Plant γ-Glutamyl Hydrolases and Folate Polyglutamates

Abstract

γ-Glutamyl hydrolase (GGH, EC 3.4.19.9) catalyzes removal of the polyglutamyl tail from folyl and p-aminobenzoyl polyglutamates. Plants typically have one or a few GGH genes; Arabidopsis has three, tandemly arranged on chromosome 1, which encode proteins with predicted secretory pathway signal peptides. Two representative Arabidopsis GGH proteins, AtGGH1 and AtGGH2 (the At1g78660 and At1g78680 gene products, respectively) were expressed in truncated form in Escherichia coli and purified. Both enzymes were active as dimers, had low Km values (0.5–2 μm) for folyl and p-aminobenzoyl pentaglutamates, and acted as endopeptidases. However, despite 80% sequence identity, they differed in that AtGGH1 cleaved pentaglutamates, mainly to di- and triglutamates, whereas AtGGH2 yielded mainly monoglutamates. Analysis of subcellular fractions of pea leaves and red beet roots established that GGH activity is confined to the vacuole and that this activity, if not so sequestered, would deglutamylate all cellular folylpolyglutamates within minutes. Purified pea leaf vacuoles contained an average of 20% of the total cellular folate compared with ∼50 and ∼10%, respectively, in mitochondria and chloroplasts. The main vacuolar folate was 5-methyltetrahydrofolate, of which 51% was polyglutamylated. In contrast, the principal mitochondrial and chloroplastic forms were 5-formyl- and 5,10-methenyltetrahydrofolate polyglutamates, respectively. In beet roots, 16–60% of the folate was vacuolar and was again mainly 5-methyltetrahydrofolate, of which 76% was polyglutamylated. These data point to a hitherto unsuspected role for vacuoles in folate storage. Furthermore, the paradoxical co-occurrence of GGH and folylpolyglutamates in vacuoles implies that the polyglutamates are somehow protected from GGH attack. γ-Glutamyl hydrolase (GGH, EC 3.4.19.9) catalyzes removal of the polyglutamyl tail from folyl and p-aminobenzoyl polyglutamates. Plants typically have one or a few GGH genes; Arabidopsis has three, tandemly arranged on chromosome 1, which encode proteins with predicted secretory pathway signal peptides. Two representative Arabidopsis GGH proteins, AtGGH1 and AtGGH2 (the At1g78660 and At1g78680 gene products, respectively) were expressed in truncated form in Escherichia coli and purified. Both enzymes were active as dimers, had low Km values (0.5–2 μm) for folyl and p-aminobenzoyl pentaglutamates, and acted as endopeptidases. However, despite 80% sequence identity, they differed in that AtGGH1 cleaved pentaglutamates, mainly to di- and triglutamates, whereas AtGGH2 yielded mainly monoglutamates. Analysis of subcellular fractions of pea leaves and red beet roots established that GGH activity is confined to the vacuole and that this activity, if not so sequestered, would deglutamylate all cellular folylpolyglutamates within minutes. Purified pea leaf vacuoles contained an average of 20% of the total cellular folate compared with ∼50 and ∼10%, respectively, in mitochondria and chloroplasts. The main vacuolar folate was 5-methyltetrahydrofolate, of which 51% was polyglutamylated. In contrast, the principal mitochondrial and chloroplastic forms were 5-formyl- and 5,10-methenyltetrahydrofolate polyglutamates, respectively. In beet roots, 16–60% of the folate was vacuolar and was again mainly 5-methyltetrahydrofolate, of which 76% was polyglutamylated. These data point to a hitherto unsuspected role for vacuoles in folate storage. Furthermore, the paradoxical co-occurrence of GGH and folylpolyglutamates in vacuoles implies that the polyglutamates are somehow protected from GGH attack. In plants, as in other organisms, tetrahydrofolate (THF) 1The abbreviations used are: THF, tetrahydrofolate; pABA, p-aminobenzoic acid; pABAGlu1, p-aminobenzoylmonoglutamate; pABAGlun, p-aminobenzoylpolyglutamate; C1, one-carbon; GGH, γ-glutamyl hydrolase; AtGGH, Arabidopsis GGH; γ-Glun, poly-γ-l-glutamate; MTHFR, methylenetetrahydrofolate reductase; PteGlu1, folic acid; PteGlun, folate polyglutamate; MES, 4-morpholineethanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; CHES, 2-(cyclohexylamino)ethanesulfonic acid; HPLC, high performance liquid chromatography. 1The abbreviations used are: THF, tetrahydrofolate; pABA, p-aminobenzoic acid; pABAGlu1, p-aminobenzoylmonoglutamate; pABAGlun, p-aminobenzoylpolyglutamate; C1, one-carbon; GGH, γ-glutamyl hydrolase; AtGGH, Arabidopsis GGH; γ-Glun, poly-γ-l-glutamate; MTHFR, methylenetetrahydrofolate reductase; PteGlu1, folic acid; PteGlun, folate polyglutamate; MES, 4-morpholineethanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; CHES, 2-(cyclohexylamino)ethanesulfonic acid; HPLC, high performance liquid chromatography. and its derivatives, collectively termed folates, usually have γ-linked polyglutamyl tails of up to about seven residues attached to the first glutamate (Fig. 1A) (1Scott J. Rébeillé F. Fletcher J. J. Sci. Food Agric. 2000; 80: 795-824Crossref Scopus (252) Google Scholar, 2Zheng L.-L. Lin Y. Lin S. Cossins E.A. Phytochemistry. 1992; 31: 2277-2282Crossref Scopus (33) Google Scholar). The tail critically affects the cofactor activity and transport of folates because most folate-dependent enzymes prefer polyglutamates, whereas most carriers prefer monoglutamates (3Shane B. Vitam. Horm. 1989; 45: 263-335Crossref PubMed Scopus (290) Google Scholar, 4Brzezińska A. Wińska P. Balińska M. Acta Biochim. Pol. 2000; 47: 735-749Crossref PubMed Scopus (67) Google Scholar). By favoring protein binding, the tail also enhances folate stability, since bound folate is less prone to oxidative degradation (1Scott J. Rébeillé F. Fletcher J. J. Sci. Food Agric. 2000; 80: 795-824Crossref Scopus (252) Google Scholar). The enzyme that adds the polyglutamyl tails, folylpolyglutamyl synthetase, has been well studied in plants and shown to have mitochondrial, plastidial, and cytosolic isoforms (5Cossins E.A. Can. J. Bot. 2000; 78: 691-708Google Scholar, 6Ravanel S. Cherest H. Jabrin S. Grunwald D. Surdin-Kerjan Y. Douce R. Rébeillé F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15360-15365Crossref PubMed Scopus (107) Google Scholar). Less is known for plants about the enzyme that removes the tails, γ-glutamyl hydrolase (GGH), although soluble GGH activity (5Cossins E.A. Can. J. Bot. 2000; 78: 691-708Google Scholar, 7Leichter J. Landymore A.F. Krumdieck C.L. Am. J. Clin. Nutr. 1979; 32: 92-95Crossref PubMed Scopus (27) Google Scholar) and GGH-like mRNAs (8Huangpu J. Pak J.H. Burkhart W. Graham M.C. Rickle S. Liu C.Y. Graham J.S. Plant Physiol. 1996; 112: 862Google Scholar, 9Rickle S.A. Xu H. Liu C.Y. Morris P.F. Graham J.S. Plant Physiol. 1998; 117: 1526Google Scholar) clearly occur widely. Biochemical characterization of plant GGHs has so far been limited to crude extracts (9Rickle S.A. Xu H. Liu C.Y. Morris P.F. Graham J.S. Plant Physiol. 1998; 117: 1526Google Scholar, 10Wu K. Cossins E.A. King J. Plant Physiol. 1994; 104: 373-380Crossref PubMed Scopus (12) Google Scholar) or a mixture of isoforms (11Lin S. Rogiers S. Cossins E.A. Phytochemistry. 1993; 32: 1109-1117Crossref Scopus (16) Google Scholar) and has produced conflicting results on the enzyme structure and mode of action. Furthermore, the subcellular location of plant GGH is unclear; cytosolic (11Lin S. Rogiers S. Cossins E.A. Phytochemistry. 1993; 32: 1109-1117Crossref Scopus (16) Google Scholar) or extracellular (12Huangpu J. Pak J.H. Graham M.C. Rickle S.A. Graham J.S. Biochem. Biophys. Res. Commun. 1996; 228: 1-6Crossref PubMed Scopus (22) Google Scholar) sites have been proposed, but the vacuole is another a priori possibility (11Lin S. Rogiers S. Cossins E.A. Phytochemistry. 1993; 32: 1109-1117Crossref Scopus (16) Google Scholar, 13Galivan J. Ryan T.J. Chave K. Rhee M. Yao R. Yin D. Pharmacol. Ther. 2000; 85: 207-215Crossref PubMed Scopus (76) Google Scholar). Wherever it is, plant GGH must be sequestered away from folate polyglutamates, since extractable GGH activities are typically enough to deglutamylate all cellular folate within minutes (7Leichter J. Landymore A.F. Krumdieck C.L. Am. J. Clin. Nutr. 1979; 32: 92-95Crossref PubMed Scopus (27) Google Scholar, 10Wu K. Cossins E.A. King J. Plant Physiol. 1994; 104: 373-380Crossref PubMed Scopus (12) Google Scholar, 11Lin S. Rogiers S. Cossins E.A. Phytochemistry. 1993; 32: 1109-1117Crossref Scopus (16) Google Scholar), and treatments that disrupt cellular compartmentation cause massive deglutamylation of folates in situ (14Melse-Boonstra A. Verhoef P. Konings E.J. Van Dusseldorp M. Matser A. Hollman P.C. Meyboom S. Kok F.J. West C.E. J. Agric. Food Chem. 2002; 50: 3473-3478Crossref PubMed Scopus (57) Google Scholar). Nor is much known about the subcellular distribution in plants of the folate polyglutamate substrates of GGH, i.e. about the glutamyl tail lengths, one-carbon (C1) substituents, and oxidation state of the folates in different organelles. The only data are for pea leaves, in which penta- and tetraglutamates of 5-formyl-THF and THF appear to predominate in mitochondria and 5-methyl-THF (of tail Rébeillé F. M. Douce R. Cossins E.A. Biochem. J. 1993; PubMed Scopus Google Scholar, Cossins E.A. Plant Physiol. PubMed Scopus Google Scholar, Cossins E.A. Scopus Google Scholar). data for pea on the distribution of total and a mainly of the and vacuole for and respectively, of leaf folate Cossins E.A. Scopus Google Scholar, B. Jabrin S. S. M. Douce R. Rébeillé F. J. Sci. Food Agric. 2001; Scopus Google Scholar, S. S. B. Douce R. Rébeillé F. Plant Physiol. PubMed Scopus Google Scholar). not folate data the of vacuoles folates or GGH occur in vacuoles is for vacuoles folates, the transport or be to folate J. R. K. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, Plant 2002; PubMed Scopus Google Scholar). However, if GGH is the be as in not folates but folate polyglutamates, and monoglutamates B. Nutr. PubMed Scopus Google Scholar, J. Chem. 1992; PubMed Google Scholar). GGH also the polyglutamates oxidative degradation of folates (11Lin S. Rogiers S. Cossins E.A. Phytochemistry. 1993; 32: 1109-1117Crossref Scopus (16) Google Scholar, Nutr. 2001; PubMed Scopus Google Scholar), p-aminobenzoyl glutamate be the first in the of for folate a but Plant 2002; PubMed Scopus Google Scholar). In this used Arabidopsis GGHs to the of this enzyme in plants and that GGH is confined to also the and tail of folates in organelles. high of 5-methyl-THF polyglutamates in and other folates, polyglutamates and were from polyglutamates were from the PubMed Scopus Google Scholar). was from and were from was from was from were from Plant plants were in in for was only red beet roots were and used within of and were on R. J. R. M. Scopus Google Scholar, K. J. Chem. PubMed Google Scholar). and a were P. S. Douce R. Biochem. Biophys. 1993; PubMed Scopus Google Scholar, Plant Physiol. 1996; PubMed Scopus Google Scholar). leaves were in the or and in and in the was of for in with The were a and on a as P. S. Douce R. Biochem. Biophys. 1993; PubMed Scopus Google Scholar), that the contained and the of the was cytosolic was as P. S. Douce R. Biochem. Biophys. 1993; PubMed Scopus Google Scholar) from in to were in for and in a of and in the was up as vacuoles were the and used for folate were with in liquid and were from in liquid and for These extracts and fractions were on in and in liquid GGH was as The enzymes and tetrahydrofolate were as Plant Physiol. 1996; PubMed Scopus Google Scholar, H. Res. 2000; PubMed Scopus Google Scholar, S. H. Y. J. Chem. PubMed Scopus Google Scholar). was in a of extracts not or in vacuoles were as D. Plant Physiol. PubMed Google Scholar) that and plant mixture was to all vacuoles were for GGH of roots were and the which was to a of and The and were in of or mixture and of The was and and the were for The were in of in or of the in were with of in and of A. were for and vacuoles were from the most of the the was to of and for The vacuole was in of or of GGH GGH in liquid and of vacuoles and of the roots they from were J. Biochem. J. 1979; PubMed Scopus Google Scholar). was from in and to were and with as R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, Biochem. PubMed Scopus Google Scholar, J. Clin. Chem. 2000; PubMed Scopus Google Scholar) with the were to with CHES, in and or with The folate was from to and the of and were were in of the was and was The and were as R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) with a or was THF, and forms of and were used to of polyglutamates. and in Escherichia for At1g78660 and At1g78680 were from the and the Arabidopsis respectively. on sequence or residues were for protein The were and the shown in and the and sites of the sequence to the of the were in coli and coli which were in and was and was for used to truncated Arabidopsis GGH sequence in a were from a were in of and in a the was to the proteins were and on in or in liquid the enzymes activity for and respectively. was the Biochem. PubMed Scopus Google Scholar) as the were a and an The proteins were and and protein were from The values for AtGGH1 and AtGGH2 respectively, from to be and In was and the of was The were were for AtGGH1 and for AtGGH2 was in with was and the were from the the the The were to the which was to The data were the and in P. Biophys. J. 2000; 78: PubMed Scopus Google Scholar) The from the were to values GGH contained folate or and enzyme in a of for the was for for was in was in the or in the were a and for or respectively. The was with a from to in and glutamate were with Y. Ryan T.J. J. Biochim. Biophys. 1993; PubMed Scopus Google Scholar), the in to a and with and of were an average of of or of had been were to enzyme and were from for the γ-glutamyl in or and the or were and the of was the of or J. Nutr. PubMed Scopus Google Scholar). These are as or The of the to the of its and so the γ-glutamyl of the GGH-like in Arabidopsis and of and and The for that plants have one or a few proteins to and appear to have one and Arabidopsis three, and Arabidopsis is, in a GGH gene The Arabidopsis are tandemly arranged on chromosome and proteins and that are to GGH and to shown in 1, the Arabidopsis proteins with a predicted secretory pathway signal H. S. J. PubMed Scopus Google Scholar, S. J. Plant PubMed Scopus Google Scholar), one J. Ryan T.J. Chave K. Rhee M. Yao R. Yin D. Pharmacol. Ther. 2000; 85: 207-215Crossref PubMed Scopus (76) Google Scholar), and the and residues that are in GGH as well as other residues that to or J. Ryan T.J. J. Chem. 2000; PubMed Scopus (22) Google Scholar, H. Ryan T.J. Chave Van P. J. Chem. 2002; PubMed Scopus Google Scholar). In to the sequence has the that have in GGHs and are from all other plant GGH to be to Arabidopsis not of it was not enzyme is also far less expressed AtGGH1 or and A. D. of GGH AtGGH1 and AtGGH2 were expressed in the signal and it with a sequence a as was for GGH J. Ryan T.J. J. Chem. 2000; PubMed Scopus (22) Google Scholar). high of proteins, which were to as Two of enzyme were in residues were results for were data only for the were which was shown to be the for enzymes for AtGGH1 and for and is also to the of the vacuole which GGHs are of GGH The mode of and of AtGGH1 and AtGGH2 were studied the of folic and as substrates (Fig. 1, and In the of AtGGH1 and whereas AtGGH2 and a of (Fig. that enzymes have an with AtGGH1 an for the and γ-glutamyl from the folate and AtGGH2 the in was also was the with an a of the γ-glutamyl AtGGH2 (Fig. The for AtGGH1 and AtGGH2 with or as are shown in The Km values for for enzymes were for a pea GGH and to for which are typically from the enzymes less of AtGGH1 and AtGGH2 with or as in a the enzymes were used to the of the (Fig. AtGGH1 acted on the but not the was to PteGlu1, so that the folate as well as and (Fig. The other were and which were not (Fig. In contrast, AtGGH2 only as the folate although (Fig. The other of AtGGH2 were and with and as the (Fig. The of is The results with as with that AtGGH1 not and AtGGH2 produced a of and not (Fig. and The of of glutamate and (Fig. and that AtGGH1 not γ-glutamyl from or whereas AtGGH2 so the of and and was as the of AtGGH2 (Fig. and was not to of the glutamate and because was in total or the (Fig. and and was from or from was GGHs J. Ryan T.J. Chave K. Rhee M. Yao R. Yin D. Pharmacol. Ther. 2000; 85: 207-215Crossref PubMed Scopus (76) Google Scholar), plant enzyme or The from the from or activity is although not for also glutamate to the was for AtGGH2 on (Fig. and enzymes were in protein yielded a in the AtGGH1 the from the was to a of the the was and the was These values are to the of the AtGGH1 and AtGGH2 and that enzymes as dimers, as GGH H. Ryan T.J. Chave Van P. J. Chem. 2002; PubMed Scopus Google Scholar). In this it is that the of the enzyme H. Ryan T.J. Chave Van P. J. Chem. 2002; PubMed Scopus Google Scholar) are in the plant of GGH was and enzyme leaves were used because they are the of for high of vacuoles and other R. J. R. M. Scopus Google Scholar, K. J. Chem. PubMed Google Scholar, P. S. Douce R. Biochem. Biophys. 1993; PubMed Scopus Google Scholar) and have been the of much in folate (11Lin S. Rogiers S. Cossins E.A. Phytochemistry. 1993; 32: 1109-1117Crossref Scopus (16) Google Scholar, Rébeillé F. M. Douce R. Cossins E.A. Biochem. J. 1993; PubMed Scopus Google Scholar, Cossins E.A. Plant Physiol. PubMed Scopus Google Scholar, Cossins E.A. Scopus Google Scholar, B. Jabrin S. S. M. Douce R. Rébeillé F. J. Sci. Food Agric. 2001; Scopus Google Scholar, S. S. B. Douce R. Rébeillé F. Plant Physiol. PubMed Scopus Google Scholar). The distribution of enzyme activities that and vacuoles were other fractions (Fig. The vacuole contained of the total activities of the and the of vacuole is to of the vacuolar folate data to be The distribution of GGH activity that of the vacuolar (Fig. that GGH is an vacuolar this vacuoles from red beet roots, a plant vacuole The GGH activity and the vacuolar J. Biochem. J. 1979; PubMed Scopus Google Scholar) in vacuoles and roots respectively, and that GGH activity is GGH activities in beet vacuoles were far in pea vacuoles of and were first polyglutamyl tails to and of the of folate THF and its The values (Fig. with total folate of pea leaves Cossins E.A. Plant Physiol. PubMed Scopus Google Scholar, Cossins E.A. Scopus Google Scholar, B. Jabrin S. S. M. Douce R. Rébeillé F. J. Sci. Food Agric. 2001; Scopus Google Scholar, S. S. B. Douce R. Rébeillé F. Plant Physiol. PubMed Scopus Google Scholar) and a distribution of folate for leaves of and other plants (5Cossins E.A. Can. J. Bot. 2000; 78: 691-708Google Scholar, Cossins E.A. Plant Physiol. PubMed Scopus Google Scholar) (the used in the to so that the The total folate in mitochondria and and respectively, of cellular also with values Cossins E.A. Plant Physiol. PubMed Scopus Google Scholar, Cossins E.A. Scopus Google Scholar, B. Jabrin S. S. M. Douce R. Rébeillé F. J. Sci. Food Agric. 2001; Scopus Google Scholar, S. S. B. Douce R. Rébeillé F. Plant Physiol. PubMed Scopus Google Scholar), as the that 5-formyl-THF is the folate in mitochondria Cossins E.A. Plant Physiol. PubMed Scopus Google Scholar, Cossins E.A. Scopus Google Scholar). The most is that vacuoles a of all as 5-methyl-THF (Fig. this is not other fractions is the enzyme data and the distribution of folate which from of other fractions (Fig. The of was to the from which vacuoles were the vacuoles for of the not the of total cellular folate and 5-methyl-THF in the folate of vacuole and the from which they were expressed to the activity of which is to vacuoles in plants S. K. J. 2000; PubMed Scopus Google Scholar). an average of 20% of total folate was vacuolar and of the 5-methyl-THF folate and of pea leaf and in a of of was for the folate in (Fig. The main mitochondrial mainly as penta- and to a Rébeillé F. M. Douce R. Cossins E.A. Biochem. J. 1993; PubMed Scopus Google Scholar) as in chloroplasts. the vacuolar 5-methyl-THF was also although the average tail was in the other organelles. The of polyglutamyl folate in the vacuole was as GGH activity is in high enough to deglutamylate most of the vacuolar folate in less a for the that the of vacuolar 5-methyl-THF polyglutamates is not because plant GGHs not compared 5-methyl-THF and as substrates for AtGGH1 and and for the GGH activity in pea vacuole the of substrates were all enzyme The activities with 5-methyl-THF and respectively) and and pea GGH, and Analysis of the of the red beet roots and The principal folate in was in with data for and other E.J. Am. J. Clin. Nutr. 2001; PubMed Scopus Google Scholar) (Fig. with pea beet vacuoles contained of 5-methyl-THF (Fig. The of 5-methyl-THF in beet vacuoles were the folate of vacuole and to In from to of the 5-methyl-THF was The vacuolar 5-methyl-THF was 76% with di- and (Fig. beet vacuoles also GGH, the in beet is that in folyl polyglutamates and the enzyme that in of red beet and vacuole were for this vacuole were for this in a results that plant GGHs are in structure and to of as other (8Huangpu J. Pak J.H. Burkhart W. Graham M.C. Rickle S. Liu C.Y. Graham J.S. Plant Physiol. 1996; 112: 862Google Scholar, 9Rickle S.A. Xu H. Liu C.Y. Morris P.F. Graham J.S. Plant Physiol. 1998; 117: 1526Google Scholar, 10Wu K. Cossins E.A. King J. Plant Physiol. 1994; 104: 373-380Crossref PubMed Scopus (12) Google Scholar, 11Lin S. Rogiers S. Cossins E.A. Phytochemistry. 1993; 32: 1109-1117Crossref Scopus (16) Google Scholar). GGHs J. Ryan T.J. Chave K. Rhee M. Yao R. Yin D. Pharmacol. Ther. 2000; 85: 207-215Crossref PubMed Scopus (76) Google Scholar, and Scholar), the Arabidopsis enzymes studied are of have and folate polyglutamates, and different despite AtGGH1 and AtGGH2 as but they prefer different and only AtGGH2 is a of AtGGH1 the enzymes in a have of plant GGHs with crude extracts or isoforms (8Huangpu J. Pak J.H. Burkhart W. Graham M.C. Rickle S. Liu C.Y. Graham J.S. Plant Physiol. 1996; 112: 862Google Scholar, 9Rickle S.A. Xu H. Liu C.Y. Morris P.F. Graham J.S. Plant Physiol. 1998; 117: 1526Google Scholar). AtGGH2 also and on The Arabidopsis GGH proteins and all other plant GGH have an in a that they as as GGHs J. Ryan T.J. Chave K. Rhee M. Yao R. Yin D. Pharmacol. Ther. 2000; 85: 207-215Crossref PubMed Scopus (76) Google Scholar). GGHs have or sites and are the proteins produced in coli have to R. Ryan T.J. J. J. Chem. 1996; PubMed Scopus Google Scholar, R. Ryan T.J. J. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar, B. Chave J. Ryan T.J. Pharmacol. 1998; Google Scholar). is, that the Arabidopsis GGHs used in the of the enzymes as they in that pea GGH activity is vacuolar is the of AtGGH2 and proteins in Arabidopsis leaf vacuoles S. J. Plant PubMed Scopus Google Scholar) and with the of signal which occur in proteins S. J. Plant PubMed Scopus Google Scholar, M. K. M. A. K. Plant Physiol. 45: PubMed Scopus Google Scholar) and in vacuolar A. 1992; PubMed Scopus Google Scholar). The vacuolar the location of GGH J. Ryan T.J. Chave K. Rhee M. Yao R. Yin D. Pharmacol. Ther. 2000; 85: 207-215Crossref PubMed Scopus (76) Google Scholar, and Scholar), because and vacuoles of plants are H. H. Plant 2002; PubMed Scopus Google Scholar). vacuolar location to with that GGH is cytosolic or but this is not the The cytosolic location was extracts which and in of the GGH activity was in the vacuole (11Lin S. Rogiers S. Cossins E.A. Phytochemistry. 1993; 32: 1109-1117Crossref Scopus (16) Google Scholar). The location was on a GGH-like protein in extracellular of GGH activity was not (12Huangpu J. Pak J.H. Graham M.C. Rickle S.A. Graham J.S. Biochem. Biophys. Res. Commun. 1996; 228: 1-6Crossref PubMed Scopus (22) Google Scholar). of the (8Huangpu J. Pak J.H. Burkhart W. Graham M.C. Rickle S. Liu C.Y. Graham J.S. Plant Physiol. 1996; 112: 862Google Scholar) and has since shown that this all other plant GGH residues and in the J. Ryan T.J. J. Chem. 2000; PubMed Scopus (22) Google Scholar). that the an GGH-like not an active GGH of folates that has a folate The mitochondrial is which is not a but an of folate-dependent enzymes P. Biochem. Sci. 1993; PubMed Scopus Google Scholar). is to the active folate a that is mitochondrial in plants S. J. Chem. 2002; PubMed Scopus Google Scholar). The folate is in is from but also in has been from the of the enzyme that S. P. Jabrin S. Rébeillé F. Douce R. J. Chem. PubMed Scopus Google Scholar). The folate of leaf and vacuoles to of a role for this a for because it is and is to other folates the in plants S. H. Y. J. Chem. PubMed Scopus Google Scholar). vacuolar of folate have not been have been predicted from the of folate and activity and the in folate from different of and The of of of and Scholar). roots are a folate are as high as in leaves and far of other roots of and The of of of and Scholar). The of vacuolar folates is paradoxical because the glutamyl tail is not to in the of the GGH activity, as the pea leaves, that the vacuole is of the H. 1994; Scopus Google Scholar) and that total folate is Cossins E.A. Scopus Google Scholar), the vacuolar and would be The vacuolar GGH activity is to vacuolar or a folate of from the vacuolar folate the folate and GGH expressed and the Km values for the Arabidopsis state the vacuolar folyl polyglutamates, have a predicted of for beet vacuoles a polyglutamate of These that pea and beet GGHs to a mixture of the Arabidopsis characterization of the activities in pea and beet vacuole extracts this The also that GGH and folyl polyglutamates to be vacuole within and mitochondria isoforms of S. Cherest H. Jabrin S. Grunwald D. Surdin-Kerjan Y. Douce R. Rébeillé F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15360-15365Crossref PubMed Scopus (107) Google Scholar) as well as the that this enzyme The of polyglutamates in is, in situ from so for which most the S. Cherest H. Jabrin S. Grunwald D. Surdin-Kerjan Y. Douce R. Rébeillé F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15360-15365Crossref PubMed Scopus (107) Google Scholar) A. U. P. Plant Physiol. 2001; PubMed Scopus Google Scholar). vacuoles it is to as for B. Nutr. PubMed Scopus Google Scholar, J. Chem. 1992; PubMed Google Scholar), of folylpolyglutamates a The co-occurrence of folylpolyglutamates and GGH in vacuoles be the of a GGH or proteins that polyglutamates from is so far for or However, GGH on folylpolyglutamates would also polyglutamate and so disrupt folate proteins a proteins have been in Nutr. PubMed Scopus Google Scholar) and shown to bound folates of the polyglutamyl tail and oxidative degradation (1Scott J. Rébeillé F. Fletcher J. J. Sci. Food Agric. 2000; 80: 795-824Crossref Scopus (252) Google Scholar, Y. Ryan T.J. J. Biochim. Biophys. 1993; PubMed Scopus Google Scholar). for on for on HPLC, and for and of the for the with