Functional analysis of folate polyglutamylation and its essential role in plant metabolism and development

Cellular folates function as co-enzymes in one-carbon metabolism and are predominantly decorated with a polyglutamate tail that enhances co-enzyme affinity, subcellular compartmentation and stability. Polyglutamylation is catalysed by folylpolyglutamate synthetases (FPGSs) that are specified by three genes in Arabidopsis, FPGS1, 2 and 3, which reportedly encode plastidic, mitochondrial and cytosolic isoforms, respectively. A mutational approach was used to probe the functional importance of folate polyglutamylation in one-carbon metabolism and development. Biochemical analysis of single FPGS loss-of-function mutants established that folate polyglutamylation is essential for organellar and whole-plant folate homeostasis. However, polyglutamylated folates were still detectable, albeit at lower levels, in organelles isolated from the corresponding isozyme knockout lines, e.g. in plastids and mitochondria of the fpgs1 (plastidial) and fpgs2 (mitochondrial) mutants. This result is surprising given the purported single-compartment targeting of each FPGS isozyme. These results indicate redundancy in compartmentalised FPGS activity, which in turn explains the lack of anticipated phenotypic defects for the single FPGS mutants. In agreement with this hypothesis, fpgs1 fpgs2 double mutants were embryo-lethal, fpgs2 fpgs3 mutants exhibited seedling lethality, and fpgs1 fpgs3 mutants were dwarfed with reduced fertility. These phenotypic, metabolic and genetic observations are consistent with targeting of one or more FPGS isozymes to multiple organelles. These data confirm the importance of polyglutamylation in folate compartmentation, folate homeostasis and folate-dependent metabolic processes, including photorespiration, methionine and pantothenate biosynthesis.     

A mutation inactivating the mitochondrial inner membrane folate transporter creates a glycine requirement for survival of chinese hamster cells

A mutant Chinese hamster ovary cell line, glyB, that required exogenous glycine for survival and growth was reported previously (Kao, F., Chasin, L., and Puck, T. T. (1969) Proc. Natl. Acad. Sci. U. S. A. 64, 1284-1291). We now report that the defect in glyB cells causative of this phenotype is a point mutation in an inner mitochondrial membrane protein required for transport of folates into mitochondria. The CHO mitochondrial folate transporter (mft) was sequenced and compared with that from glyB cells. The hamster sequence was nearly identical to that of the recently reported human mitochondrial folate transporter. The corresponding cDNA from glyB cells contained a single nucleotide change that introduced a glutamate in place of the glycine in wild-type hamster MFT at codon 192 in a predicted transmembrane domain. Transfection of the wild-type hamster cDNA into glyB cells allowed cell survival in the absence of glycine and the accumulation of folates in mitochondria, whereas transfection of the Glu-192 cDNA did not. Genomic sequence analysis and fluorescence in situ hybridization demonstrated a single mutated allele of the mft gene in glyB cells, whereas there were two alleles in CHO cells. We conclude that we have defined the cause of the glyB auxotrophy and that the glyB mft mutation identified a region of this mitochondrial folate carrier vital to its transport function.     

Retrovirally mediated complementation of the glyB phenotype. Cloning of a human gene encoding the carrier for entry of folates into mitochondria

The transduction of a human placental cDNA retroviral library into glyB cells, a Chinese hamster ovary K1 subline that is deficient in the transport of folates into mitochondria, resulted in the complementation of glycine auxotrophy of these cells. A 2.6-kilobase pair cDNA insert flanked by retroviral sequences had integrated into genomic DNA in rescued cells. An open reading frame in this cDNA encoded a 35-kDa protein homologous to several inner mitochondrial wall transporters for intermediate metabolites. The subcloned cDNA complemented the glycine auxotrophy of glyB cells and reinstated folate accumulation in the mitochondria of transfected cells. The human origin, chromosomal location, and intron-exon organization of the isolated mitochondrial folate transporter gene were deduced from the expressed sequence tag database and human genome project data.     

Both metaxin and Tom20 together with two mitochondria-specific motifs support mitochondrial targeting of dual-targeting AtSufE1

Plant cells have two endosymbiotic organelles, chloroplasts, and mitochondria. These organelles perform specific functions that depend on organelle-specific proteins. The majority of chloroplast and mitochondrial proteins are specifically imported by the transit peptide and presequence, respectively. However, a significant number of proteins are also dually targeted to these two organelles. Currently, it is not fully understood how proteins are dually targeted to both chloroplasts and mitochondria. In this study, the mechanism underlying mitochondrial targeting of dual targeting AtSufE1 in Arabidopsis was elucidated. The N-terminal fragment containing 80 residues of AtSufE1 (AtSufE1N80) was sufficient to confer dual targeting of reporter protein, AtSufE1N80:GFP, in protoplasts. Two sequence motifs, two arginine residues at 15^th and 21^st positions, and amino acid (aa) sequence motif AKTLLLRPLK from the 31^st to 40^th aa position, were responsible for targeting to mitochondria a portion of reporter proteins amid the chloroplast targeting. The sequence motif PSEVPFRRT from the 41^st to 50^th aa position constitutes a common motif for targeting to both chloroplasts and mitochondria. For mitochondrial import of AtSufE1:N80, Metaxin played a critical role. In addition, BiFC and protein pull-down experiments showed that AtSufE1N80 specifically interacts with import receptors, Metaxin and Tom20. The interaction of AtSufE1N80 with Metaxin was required for the interaction with Tom20. Based on these results, we propose that mitochondrial targeting of dual-targeting AtSufE1 is mediated by both mitochondria-specific and common sequence motifs in the signal sequence through the interaction with import receptors, Metaxin and Tom20, in a successive manner.     

Depleted folate pool and dysfunctional mitochondria associated with defective mitochondrial folate proteins sensitize Chinese ovary cell mutants to tert-butylhydroperoxide-induced oxidative stress and apoptosis

The functional role of mitochondrial (mt) folate-associated proteins in mammalian cells is not clearly understood. We investigated the respiratory function and apoptosis phenotype of Chinese hamster ovary (CHO) mutant cells with defective mt serine hydroxymethyltransferase (SHMT) activities (glyA) or with defective mt folate transporter (glyB) in the absence/presence of oxidant challenge. The mechanisms underlying their aberrant phenotypes were explored. Compared with CHOK1 wild-type cells, both mutants carried dysfunctional mitochondria with reduced respiratory complex IV activity and depolarized mt membrane potential (P<.05). Elevated superoxide levels and accumulated mtDNA large deletions were observed in glyB in association with a depleted compartmental folate pool (P<.05). tert-Butylhydroperoxide (tBH) treatment at 50 μM for 72 h significantly depleted mt and cytosolic folate levels, impaired antioxidant defenses, and aggravated mt oxidative dysfunction in both mutants (P<.05), more severely in glyB. Only tBH-treated glyB cells displayed an elevated ratio of mt Bax/Bcl-2, activation of procaspases 9 and 3, and apoptosis promotion. The apoptotic phenotype of tBH-treated glyB could be partially corrected by folate supplementation (10–1000 μM), which enriched compartmental folate levels, restored antioxidant defenses, eliminated mt oxidative injuries, and normalized mt membrane function. Our data identify previously unrecognized roles of mt folate-associated proteins in the protection of mitochondria against oxidative insults. Defective mt folate transporter sensitized glyB cells to elevated oxidative stress and tBH-induced apoptosis, partly mediated by depleted compartmental folate and mt dysfunction. Defective mt SHMT sensitized glyA to respiratory dysfunction and tBH-induced oxidative injury without apoptosis promotion.     

Dual intracellular localization and targeting of aminoimidazole ribonucleotide synthetase in cowpea

De novo purine biosynthesis is localized to both mitochondria and plastids isolated from Bradyrhizobium sp.-infected cells of cowpea (Vigna unguiculata L. Walp) nodules, but several of the pathway enzymes, including aminoimidazole ribonucleotide synthetase (AIRS [EC 6.3.3.1], encoded by Vupur5), are encoded by single genes. Immunolocalization confirmed the presence of AIRS protein in both organelles. Enzymatically active AIRS was purified separately from nodule mitochondria and plastids. N-terminal sequencing showed that these two isoforms matched the Vupur5 cDNA sequence but were processed at different sites following import; the mitochondrial isoform was five amino acids longer than the plastid isoform. Electrospray tandem mass spectrometry of a trypsin digest of mitochondrial AIRS identified two internal peptides identical with the amino acid sequence deduced from Vupur5 cDNA. Western blots of proteins from mitochondria and plastids isolated from root tips showed a single AIRS protein present at low levels in both organelles. (35)S-AIRS protein translated from a Vupur5 cDNA was imported into isolated pea (Pisum sativum) leaf chloroplasts in vitro by an ATP-dependent process but not into import-competent mitochondria from several plant and non-plant sources. Components of the mature protein are likely to be important for import because the N-terminal targeting sequence was unable to target green fluorescent protein to either chloroplasts or mitochondria in Arabidopsis leaves. The data confirm localization of the protein translated from the AIRS gene in cowpea to both plastids and mitochondria and that it is cotargeted to both organelles, but the mechanism underlying import into mitochondria has features that are yet to be identified.     

Arabidopsis non-host resistance PSS30 gene enhances broad-spectrum disease resistance in the soybean cultivar Williams 82

Non-host resistance (NHR), which protects all members of a plant species from non-adapted or non-host plant pathogens, is the most common form of plant immunity. NHR provides the most durable and robust form of broad-spectrum immunity against non-adaptive pathogens pathogenic to other crop species. In a mutant screen for loss of Arabidopsis (Arabidopsis thaliana) NHR against the soybean (Glycine max (L.) Merr.) pathogen Phytophthora sojae, the Phytophthora sojae-susceptible 30 (pss30) mutant was identified. The pss30 mutant is also susceptible to the soybean pathogen Fusarium virguliforme. PSS30 encodes a folate transporter, AtFOLT1, which was previously localized to chloroplasts and implicated in the transport of folate from the cytosol to plastids. We show that two Arabidopsis folate biosynthesis mutants with reduced folate levels exhibit a loss of non-host immunity against P. sojae. As compared to the wild-type Col-0 ecotype, the steady-state folate levels are reduced in the pss1, atfolt1 and two folate biosynthesis mutants, suggesting that folate is required for non-host immunity. Overexpression of AtFOLT1 enhances immunity of transgenic soybean lines against two serious soybean pathogens, the fungal pathogen F. virguliforme and the soybean cyst nematode (SCN) Heterodera glycines. Transgenic lines showing enhanced SCN resistance also showed increased levels of folate accumulation. This study thus suggests that folate contributes to non-host plant immunity and that overexpression of a non-host resistance gene could be a suitable strategy for generating broad-spectrum disease resistance in crop plants.     

A novel in vitro system for simultaneous import of precursor proteins into mitochondria and chloroplasts

Most chloroplast and mitochondrial precursor proteins are targeted specifically to either chloroplasts or mitochondria. However, there is a group of proteins that are dual targeted to both organelles. We have developed a novel in vitro system for simultaneous import of precursor proteins into mitochondria and chloroplasts (dual import system). The mitochondrial precursor of alternative oxidase, AOX was specifically targeted only to mitochondria. The chloroplastic precursor of small subunit of pea ribulose bisphosphate carboxylase/oxygenase, Rubisco, was mistargeted to pea mitochondria in a single import system, but was imported only into chloroplasts in the dual import system. The dual targeted glutathione reductase GR precursor was targeted to both mitochondria and chloroplasts in both systems. The GR pre-sequence could support import of the mature Rubisco protein into mitochondria and chloroplasts in the single import system but only into chloroplasts in the dual import system. Although the GR pre-sequence could support import of the mature portion of the mitochondrial FAd subunit of the ATP synthase into mitochondria and chloroplasts, mature AOX protein was only imported into mitochondria under the control of the GR pre-sequence in both systems. These results show that the novel dual import system is superior to the single import system as it abolishes mistargeting of chloroplast precursors into pea mitochondria observed in a single organelle import system. The results clearly show that although the GR pre-sequence has dual targeting ability, this ability is dependent on the nature of the mature protein.     

Mitochondrial and plastidial COG0354 proteins have folate-dependent functions in iron-sulphur cluster metabolism

COG0354 proteins have been implicated in synthesis or repair of iron/sulfur (Fe/S) clusters in all domains of life, and those of bacteria, animals, and protists have been shown to require a tetrahydrofolate to function. Two COG0354 proteins were identified in Arabidopsis and many other plants, one (At4g12130) related to those of α-proteobacteria and predicted to be mitochondrial, the other (At1g60990) related to those of cyanobacteria and predicted to be plastidial. Grasses and poplar appear to lack the latter. The predicted subcellular locations of the Arabidopsis proteins were validated by in vitro import assays with purified pea organelles and by targeting assays in Arabidopsis and tobacco protoplasts using green fluorescent protein fusions. The At4g12130 protein was shown to be expressed mainly in flowers, siliques, and seeds, whereas the At1g60990 protein was expressed mainly in young leaves. The folate dependence of both Arabidopsis proteins was established by functional complementation of an Escherichia coli COG0354 (ygfZ) deletant; both plant genes restored in vivo activity of the Fe/S enzyme MiaB but restoration was abrogated when folates were eliminated by deleting folP. Insertional inactivation of At4g12130 was embryo lethal; this phenotype was reversed by genetic complementation of the mutant. These data establish that COG0354 proteins have a folate-dependent function in mitochondria and plastids, and that the mitochondrial protein is essential. That plants retain mitochondrial and plastidial COG0354 proteins with distinct phylogenetic origins emphasizes how deeply the extant Fe/S cluster assembly machinery still reflects the ancient endosymbioses that gave rise to plants.     

A central role for gamma-glutamyl hydrolases in plant folate homeostasis

Most cellular folates carry a short poly-γ-glutamate tail, and this tail is believed to affect their efficacy and stability. The tail can be removed by γ-glutamyl hydrolase (GGH; EC 3.4.19.9), a vacuolar enzyme whose role in folate homeostasis remains unclear. In order to probe the function of GGH, we modulated its level of expression and subcellular location in Arabidopsis plants and tomato fruit. Three-fold overexpression of GGH in vacuoles caused extensive deglutamylation of folate polyglutamates and lowered the total folate content by approximately 40% in Arabidopsis and tomato. No such effects were seen when GGH was overexpressed to a similar extent in the cytosol. Ablation of either of the major Arabidopsis GGH genes (AtGGH1 and AtGGH2) alone did not significantly affect folate status. However, a combination of ablation of one gene plus RNA interference (RNAi)-mediated suppression of the other (which lowered total GGH activity by 99%) increased total folate content by 34%. The excess folate accumulated as polyglutamate derivatives in the vacuole. Taken together, these results suggest a model in which: (i) folates continuously enter the vacuole as polyglutamates, accumulate there, are hydrolyzed by GGH, and exit as monoglutamates; and (ii) GGH consequently has an important influence on polyglutamyl tail length and hence on folate stability and cellular folate content.     

Characterization of folate uptake by choroid plexus epithelial cells in a rat primary culture model

Reduced derivatives of folic acid (folates) play a critical role in the development, function and repair of the CNS. However, the molecular systems regulating folate uptake and homeostasis in the central nervous system remain incompletely defined. Choroid plexus epithelial cells express high levels of folate receptor α (FRα) suggesting that the choroid plays an important role in CNS folate trafficking and maintenance of CSF folate levels. We have characterized 5-methyltetrahydrofolate (5-MTHF) uptake and metabolism by primary rat choroid plexus epithelial cells in vitro . Two distinct processes are apparent; one that is FRα dependent and one that is independent of the receptor. FRα binds 5-MTHF with high affinity and facilitates efficient uptake of 5-MTHF at low extracellular folate concentrations; a lower affinity FRα independent system accounts for increased folate uptake at higher concentrations. Cellular metabolism of 5-MTHF depends on the route of folate entry into the cell. 5-MTHF taken up via a non-FRα -mediated process is rapidly metabolized to folylpolyglutamates, whereas 5-MTHF that accumulates via FRα remains non-metabolized, supporting the hypothesis that FRα may be part of a pathway for transcellular movement of the vitamin. The proton-coupled folate transporter, proton-coupled folate transporter (PCFT), mRNA was also shown to be expressed in choroid plexus epithelial cells. This is consistent with the role we have proposed for proton-coupled folate transporter in FRα-mediated transport as the mechanism of export of folates from the endocytic compartment containing FRα.     

Rodent intestinal folate transporters (SLC46A1): secondary structure, functional properties, and response to dietary folate restriction

This laboratory recently identified a human gene that encodes a novel folate transporter [Homo sapiens proton-coupled folate transporter (HsPCFT); SLC46A1] required for intestinal folate absorption. This study focused on mouse (Mus musculus) PCFT (MmPCFT) and rat (Rattus norvegicus) PCFT (RnPCFT) and addresses their secondary structure, specificity, tissue expression, and regulation by dietary folates. Both rodent PCFT proteins traffic to the cell membrane with the NH(2)- and COOH-termini accessible to antibodies targeted to these domains only in permeabilized HeLa cells. This, together with computer-based topological analyses, is consistent with a model in which rodent PCFT proteins likely contain 12 transmembrane domains. Transport of [(3)H]folates was optimal at pH 5.5 and decreased with increasing pH due to an increase in K(m) and a decrease in V(max). At pH 7.0, folic acid and methotrexate influx was negligible, but there was residual (6S)5-methyltetrahydrofolate transport. Uptake of folates in PCFT-injected Xenopus oocytes was electrogenic and pH dependent. Folic acid influx K(m) values of MmPCFT and RnPCFT, assessed electrophysiologically, were 0.7 and 0.3 microM at pH 5.5 and 1.1 and 0.8 microM at pH 6.5, respectively. Rodent PCFTs were highly specific for monoglutamyl but not polyglutamyl methotrexate. MmPCFT mRNA was highly expressed in the duodenum, proximal jejunum, liver, and kidney with lesser expression in the brain and other tissues. MmPCFT protein was localized to the apical brush-border membrane of the duodenum and proximal jejunum. MmPCFT mRNA levels increased approximately 13-fold in the proximal small intestine in mice fed a folate-deficient vesus folate-replete diet, consistent with the critical role that PCFT plays in intestinal folate absorption.     

The enteropathogenic Escherichia coli (EPEC) Map effector is imported into the mitochondrial matrix by the TOM/Hsp70 system and alters organelle morphology

Enteropathogenic Escherichia coli (EPEC) is a human intestinal pathogen and a major cause of diarrhoea, particularly among infants in developing countries. EPEC target the Map and EspF multifunctional effector proteins to host mitochondria - organelles that play crucial roles in regulating cellular processes such as programmed cell death (apoptosis). While both molecules interfere with the organelles ability to maintain a membrane potential, EspF plays the predominant role and is responsible for triggering cell death. To learn more about the Map-mitochondria interaction, we studied Map localization to mitochondria with purified mitochondria (from mammalian and yeast cells) and within intact yeast. This revealed that (i) Map targeting is dependent on the predicted N-terminal mitochondrial targeting sequence, (ii) the N-terminal 44 residues are sufficient to target proteins to mitochondria and (iii) Map import involves the mitochondrial outer membrane translocase (Tom22 and Tom40), the mitochondrial membrane potential, and the matrix chaperone, mtHsp70. These results are consistent with Map import into the mitochondria matrix via the classical import mechanism. As all known, Map-associated phenotypes in mammalian cells are independent of mitochondrial targeting, this may indicate that import serves as a mechanism to remove Map from the cytoplasm thereby regulating cytoplasmic function. Intriguingly, Map, but not EspF, alters mitochondrial morphology with deletion analysis revealing important roles for residues 101-152. Changes in mitochondrial morphology have been linked to alterations in the ability of these organelles to regulate cellular processes providing a possible additional role for Map import into mitochondria.     

Mammalian Mitochondrial and Cytosolic Folylpolyglutamate Synthetase Maintain the Subcellular Compartmentalization of Folates

Folylpoly-γ-glutamate synthetase (FPGS) catalyze the addition of multiple glutamates to tetrahydrofolate derivatives. Two mRNAs for the fpgs gene direct isoforms of FPGS to the cytosol and to mitochondria in mouse and human tissues. We sought to clarify the functions of these two compartmentalized isoforms. Stable cell lines were created that express cDNAs for the mitochondrial and cytosolic isoforms of human FPGS under control of a doxycycline-inducible promoter in the AUXB1 cell line. AUXB1 are devoid of endogenous FPGS activity due to a premature translational stop at codon 432 in the fpgs gene. Loss of folates was not measurable from these doxycycline-induced cells or from parental CHO cells over the course of three CHO cell generations. Likewise, there was no detectable transfer of folate polyglutamates either from the cytosol to mitochondria, or from mitochondria to the cytosol. The cell line expressing cytosolic FPGS required exogenous glycine but not thymidine or purine, whereas cells expressing the mitochondrial isoform required exogenous thymidine and purine but not glycine for optimal growth and survival. We concluded that mitochondrial FPGS is required because folate polyglutamates are not substrates for transport across the mitochondrial membrane in either direction and that polyglutamation not only traps folates in the cytosol, but also in the mitochondrial matrix.