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.
Raichaudhuri
Ayan
A
Plant Science Institute, Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Peng
Mingsheng
M
Naponelli
Valeria
V
Chen
Sixue
S
Sánchez-Fernández
Rocío
R
Gu
Honglan
H
Gregory
Jesse F
JF
Hanson
Andrew D
AD
Rea
Philip A
PA
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D027461:Beta vulgaris / Q000235:genetics / Q000378:metabolism; D018432:Drug Resistance, Multiple / Q000187:drug effects / Q000235:genetics; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D005492:Folic Acid / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D008727:Methotrexate / Q000378:metabolism* / Q000494:pharmacology; D018532:Pisum sativum / Q000235:genetics / Q000378:metabolism; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism; D018517:Plant Roots / Q000235:genetics / Q000378:metabolism; D011523:Protoplasts / Q000378:metabolism; D014617:Vacuoles / Q000235:genetics / Q000378:metabolism*; D014803:Vitamin B Complex / Q000378:metabolism / Q000494:pharmacology
D018528:ATP-Binding Cassette Transporters; D029681:Arabidopsis Proteins; D004791:Enzyme Inhibitors; C495596:MRP1 protein, Arabidopsis; D014803:Vitamin B Complex; D005492:Folic Acid; D008727:Methotrexate