Reference "Plant gamma-glutamyl hydrolases and folate polyglutamates: characterization, compartmentation, and co-occurrence in vacuoles"

Reference ID

54968

Title

Plant gamma-glutamyl hydrolases and folate polyglutamates: characterization, compartmentation, and co-occurrence in vacuoles

Source

J Biol Chem, 2005, vol. 280, pp. 28877-28884

Authors (8)

Orsomando-G	de-la-Garza-R-D
Green-B-J	Peng-M
Rea-P-A	Ryan-T-J
Gregory-J-F-3rd	Hanson-A-D

Abstract

gamma-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 microm) 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
approximately 50 and approximately 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.