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

The vacuoles of pea (Pisum sativum) leaves and red beet (Beta vulgaris) storage root are major sites for the intracellular compartmentation of folates. In the light of these findings and preliminary experiments indicating that some plant multidrug resistance-associated protein (MRP) subfamily ATP-binding cassette transporters are able to transport compounds of this type, the Arabidopsis thaliana vacuolar MRP, AtMRP1 (AtABCC1), and its functional equivalent(s) in vacuolar membrane vesicles purified from red beet storage root were studied. In so doing, it has been determined that heterologously expressed AtMRP1 and its equivalents in red beet vacuolar membranes are not only competent in the transport of glutathione conjugates but also folate monoglutamates and antifolates as exemplified by pteroyl-l-glutamic acid and methotrexate (MTX), respectively. In agreement with the results of these in vitro transport measurements, analyses of atmrp1 T-DNA insertion mutants of Arabidopsis ecotypes Wassilewskia and Columbia disclose an MTX-hypersensitive phenotype. atmrp1 knock-out mutants are more sensitive than wild-type plants to growth retardation by nanomolar concentrations of MTX, and this is associated with impaired vacuolar antifolate sequestration. The vacuoles of protoplasts isolated from the leaves of Wassilewskia atmrp1 mutants accumulate 50% less [(3)H]MTX than the vacuoles of protoplasts from wild-type plants when incubated in media containing nanomolar concentrations of this antifolate, and vacuolar membrane-enriched vesicles purified from the mutant catalyze MgATP-dependent [(3)H]MTX uptake at only 40% of the capacity of the equivalent membrane fraction from wild-type plants. AtMRP1 and its counterparts in other plant species therefore have the potential for participating in the vacuolar accumulation of folates and related compounds.

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

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.

Alternate energy-dependent pathways for the vacuolar uptake of glucose and glutathione conjugates.

Through the development and application of a liquid chromatography-mass spectrometry-based procedure for measuring the transport of complex organic molecules by vacuolar membrane vesicles in vitro, it is shown that the mechanism of uptake of sulfonylurea herbicides is determined by the ligand, glucose, or glutathione, to which the herbicide is conjugated. ATP-dependent accumulation of glucosylated chlorsulfuron by vacuolar membrane vesicles purified from red beet (Beta vulgaris) storage root approximates Michaelis-Menten kinetics and is strongly inhibited by agents that collapse or prevent the formation of a transmembrane H(+) gradient, but is completely insensitive to the phosphoryl transition state analog, vanadate. In contrast, ATP-dependent accumulation of the glutathione conjugate of a chlorsulfuron analog, chlorimuron-ethyl, is incompletely inhibited by agents that dissipate the transmembrane H(+) gradient but completely abolished by vanadate. In both cases, however, conjugation is essential for net uptake because neither of the unconjugated parent compounds are accumulated under energized or nonenergized conditions. That the attachment of glucose to two naturally occurring phenylpropanoids, p-hydroxycinnamic acid and p-hydroxybenzoic acid via aromatic hydroxyl groups, targets these compounds to the functional equivalent of the transporter responsible for chlorsulfuron-glucoside transport, confirms the general applicability of the H(+) gradient dependence of glucoside uptake. It is concluded that H(+) gradient-dependent, vanadate-insensitive glucoside uptake is mediated by an H(+) antiporter, whereas vanadate-sensitive glutathione conjugate uptake is mediated by an ATP-binding cassette transporter. In so doing, it is established that liquid chromatography-mass spectrometry affords a versatile high-sensitivity, high-fidelity technique for studies of the transport of complex organic molecules whose synthesis as radiolabeled derivatives is laborious and/or prohibitively expensive.