Caco-2 cells involves the Reduced Folate Transporter (but not the Organic Anion Transporter), and that Multidrug Resistance Protein and/or Organic Anion Transporter (but not Breast Cancer Resistance Protein) may mediate apical efflux of [ 3H]folate.

Fluorescein-methotrexate transport in brush border membrane vesicles from rat small intestine

The transport characteristics of fluorescein-methotrexate (F-MTX) in isolated brush border membrane vesicles (BBMVs) from rat small intestine were studied. F-MTX uptake in BBMVs was measured by a rapid filtration technique. Our results demonstrated that F-MTX uptake into vesicles was 1) significantly increased under the experimental conditions of an outwardly directed OH − gradient or an inwardly directed H +gradient, 2) sensitive to temperature, 3) increased with decreasing pH of the incubation buffer, 4) significantly inhibited by 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) at the early stage of the uptake, and 5) significantly inhibited by methotrexate (MTX). Thus, the transport of F-MTX in BBMVs was shown to be mediated in part by the reduced folate transporter (RFC) which was known to transport MTX through the epithelium of small intestine.

Transcellular transfer of folate across the retinal pigment epithelium

The differential polarized distribution of the folate receptor a (FRa) in the basal membrane and reduced-folate transporter (RFT-1) in the apical membrane was demonstrated previously in the retinal pigment epithelium (RPE). Based on this, we hypothesized that folate would enter the RPE via FRa and exit the cell via RFT-1. To test this, we performed in vitro transport assays using ARPE-19 cells cultured on permeable supports. The cells were grown for 4 weeks and electron microscopic analysis indicated that the cells have the phenotypic features of normal RPE cells including apical microvillous processes and junctional complexes. Measurement of transepithelial resistance showed that the resistance increases in the cells as they differentiate over several weeks. Transport assays showed that ARPE-19 cells transport folate in a basal-to-apical direction, but do not transport this vitamin significantly in the apical-to basal-direction. This was not a diffusional process, as the paracellular markers inulin and sucrose were transferred across the cell monolayer at a much lower level. The presence of FRa in the basal membrane was demonstrable by folate binding and that of RFT-1 in the apical membrane by blockade of folate transport by RFT-1-specific antibody. This study represents the first in vitro demonstration of transcellular transfer of folate across RPE and suggests that folate is transported from the choriocapillaris to the adjacent photoreceptor cells in vivo by the concerted action of FRa in the basal membrane and RFT-1 in the apical membrane.

Identification and Characterization of the Human and Mouse SLC19A3 Gene: A Novel Member of the Reduced Folate Family of Micronutrient Transporter Genes

We report here the isolation, characterization, and chromosomal localization of the genes encoding the human and corresponding murine orthologue of solute carrier family 19A member 3 (SLC19A3). Human SLC19A3 encodes a 496-amino-acid residue protein with a predicted molecular weight of 56 kDa that shares sequence similarity to both SLC19A1 (reduced folate transporter (RFC-1)) and SLC19A2 (high affinity thiamine transporter (THTR-1)). Like the SLC19A1 and SLC19A2 proteins, SLC19A3 contains 12 putative transmembrane domains. The human SLC19A3 gene is widely expressed, with the most abundant expression observed in placenta, kidney, and liver, and has been mapped to chromosome 2q37. The murine SLC19A3 gene maps to central chromosome 1 in the region defined as a seizure susceptibility locus in the DBA/2J mouse strain. This article describes the identification of SLC19A3, a gene encoding a novel solute transporter, and establishes murine SLC19A3 as a candidate gene for seizures in the DBA/2J mouse.

Thiamine intestinal transport and related issues: recent aspects.

In the intestinal lumen thiamine is in free form and very low concentrations. Absorption takes place primarily in the proximal part of the small intestine by means of a dual mechanism, which is saturable at low (physiological) concentrations and diffusive at higher. Thiamine undergoes intracellular phosphorylation mainly to thiamine pyrophosphate, while at the serosal side only free thiamine is present. Thiamine uptake is enhanced by thiamine deficiency, and reduced by thyroid hormone and diabetes. The entry of thiamine into the enterocyte, as evaluated in brush border membrane vesicles of rat small intestine in the absence of H+ gradient, is Na+- and biotransformation-independent, completely inhibited by thiamine analogs and reduced by ethanol administration and aging. The transport involves a saturable mechanism at low concentrations of vitamin and simple diffusion at higher. Outwardly oriented H+ gradients enhance thiamine transport, whose saturable component is a Na+-independent electroneutral uphill process utilizing energy supplied by the H+ gradient, and involving a thiamine/ H+ 1:1 stoichiometric exchange. The exit of thiamine from the enterocyte, as evaluated in basolateral membrane vesicles, is Na+-dependent, directly coupled to ATP hydrolysis by Na+-K+-ATPase, and inhibited by thiamine analogs. Transport of thiamine by renal brush border membrane vesicles is similar to the intestinal as far as both H+ gradient influence and specificity are concerned. In the erythrocyte thiamine transport is a Na+-independent, electroneutral process yet with two components: saturable, prevailing at low thiamine concentrations, and diffusive at higher. The saturable (specific) component is missing in patients of the rare disease known as thiamine-responsive megaloblastic anaemia (TRMA), producing a general disturbance of thiamine transport up to thiamine deficiency. The TRMA gene is located in chromosome 1q23.3. Recently, the thiamine transporter has been cloned: it is a protein of 497 amino acid residues with high homology with the reduced-folate transporter.

Mutations in the reduced-folate carrier affect protein localization and stability

The reduced-folate-carrier (rfc) gene has been shown to be functionally important for reduced-folate transport in mammalian cells. In the present paper we describe the identification of alterations in both alleles of the rfc gene in a mutant Chinese-hamster ovary cell line deficient in methotrexate transport. One allele of the rfc gene contains a point mutation resulting in a Gly345 → Arg substitution in the predicted amino acid sequence. In this case, a protein of similar size to the wild-type protein is produced, although it remains as an immature, core-glycosylated, form. The second allele contains a point mutation in the last base of intron 5 that results in the utilization of a cryptic splice site leading to a seven-base deletion in the mRNA. The use of an alternate splice site changes the reading frame to yield a truncated protein with 68 different C-terminal amino acids as compared with the wild-type. Both of these altered gene products were monitored by fusion with green fluorescent protein and found to be non-functional with an increased rate of turnover. The protein with the point mutation is trapped in the endoplasmic reticulum with subsequent degradation, whereas the product of the splice mutation is not membrane-associated and is partially degraded. Thus mutations in both alleles of the rfc gene in this resistant cell line account for the loss of reduced-folate transport. The observations made regarding the degradation of these mutant gene products also provide support for putative checkpoints in the endoplasmic reticulum.