Hereditary folate malabsorption: A positively charged amino acid at position 113 of the proton-coupled folate transporter (PCFT/SLC46A1) is required for folic acid binding

The proton-coupled folate transporter (PCFT/SLC46A1) mediates intestinal folate uptake at acidic pH. Some loss of folic acid (FA) transport mutations in PCFT from hereditary folate malabsorption (HFM) patients cluster in R113, thereby suggesting a functional role for this residue. Herein, unlike non-conservative substitutions, an R113H mutant displayed 80-fold increase in the FA transport Km while retaining parental Vmax, hence indicating a major fall in folate substrate affinity. Furthermore, consistent with the preservation of 9% of parental transport activity, R113H transfectants displayed a substantial decrease in the FA growth requirement relative to mock transfectants. Homology modeling based on the crystal structures of the Escherichia coli transporter homologues EmrD and glycerol-3-phosphate transporter revealed that the R113H rotamer properly protrudes into the cytoplasmic face of the minor cleft normally occupied by R113. These findings constitute the first demonstration that a basic amino acid at position 113 is required for folate substrate binding.     

A high-affinity folate binding protein in human semen

The presence of a folate binding protein of high-affinity type (affinity constant 3.10¹⁰M^–1, maximum folate binding 1.4 nM) in human semen was demonstrated in equilibrium dialysis experiments (37°C, pH 7.4) with the radioligand³H-folate. Radioligand dissociation from the binding protein was slow at pH 7.4, but rapid at pH 3.5. By use of rabbit antibodies against 25 kDa human milk folate binding protein we determined the concentration of folate binding protein in 16 speciments of human semen in an enzyme-linked immunosorbent assay. The concentration of immunoreactive folate binding protein was independent of the number of spermatozoa in individual specimens. Gel filtration showed that immunoreactive and radioligand bound folate binding protein coeluted in two peaks: a major one of 100 kDa and a minor one of 25 kDa.     

Complex interaction between serum folate levels and genetic polymorphisms in folate pathway genes: biomarkers of prostate cancer aggressiveness

Little is known about the role of folate and polymorphisms associated with folate metabolism on prostate cancer risk in populations of African origin. We examined the relationship between serum folate and prostate cancer and whether any association was modified by genetic polymorphisms for folate metabolism. The study was case–control in design and consisted of 218 men 40–80 years old with newly diagnosed, histologically confirmed prostate cancer and 236 cancer-free men attending the same urology clinics in Jamaica, March 2005–July 2007. Serum folate was measured by an immunoassay method and genomic DNA evaluated for MTHR (C677T and A1298C), MTRR A66G, and MTR A2756G polymorphisms. Mean serum folate concentration was higher among cases (12.3 ± 4.1 nmol/L) than controls (9.7 ± 4.2 nmol/L). Serum folate concentration showed a positive association with prostate cancer (OR, 4.41; CI, 2.52–7.72 per 10 nmol/L) regardless of grade. No interactions were observed between genotype and folate concentration, but a weak gene effect was observed for MTHFR A1298C and low-grade prostate cancer. Larger studies to investigate the role of gene–gene/gene–diet interactions in Black men are needed.     

Mediated uptake of folate by a high-affinity binding protein in sublines of L1210 cells adapted to nanomolar concentrations of folate

Summary An L1210 cell line (JT-1), which can grow in medium supplemented with 1nm folate, has been isolated. These cells exhibit a slower growth rate than folate-replete parental cells and have a lower ability to transport folate or methotrexate via the reduced folate transport system. Measurements at nanomolar concentrations of folate revealed that the adapted cells have acquired a high-affinity folate-binding protein. Binding to this component at 37°C was rapid and reached a maximum value after 30 min which corresponded in amount to 0.23±0.3 pmol/mg protein, and excess unlabeled folate added 30 min subsequent to the [³H]folate led to a rapid release of the bound substrate. Radioactivity bound to or released from the cells after 30 min at 37°C remained as unmetabolized folic acid. Binding was also rapid at 0°C but uptake at the plateau was only one-half the value obtained at 37°C. Half-maximal saturation of the binding component (K _D) occurred at a folate concentration of 0.065nm at pH 7.4, while the affinity for folate decreased 30-fold when the pH was reduced to 6.2 (K _D=2.0nm). 5-Methyltetrahydrofolate was also bound by this component (K _i=13nm at pH 7.4) but with a much lower affinity than for folate, while progressively weaker interactions were observed with 5-formyltetrahydrofolate (K _i=45nm) and methotrexate (K _i=325nm). When the same adaptation procedure was performed with limiting amounts of 5-formyltetrahydrofolate, two additional cell lines, JT-2 and JT-3, were isolated which expressed elevated levels of the folate-binding protein. The binding activity of the latter cells was 0.46 and 1.4 pmol/mg protein, respectively. When the level of binding protein was compared in cells grown at different concentrations of folate, an increase in medium folate from 1 to 500nm caused a sevenfold reduction in binding activity in the JT-3 cell line, while these same growth conditions had no effect on binding by the other cells. These results indicate that L1210 cells adapted to low concentrations of folate or 5-formyltetrahydrofolate contain elevated levels of a high-affinity binding protein and that this protein is able to mediate the intracellular accumulation of folate compounds. L1210 cells thus appear to have two potential uptake routes for folate compounds, the previously characterized anion-exchange system and a second route mediated by a high-affinity binding protein. An additional low-affinity, high-capacity transport system for folate that had been proposed previously was not observed under a variety of experimental conditions in either the adapted or parental cells.     

Reduced folate carrier A80G polymorphism and susceptibility to neural tube defects: A meta-analysis

The reduced folate carrier (RFC1) plays a crucial role in mediating folate delivery into a variety of cells. RFC1 polymorphism (A80G) has been reported to be associated with increased risk of neural tube defects (NTDs). However, results derived from individually underpowered studies are conflicting. We performed a systematic search of MEDLINE and EMBASE databases and carried out a meta-analysis on the association between RFC1 polymorphism (A80G) and NTDs risk. Overall, a significant correlation between RFC1 A80G polymorphism and NTDs risk was found neither in infants nor in maternal (allele contrast in infants: OR_RE = 1.15, 95% CI: 0.92–1.45; allele contrast in mothers: OR_RE = 1.24, 95% CI: 0.98–1.56). The present meta-analysis failed to support a positive association between RFC1 A80G polymorphism and susceptibility to NTDs. It is important to realize, however, that socio-economic factors, and gene–environment and gene–gene interactions, could have influenced the outcome of our meta-analysis. For this reason, a relationship between the A80G polymorphism and NTD risk cannot be entirely discounted.     

Anion specificity of the jejunal folate carrier: Effects of reduced folate analogues on folate uptake and efflux

Summary We previously reported that³H-folate uptake by rabbit jejunal brush-border membrane (BBM) vesicles was markedly stimulated by an outwardly directed OH^– gradient (pH_in 7.7, pH_out 5.5), inhibited by anion exchange inhibitors (DIDS, SITS, furosemide), and saturable (folateK _m=0.19 m) suggesting carrier-mediated folate/OH^– exchange (or H⁺/folate cotransport). In the present study, the anion specificity of this transport process was examined. Under conditions of an outwardly directed OH^– gradient, DIDS-sensitive folate uptake wascis inhibited (>90%) by reduced folate analogues: dihydrofolate (IC₅₀=0.40 m), folinic acid (IC₅₀=0.50 m), 5-methyltetrahydrofolate (IC₅₀=0.53 m), and (+)amethopterin (IC₅₀=0.93 M). In contrast, 10 m (–)amethopterin had only a modest effect on folate uptake (18% inhibition) suggesting stereospecificity of the folate/OH^– exchanger. The nonpteridine compounds which are transported by the folate carrier in L1210 leukemic cells (phthalate, thiamine pyrophosphate, and PO ₄ ^–3 ) did not inhibit jejunal folate uptake. Furthermore, folate uptake was not inhibited by SO ₄ ^–2 (4mm) or oxalate (4mm) thereby distinguishing this carrier from the previously described intestinal SO ₄ ^–2 /OH^– and oxalate/Cl^– exchangers. After BBM vesicles were loaded with³H-folate, the initial velocity of³H-folate efflux was stimulated by unlabeled folate in the efflux medium. The transstimulation of³H-folate efflux by unlabeled folate was furosemide (or DIDS) inhibitable and temperature sensitive. Half-maximal stimulation of furosemide-sensitive³H-folate efflux was observed with 0.25±0.05 m unlabeled folate, a concentration similar to theK _m for folate uptake. These data suggest that folate-stimulated³H-folate efflux is mediated by the folate/OH^– exchanger. With the exception of (–) amethopterin, reduced folate analogues also transstimulated furosemide-sensitive³H-folate efflux in a concentration-dependent manner suggesting stereospecific transport of these analogues by the folate/OH^– exchanger. In summary, folate transport by the jejunal folate/OH^– exchanger demonstrates bothcis inhibition and transstimulation by reduced folate analogues, but not by other inorganic or organic anions suggesting bidirectional transport of folate and a high degree of anion specificity.     

Effect of genetic polymorphisms involved in folate metabolism on the concentration of serum folate and plasma total homocysteine (p-tHcy) in healthy subjects after short-term folic acid supplementation: a randomized,double blind,crossover study

Data on the effect of combined genetic polymorphisms, involved in folate metabolism, on the concentration of serum folate after folic acid supplementation are scarce. Therefore, we investigated the impact of seven gene polymorphisms on the concentration of serum folate and p-tHcy in healthy subjects after short-term folic acid supplementation. In a randomized, double blind, crossover study, apparently healthy subjects were given either 0.8 mg folic acid per day (n = 46) or placebo (n = 45) for 14 days. The washout period was 14 days. Fasting blood samples were collected on day 1, 15, 30 and 45. Data on subjects on folic acid supplementation (n = 91) and on placebo (n = 45) were used for the statistical analysis. The concentration of serum folate increased higher in subjects with higher age (53.5 ± 7.0 years) than in subjects with lower age (24.3 ± 3.2 years) after folic acid supplementation (p = 0.006). The baseline concentration of serum folate in subjects with polymorphism combination, reduced folate carrier protein, RFC1-80 GA and methylenetetrahydrofolate reductase, MTHFR677 CT+TT, was lower than RFC1-80 AA and MTHFR677 CT+TT (p = 0.002). After folic acid supplementation, a higher increase in the concentration of serum folate was detected in subjects with polymorphism combination RFC1-80 GA and MTHFR677 CC than RFC1-80 GG and MTHFR CT+TT combination (p < 0.0001). The baseline concentration of plasma total homocysteine (p-tHcy) was altered by combined polymorphisms in genes associated with folate metabolism. After folic acid supplementation, in subjects with combined polymorphisms in methylenetetrahydrofolate dehydrogenase, MTHFD1-1958 and MTHFR-677 genes, the concentration of p-tHcy was changed (p = 0.002). The combination of RFC1-80 and MTHFR-677 polymorphisms had a profound affect on the concentration of serum folate in healthy subjects before and after folic acid supplementation.     

Erythrocyte folate concentrations,CpG methylation at genomically imprinted domains,and birth weight in a multiethnic newborn cohort

Epigenetic mechanisms are proposed to link maternal concentrations of methyl group donor nutrients with the risk of low birth weight. However, empirical data are lacking. We have examined the association between maternal folate and birth weight and assessed the mediating role of DNA methylation at nine differentially methylated regions (DMRs) of genomically imprinted genes in these associations. Compared with newborns of women with folate levels in the lowest quartile, birth weight was higher in newborns of mothers in the second (β = 143.2, se = 63.2, P = 0.02), third (β = 117.3, se = 64.0, P = 0.07), and fourth (β = 133.9, se = 65.2, P = 0.04) quartiles, consistent with a threshold effect. This pattern of association did not vary by race/ethnicity but was more apparent in newborns of non-obese women. DNA methylation at the PLAGL1, SGCE, DLK1/MEG3 and IGF2/H19 DMRs was associated with maternal folate levels and also birth weight, suggestive of threshold effects. MEG3 DMR methylation mediated the association between maternal folate levels and birth weight (P =0.06). While the small sample size and partial scope of examined DMRs limit our conclusions, our data suggest that, with respect to birth weight, no additional benefits may be derived from increased maternal folate concentrations, especially in non-obese women. These data also support epigenetic plasticity as a key mechanistic response to folate availability during early fetal development.     

High-affinity folate binding in human mammary gland

High-affinity³H-folate binding in Triton X-100 solubilized human mammary gland tissue displayed characteristics, e.g. apparent positive cooperativity and increasing affinity with decreasing concentration of folate binding protein, shown to be typical of specific folate binding. Radioligand dissociation was slow at pH 7.4. A major fraction of the bound radioligand dissociated rapidly at pH 3.5, while a residual binding of 20% persisted even after prolonged dialysis at pH 3.5. Gel chromatography revealed two major folate binding proteins (M_r100 kDa and 25 kDa). However, only one single band was detectable on SDS-PAGE immunoblotting. The highest folate binding activity per g protein was associated with the upper triglyceride-containing layer of the 1000 g supernatant of the homogenate. The folate binding protein extracted from this layer had a low cross-reactivity (<5%) with rabbit antibodies against 25 kDa human milk folate binding protein. The folate binding protein in the 1000 g pellet and the aqueous phase of the 1000 g supernatant was present at a low concentration and had a cross-reactivity of 100%.     

Embryonic development in the reduced folate carrier knockout mouse is modulated by maternal folate supplementation

BACKGROUND: The reduced folate carrier (RFC1) is a ubiquitously expressed integral membrane protein that mediates delivery of 5‐methyltetrahydrofolate into mammalian cells. In this study, embryonic/fetal development is characterized in an RFC1 knockout mouse model in which pregnant dams receive different levels of folate supplementation. METHODS: RFC1^+/? males were mated to RFC1^+/? females, and pregnant dams were treated with vehicle (control) or folic acid (25 or 50 mg/kg) by daily subcutaneous injection (0.1 mL/10 g bwt), beginning on E0.5 and continuing throughout gestation until the time of sacrifice. RESULTS: Without maternal folate supplementation, RFC1 nullizygous embryos die shortly postimplantation. Supplementation of pregnant dams with 25 mg/kg/day folic acid prolongs survival of mutant embryos until E9.5–E10.5, but they are developmentally delayed relative to wild‐type littermates, display a marked absence of erythropoiesis, severe neural tube and limb bud defects, and failure of chorioallantoic fusion. Fgfr2 protein levels are significantly reduced or absent in the extraembryonic membranes of RFC1 nullizygous embryos. Maternal folate supplementation with 50 mg/kg/day results in survival of 22% of RFC1 mutants to E18.5, but they develop with multiple malformations of the eyelids, lungs, heart, and skin. CONCLUSIONS: High doses of daily maternal folate supplementation during embryonic/fetal development are necessary for early postimplantation embryonic viability of RFC1 nullizygous embryos, and play a critical role in chorioallantoic fusion, erythropoiesis, and proper development of the neural tube, limbs, lungs, heart, and skin. Birth Defects Research (Part A), 2008. © 2008 Wiley‐Liss, Inc.     

A high-affinity folate binding protein in human amniotic fluid. Radioligand binding characteristics,immunological properties and molecular size

The presence of a folate binding protein of high-affinity type (affinity constant 5 · 10⁹M^–1, maximum folate binding 3 nM) in human amniotic fluid was demonstrated in equilibrium dialysis experiments (37°C, pH 7.4) with the radioligand³H-folate. Dissociation of³H-folate from the binding protein was slow at pH 7.4 but rapid at pH 3.5. By use of rabbit antibodies against low molecular weight folate binding protein from human milk we determined the concentration of folate binding protein in 5 amniotic fluids (range 1.5–2.3 nM) in an Enzyme-Linked Immunosorbent Assay (ELISA). ultrogel AcA 44 chromatography of amniotic fluid showed that immunoreactive and radioligand bound folate binding protein coeluted in two peaks: a major one (M _r 25 000) and a minor one (M _r 100 000).     

The potential of bifidobacteria as a source of natural folate

Aims: To screen 19 strains of bifidobacteria for main folate forms composition in synthetic folate‐free and complex folate‐containing media. Methods and Results: HPLC was used to analyse deconjugated folates extracted from bacterial biomass. Most strains had a total folate content above 4000 μg per 100 g dry matter (DM). The highest value of 9295 μg per 100 g DM was found in Bifidobacterium catenulatum ATCC 27539 and the lowest in Bifidobacterium animalis ssp. animalis ATCC 25527 containing 220 μg per 100 g DM. Ten strains grew in a synthetic folate‐free medium (FFM), showing folate autotrophy and suggesting folate auxotrophy of the remaining nine. In the autotrophic strains, a consistently higher folate level was found in FFM as compared to a more complex folate‐containing medium, suggesting reduced requirements for folates in the presence of growth factors otherwise requiring folates for synthesis. The contents of total folate, 5‐CH₃‐H₄folate and H₄folate were strain dependent. 5‐CH₃‐H₄folate dominated in most strains. Conclusions: Our results show that bifidobacteria folate content and composition is dynamic, is strain specific and depends on the medium. Suitable selection of the growth conditions can result in high levels of folate per cell unit biomass. Significance and Impact of the Study: This suggests that certain bifidobacteria may contribute to the folate intake, either directly in foods, such as fermented dairy products, or in the intestine as folate‐trophic probiotics or part of the natural microbiota.     

Synthesis of folate derivatives in aerated storage tissue disks

Microbiological assay of extracts prepared from carrot, potato, turnip and beet storage tissue disks revealed that folate derivatives were synthesized during a 48 hr aeration period in sterile distilled water. The composition of the folate pool in carrot was examined by DEAE—cellulose column chromatography, γ-glutamylcarboxypeptidase treatment and differential assay of individual derivatives using Lactobacillus casei and Streptococcus faecalis. The principal folates were polyglutamates of formyl and methyl tetrahydrofolate. Smaller quantities of the corresponding mono- and di-glutamates were also detected. The latter derivatives occurred in pools having a high degree of metabolic turnover. The specific activities of three enzymes catalyzing production of these derivatives from tetrahydrofolate increased during the first 12 hr of aeration. Amino acid analyses revealed that folate synthesis in carrot disks was accompanied by depletion of free serine and by net synthesis of free and protein methionine.     

A high-affinity folate binding protein in human urine. Radioligand binding characteristics,immunological properties and molecular size

High-affinity binding of [³H]folate in human urine displayed characteristics, e.g. apparent positive cooperativity, which are typical of specific folate binding. By means of a two-site enzyme-linked immunosorbent assay (ELISA) with rabbit antibodies against the low molecular weight folate binding protein from human milk, we measured folate binding protein concentrations in the range of 0.51 to 4.13 nM in urine samples from 16 apparently healthy individuals. Ultrogel AcA 44 chromatography of the urine showed that immunoreactive and radioligand bound folate binding protein coeluted in one large peak (M_r25,000).     

Simultaneous activity of two different mechanisms of folate transport in ovarian carcinoma cell lines

We investigated whether the folate receptor α-isoform (FRα), which is overexpressed on ovarian carcinoma cells, is functionally active in internalizing the physiological form of folate, 5-methyl tetrahydrofolate (THF). Six ovarian tumor cell lines, expressing different levels of FRα (COR ≫ OVCAR3 > IGROV1 > OVCAR4 > SKOV3 > OVCAR5), were maintained in folate-depleted medium and internalization of 10 nM evaluated as acid-resistant radioactivity at 0° and 37°C. The amount of 5-methyl[³H]THF present in this fraction was not strictly related to the number of membrane receptors, since even cell lines with low FRα expression, e.g., OVCAR4, showed efficient internalization. Time-course studies indicated that, whereas no uptake was detected at 0°C, at 37°C the internalized fraction showed a slow and constant increase, until 4 h. At this time, the internalized radioactivity represented <50% of the total bound in COR, OVCAR3 and IGROV1 cells, whereas the other cell lines tested internalized fourfold more folate than their surface binding capacity. The incubation in the presence of a concentration (50 nM) of 5-methyl[³H]THF, which best ensures receptors saturation on cells with highest FR levels (COR and OVCAR3), had slight effect on surface binding of all the tested cell lines, including IGROV1 and SKOV3. In contrast, the increase of the uptake was more pronounced, particularly in SKOV3 cells. These results, together with the accumulation curves of folic acid (FA) and 5-methylTHF at 37°C, suggested the presence of a molecule on ovarian carcinoma cells with high affinity for reduced folates, possibly a reduced folate carrier (RFC). Measurement of radioactivity present in the supernatant of IGROV1 and SKOV3 cells, subjected to hypotonic lysis and cell fractionation, further indicated that 5-methyl[³H]THF was translocated to the cytosol and, despite differences in membrane levels of FRα expression this internalized fraction was similar in both cell lines. Inhibition experiments to selectively block FRα or RFC activity showed a differential sensitivity of the two pathways depending on the cell line examined. Internalization was more consistently inhibited on IGROV1 than on SKOV3 cells by treatments that disrupt FRα activity, e.g., incubation with excess FA and phosphatidylinositol specific phospholipase C, whereas Probenecid, which preferentially inhibits the carrier-mediated pathway, showed a strong inhibitory effect on both cell lines. These findings suggest that the internalization of 5-methylTHF in these tumor cells depends not only on the level of overexpressed FRα, but another transport route, with features characteristic for RFC, is functional and participates in folate uptake. J. Cell. Biochem. 65:479–491. © 1997 Wiley-Liss Inc.     

Quantification of key folate forms in serum using stable-isotope dilution ultra performance liquid chromatography–tandem mass spectrometry

Folates act as essential coenzymes in many biological pathways. Alteration in folate form distribution might have biological significance, especially in relation to certain genetic polymorphisms. We developed a stable-isotope dilution ultra performance liquid chromatography–mass spectrometry (UPLC–MS/MS) method for quantification of the folate forms 5-methyltetrahydrofolate (5-methylTHF), 5-formylTHF, 5,10-methenylTHF, THF, and folic acid in serum. After extraction using an ion exchange and mixed mode solid-phase, samples were separated and detected using an UPLC–MS/MS system. The quantification limits were between 0.17 nmol/L (5-formylTHF) and 1.79 nmol/L (THF), and the assay was linear up to 100 nmol/L (5-methylTHF) and 10 nmol/L (5-formylTHF, 5,10-methenylTHF, THF, and folic acid). The intraassay CVs for 5-methylTHF and 5-formylTHF were 2.0% and 7.2%, respectively. Mean recoveries were between 82.3% for THF and 110.8% for 5,10-methenylTHF. Concentrations of total folate measured by the new method showed a strong correlation with those measured by an immunologic assay (r = 0.939; p < 0.001). The mean total folate from 32 apparently healthy subjects was 18.09 nmol/L, of which 87.23% was 5-methylTHF. Concentrations of homocysteine showed a better correlation to the total folate measured by the new method compared to that obtained by an immunologic assay. We also confirmed that MTHFR polymorphism has a significant effect on folate distribution in this small population of non-supplemented subjects.     

Impact of folic acid fortification on global DNA methylation and one-carbon biomarkers in the Women's Health Initiative Observational Study cohort

DNA methylation is an epigenetic mechanism that regulates gene expression and can be modified by one-carbon nutrients. The objective of this study was to investigate the impact of folic acid (FA) fortification of the US food supply on leukocyte global DNA methylation and the relationship between DNA methylation, red blood cell (RBC) folate, and other one-carbon biomarkers among postmenopausal women enrolled in the Women's Health Initiative Observational Study. We selected 408 women from the highest and lowest tertiles of RBC folate distribution matching on age and timing of the baseline blood draw, which spanned the pre- (1994–1995), peri- (1996–1997), or post-fortification (1998) periods. Global DNA methylation was assessed by liquid chromatography-tandem mass spectrometry and expressed as a percentage of total cytosine. We observed an interaction (P = 0.02) between fortification period and RBC folate in relation to DNA methylation. Women with higher (vs. lower) RBC folate had higher mean DNA methylation (5.12 vs. 4.99%; P = 0.05) in the pre-fortification period, but lower (4.95 vs. 5.16%; P = 0.03) DNA methylation in the post-fortification period. We also observed significant correlations between one-carbon biomarkers and DNA methylation in the pre-fortification period, but not in the peri- or post-fortification period. The correlation between plasma homocysteine and DNA methylation was reversed from an inverse relationship during the pre-fortification period to a positive relationship during the post-fortification period. Our data suggest that (1) during FA fortification, higher RBC folate status is associated with a reduction in leukocyte global DNA methylation among postmenopausal women and; (2) the relationship between one-carbon biomarkers and global DNA methylation is dependent on folate availability.     

An apicoplast‐resident folate transporter is essential for sporogony of malaria parasites

Malaria parasites are fast replicating unicellular organisms and require substantial amounts of folate for DNA synthesis. Despite the central role of this critical co‐factor for parasite survival, only little is known about intraparasitic folate trafficking in Plasmodium. Here, we report on the expression, subcellular localisation and function of the parasite's folate transporter 2 (FT2) during life cycle progression in the murine malaria parasite Plasmodium berghei. Using live fluorescence microscopy of genetically engineered parasites, we demonstrate that FT2 localises to the apicoplast. In invasive P. berghei stages, a fraction of FT2 is also observed at the apical end. Upon genetic disruption of FT2, blood and liver infection, gametocyte production and mosquito colonisation remain unaltered. But in the Anopheles vector, FT2‐deficient parasites develop inflated oocysts with unusual pulp formation consisting of numerous single‐membrane vesicles, which ultimately fuse to form large cavities. Ultrastructural analysis suggests that this defect reflects aberrant sporoblast formation caused by abnormal vesicular traffic. Complete sporogony in FT2‐deficient oocysts is very rare, and mutant sporozoites fail to establish hepatocyte infection, resulting in a complete block of parasite transmission. Our findings reveal a previously unrecognised organellar folate transporter that exerts critical roles for pathogen maturation in the arthropod vector.     

Irreversible inhibition of folate transport in Lactobacillus casei by covalent modification of the binding protein with carbodiimide-activated folate

Activated folate formed by reaction of folic acid and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide irreversibly inhibits the folate transport system of Lactobacillus casei. Complete inhibition of both folate binding to the carrier protein and folate transport was achieved by pretreatment of the cells at low temperature (4 °C) and at neutral pH with 200 nm activated folate. Fifty percent inhibition of binding and transport occurred at 35 and 40 nm activated folate, respectively. Specificity was demonstrated by the fact that excess nonactivated folate added during the pretreatment step afforded complete protection of the binding protein against inhibition, and that activated folate had no effect on the binding or transport of thiamine. Rapid measurements at 4 °C were employed to show that, prior to the appearance of irreversible inhibition, activated folate (K_i = 15 nM) interacted reversibly with the binding site for folate (K_d = 0.8 nM). Cells treated with activated [³H]folate incorporated 1 mol of folate per mole of binding protein. Purification of the labeled protein followed by digestion with Pronase led to the isolation of a compound identified as ?-N-folyl lysine. The ?-amino group of a lysyl residue thus appears to be the nucleophilic group at the binding site that reacts with activated folate.