Plant γ-Glutamyl Hydrolases and Folate Polyglutamates

Abstract

␥-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 K m values (0.5-2 M) 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 ϳ50 and ϳ10%, 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.

Metabolism of the Folate Precursor p-Aminobenzoate in Plants

“…Leaflets were incubated in Petri dishes for 3 h at 24°C in light (150 mol m Ϫ2 s Ϫ1 ). After the incubation, protoplasts were prepared from the [ 14 C]pABA-fed leaflets together with unlabeled pea leaflets (24 g) following a 5-h digestion as described (20) except that the centrifugation step used to harvest protoplasts included a cushion of 50% (v/v) Percoll in 10 mM MESNaOH, pH 7.0, 0.5 M sorbitol, 5 mM CaCl 2 , 0.5 mg/ml polyvinylpyrrolidone-40. The protoplast layers were adjusted to 13% Percoll in the same buffer and purified on a three-step sucrosesorbitol gradient as described (21).…”

“…The protoplast layers were adjusted to 13% Percoll in the same buffer and purified on a three-step sucrosesorbitol gradient as described (21). Vacuoles were prepared from lysed protoplasts (20) after adding to the lysate unlabeled folic acid to a final concentration of 50 M and 2-25 Ci (0.05-0.58 nmol) of [ 3 H]folic acid. Marker enzymes were extracted and assayed as described (20).…”

“…Vacuoles were prepared from lysed protoplasts (20) after adding to the lysate unlabeled folic acid to a final concentration of 50 M and 2-25 Ci (0.05-0.58 nmol) of [ 3 H]folic acid. Marker enzymes were extracted and assayed as described (20). Vacuolar yield was quantified by counting protoplast and vacuole preparations using a hemocytometer, and by measuring ␣-mannosidase activity (22).…”

Identification of Transport-critical Residues in a Folate Transporter from the Folate-Biopterin Transporter (FBT) Family

“…Folates are made in mitochondria and exported to the cytosol, plastids, and vacuole (5)(6)(7). This implies the existence of several organellar folate transporters.…”

Plant Vacuolar ATP-binding Cassette Transporters That Translocate Folates and Antifolates in Vitro and Contribute to Antifolate Tolerance in Vivo

“…red beet (Beta vulgaris) storage roots (3). A hitherto unsuspected role for plant vacuoles in folate storage is implicated, which in turn necessitates a mechanism or mechanisms for the transport of these compounds into this compartment.…”