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

Plant Vacuolar ATP-binding Cassette Transporters ThatTranslocate Folates and Antifolates in Vitro and Contribute toAntifolate Tolerance in Vivo*

Received for publication, November 13, 2008, and in revised form, January 5, 2009 Published, JBC Papers in Press, January 9, 2009, DOI 10.1074/jbc.M808632200

Ayan Raichaudhuri‡, Mingsheng Peng‡, Valeria Naponelli§, Sixue Chen‡1, Rocío Sanchez-Fernandez‡2,Honglan Gu‡, Jesse F. Gregory III§, Andrew D. Hanson¶, and Philip A. Rea‡3

From the ‡Plant Science Institute, Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104 andthe §Food Science and Human Nutrition Department and ¶Horticultural Sciences Department, University of Florida,Gainesville, Florida 32611

The vacuoles of pea (Pisum sativum) leaves and red beet (Betavulgaris) storage root are major sites for the intracellular com-partmentation of folates. In the light of these findings and pre-liminary experiments indicating that some plant multidrugresistance-associated protein (MRP) subfamily ATP-bindingcassette transporters are able to transport compounds of this type,theArabidopsis thalianavacuolarMRP,AtMRP1(AtABCC1), andits functional equivalent(s) in vacuolarmembrane vesicles purifiedfrom red beet storage root were studied. In so doing, it has beendetermined that heterologously expressed AtMRP1 and its equiv-alents in red beet vacuolar membranes are not only competent inthe transport of glutathione conjugates but also folatemonogluta-matesandantifolatesasexemplifiedbypteroyl-L-glutamicacidandmethotrexate (MTX), respectively. Inagreementwith the resultsofthese in vitro transportmeasurements, analyses ofatmrp1T-DNAinsertion mutants of Arabidopsis ecotypes Wassilewskia andColumbia disclose an MTX-hypersensitive phenotype. atmrp1knock-out mutants are more sensitive than wild-type plants togrowth retardationbynanomolar concentrationsofMTX,and thisis associated with impaired vacuolar antifolate sequestration. Thevacuoles of protoplasts isolated from the leaves of Wassilewskiaatmrp1mutants accumulate 50% less [3H]MTX than the vacuolesof protoplasts fromwild-typeplantswhen incubated inmedia con-taining nanomolar concentrations of this antifolate, and vacuolarmembrane-enriched vesicles purified from the mutant catalyzeMgATP-dependent [3H]MTXuptakeatonly40%of thecapacityofthe equivalentmembrane fraction fromwild-type plants. AtMRP1and its counterparts in other plant species therefore have thepotential for participating in the vacuolar accumulation of folatesand related compounds.

Tetrahydrofolate (THF)4 and its derivatives, “folates,” areessential cofactors for one-carbon transfer reactions, forinstance those crucial for nucleotide biosynthesis and aminoacid metabolism. In humans and other animals who cannotsynthesize folates de novo, these cofactors must be obtainedfrom dietary sources, principally plantmaterials. The repercus-sions of dietary folate deficiencies range from an increased pre-disposition tomegaloblastic anemia and birth defects to cardio-vascular disease and certain cancers (1).Folates are tripartite molecules, consisting of pteridine and

p-aminobenzoate, which together constitute the pteroyl moi-ety, and one ormore glutamate residues (Fig. 1A). In plants andmost other organisms, the parent folate molecule, pteroylmonoglutamate (PteGlu1), is poly-�-glutamylated to yield pter-oyl polyglutamates (PteGlun) containing 1–7 additional gluta-mate residues (2).The experiments described here were directed at determin-

ing whether a multidrug resistance-associated protein (MRP)-typeATP-binding cassette (ABC) transportermight participatein vacuolar folate uptake. The reasons for conducting thesestudies were 2-fold. The first was recognition that a significantfraction of total cellular folate localizes to the vacuolar com-partment of plant cells. Vacuoles purified from pea (Pisum sati-vum) leaves contain an average of 20% of the total cellularfolate, compared with�50 and 10%, respectively, in mitochon-dria and chloroplasts (3). Approximately 50% of the principalvacuolar folate in this system, 5-methyl-THF, is polyglutamy-lated, whereas the principal mitochondrial and plastidial formsare polyglutamylated derivatives of 5-formyl-THF and 5–10-methenyl-THF, respectively (3). This is probably a general phe-nomenon in that between 20 and 60% of total tissue folate,mainly in the form of 5-methyl-THF, of which about 80% ispolyglutamylated, can be recovered in the vacuolar fraction of

* This work was supported by United States Department of Energy GrantDE-FG02-91ER20055 (to P. A. R.) and in part by National Institutes of HealthGrant RO1 GM071382 (to A. D. H.). The costs of publication of this articlewere defrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18 U.S.C.Section 1734 solely to indicate this fact.

1 Present address: Botany Dept., Genetics Institute, University of Florida,Gainesville, FL 32611.

2 Present address: SunGene GmbH, Correnstrasse 3, D-06466 Gatersleben,Germany.

3 To whom correspondence should be addressed: Plant Science Institute,Dept. of Biology, Carolyn Hoff Lynch Biology Laboratory, 433 South Univer-sity Ave., University of Pennsylvania, Philadelphia, PA 19104. Tel.: 215-898-0807; Fax: 215-898-8780; E-mail: [email protected].

4 The abbreviations used are: THF, tetrahydrofolate; ABA, abscisic acid; ABC,ATP-binding cassette; AtMRP, A. thaliana MRP; Bn-NCC-1, Brassica napusnonfluorescent chlorophyll catabolite 1; Bistris propane, 1,3-bis[tris(hy-droxymethyl)methylamino]propane; CDNB, 1-chloro-2,4-dinitrobenzene;DNP-GS, S-(2,4-dinitrophenyl)-GS; E217�G, 17�-estradiol 17-(�-D-glucuro-nide); FBP, folate-binding protein; FPGS, folylpolyglutamate synthetase;GGH, �-glutamyl hydrolase; GST, glutathione S-transferase; HPLC, highperformance liquid chromatography; Mes, 2-(N-morpholino)ethanesulfo-nic acid; MRP, multidrug resistance-associated protein; MTX, methotrex-ate; NEM-GS, N-ethylmaleimide-GS; p-ABA, p-aminobenzoate; Pte, pteroyl;PteGlu1, pteroyl monoglutamate; PteGlun, pteroyl polyglutamate; RT,reverse transcription; WS, Wassilewskia; Col-0, Columbia.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 13, pp. 8449 –8460, March 27, 2009© 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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red beet (Beta vulgaris) storage roots (3). A hitherto unsus-pected role for plant vacuoles in folate storage is implicated,which in turn necessitates a mechanism or mechanisms for thetransport of these compounds into this compartment. At thetime of writing, the only plant folate transporters to have beenidentified are those that localize to plastid membranes (4, 5).A second reason for these investigations was an appreciation

that some of the ABC transporters from mammalian sources,specifically some members of the MRP subfamily, are compe-tent in the transport of the chemotherapeutic antifolate meth-otrexate (MTX; Fig. 1B) and physiological folates (6–8).The cellular pharmacology of MTX is reasonably well

defined in mammals. It has been determined that cellularuptake occurs primarily by facilitated diffusion and that onceinside the cellMTX acts as a potent competitive inhibitor of theenzyme dihydrofolate reductase, responsible for the regenera-tion of reduced THF.MTX thereby retards cell proliferation bydepleting intracellular reduced folate pools essential for thebiosynthesis of the purines required for DNA replication. SomemammalianMRPs contribute to the detoxification ofMTXandcompromise chemotherapy by participating in the active extru-sion of this drug across the plasma membrane (8). BecauseMRPs represent the second largest subfamily of full moleculeABC transporters from plant sources, a potentially significantimplication follows. If some of the MRPs from mammaliansources can transport MTX, a strict structural analog of Pte-Glu1, aswell as PteGlu1 itself (7), there is a possibility that one ormore members of the same subfamily from plant sources sharethis property.Many early studies of plant MRP-type transport processes

focused on the MgATP-energized, vanadate-inhibitable, un-coupler-insensitive uptake of two model glutathione (GSH)conjugates (GS-conjugates), N-ethylmaleimide-GS (NEM-GS)and S-(2,4-dinitrophenyl)-GS (DNP-GS), and the glutathiony-lated chloroacetamide herbicide, metolachlor-GS, by isolatedvacuoles (9) and vacuolar membrane vesicles purified fromplants (10). In this way it was recognized that the transportersresponsible bear a close functional resemblance to the GS-con-jugate transporting ATPases of mammalian cells and probablybelong to the MRP subfamily of ABC transporters (11, 12).

Of the 15 uniqueMRPs encoded by the genome ofArabidop-sis thaliana, five (AtMRPs 1–5) have been cloned and shown toencode functional transporters after heterologous expressionin Saccharomyces cerevisiae ycf1� strains from which the geneencoding the endogenous vacuolarMRP, yeast cadmium factor1 (ycf 1), has been disrupted (reviewed in Ref. 13). All five arecompetent in the transport of GS-conjugates. In addition, sev-eral are able to transport other amphipathic anions, includingglucuronate conjugates and linearized chlorophyll (tetrapyr-role) catabolites (13).If plantMRPs are to contribute to the vacuolar sequestration

of folates, the membrane localization of these transporters andthe form in which the folates are transported and stored mustbe addressed. Regarding membrane localization, two membersof the Arabidopsis MRP subfamily, AtMRP1 and AtMRP2(AtABCC1 and AtABCC2, respectively, according to the newnomenclature described in Ref. 14), have been clearly localizedto the vacuolar membrane in planta (15, 16). However, on thebasis of pilot experiments, only AtMRP1 catalyzes appreciablefolate andMTX transport in vitro. AlthoughAtMRP4 (ABCC4)is also able to transport these compounds at high capacity (17),its exact membrane localization is unclear. On the one hand, itlocalizes to the plasma membrane rather than the vacuolarmembrane when its partial translation product is fused withgreen fluorescent protein (18). On the other hand, the results ofrecent Arabidopsis organellar proteomic analyses and studiesof constructs inwhich green fluorescent protein is C-terminallyfused to the full-length translation product are consistent withlocalization to the vacuolar membrane (19). Pending furtherinsight into the membrane localization of AtMRP4, the inves-tigations described here are specifically directed at elucidatingthe role AtMRP1 might play in vacuolar folate uptake.Two factors are crucial when considering the form in which

folates are transported. The first is that although it has beenestablished that a subset of human and rodent MRPs conferresistance to MTX and have the capacity to transport this andphysiological folates, they only do so when these compoundsare monoglutamylated; polyglutamylation essentially abolishestransport (7, 8). The second factor is that in plants, as in otherorganisms, folates are usually polyglutamylated with up toseven �-linked glumate residues (2) that serve to enhancecofactor activity and stability. More than 50% of the extractablevacuolar pool of folates is polyglutamylated (3). When accountis taken of the likelihood that the vacuole lacks the machineryfor folate polyglutamylation, the enzyme folylpolyglutamatesynthetase (FPGS) and the energy source ATP (3, 20, 21), theimplication is clear. If plantMRPs do participate in the vacuolarlocalization of folates, they are responsible for delivery of only asubfraction of this pool, the monoglutamylated component, intothis compartment or, unlike their mammalian counterparts, areable to transport polyglutamates as well as monoglutamates. Aprimary objective of the investigations reported here was to iden-tify the transport formofvacuolar folates,namelywhether theyaretransported as monoglutamates or polyglutamates, and to deter-mine whether a vacuolar membrane-associated MRP-type func-tionality, as exemplified by AtMRP1, might be responsible.To assess the general applicability of the properties of heter-

ologously expressed AtMRP1 and Arabidopsis T-DNA knock-

FIGURE 1. Structures of folic acid (A) and methotrexate (MTX) (B). R instructure A is OH in folate monoglutamate (pteroyl-L-glutamic acid, PteGlu1)and n glutamate residues in folate polyglutamates (PteGlun).

Plant MRP-mediated Vacuolar Folate Uptake

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out mutants for this transporter to native membranes fromother plant sources, parallel experiments were performed onvacuolar membrane vesicles purified from red beet storageroot. This systemwas considered to be particularly appropriatefor these investigations for three reasons. First, red beet storageroot is a rich source of high purity transport-competent vesiclesderived from the membrane bounding the vacuole (22). Sec-ond, previous investigations have established that vacuolarmembrane vesicles purified from this source contain an MRP-like functionality or functionalities capable of catalyzing thetransport of GS-conjugates (10, 23, 24). Third, red beet storageroot is listed as one of the richest natural sources of folates (25),a sizeable fraction of which localize to the vacuole (3). Thefolate content of red beet storage root is as high as those ofgreen leaves, for instance those of spinach which is consideredto be one of the richest sources of this class of vitamins (25).

MATERIALS AND METHODS

Chemicals—ATP, creatine kinase (type I from rabbit mus-cle), creatine phosphate, and N-ethylmaleimide (NEM) werepurchased from Sigma. Mixed cellulose ester membrane filters(HAWP filters, 0.45-�m pore size) and hydrophilic polyvinyli-dene difluoride (Durapore) membrane filters (GVWP filters,0.22-�m pore diameter) were purchased from Millipore Corp.[glycine-2-3H]Glutathione ([3H]GSH) (41.5 Ci/mmol) was pur-chased fromPerkinElmer Life Sciences. [3�,5�,7,9-3H]Folic acid([3H]PteGlu1) (42.8 Ci/mmol) and [3�,5�,7-3H]methotrexate([3H]MTX) (12 Ci/mmol) were purchased from Moravek Bio-chemicals and Radiochemicals (Brea, CA). Radiolabeled poly-glutamylated folate ([3H]PteGlun) was synthesized as described(26). Briefly, 2 nmol of [3H]PteGlu1 were incubated with 25 �gof Escherichia coli FPGS for 24 h at 37 °C in 50 �l of 50 mMTris-HCl buffer (pH 8.6) containing 10 mMMgCl2, 50 mM KCl,5 mM ATP, 25 �g of bovine serum albumin, and 20 mM L-glu-tamate. The reaction was terminated by boiling the mixture for5 min and subsequent centrifugation at 20,000 � g for 10 min.The products of the reaction were purified by FBP-affinitychromatography as described below for the purification of[3H]PteGlu1, and quantitated and identified by HPLC withelectrochemical detection (26). For the investigations describedhere, the [3H]PteGlun preparations consisted of 45% [3H]Pte-Glu3, 55% [3H]PteGlu4, and 5% [3H]PteGlu5. [3H]NEM-GSwassynthesized by mixing reduced [glycine-2-3H]GSH with NEMat a 1.1:1.0 molar ratio in Hepes/Bistris propane buffer (pH 8.0)(27). All of the molecular biology reagents, including TRIzol,Superscript II RNase H� RT ,and the DNeasy plant mini kitswere purchased from Invitrogen and Qiagen. All of the otherreagents were of analytical grade and purchased from Fisher,Research Organics, Inc., or Sigma.Plant Materials—For the majority of the investigations of A.

thaliana, ecotypeWassilewskia (WS) was employed except forthe analyses of atmrp1-2 knock-out mutants, which were per-formed on ecotype Columbia (Col-0). Fresh red beet (Beta vul-garis) storage roots were purchased locally, stored at 4 °C, andused within 2 days of purchase.Isolation of AtMRP1 T-DNA Insertion Mutants—For the

experiments described here, two AtMRP1 insertion mutants,atmrp1-1 and atmrp1-2, respectively, from Arabidopsis eco-

types WS and Col-0 were obtained. TheWS allele was isolatedby PCR-based reverse genetic screens of the Feldmann collec-tion of T-DNA insertion lines (28) using appropriate AtMRP1-specific and T-DNA left or right border-specific primer pairs.The Col-0 atmrp1-2 allele was identified using the SIGnALArabidopsis genemapping tool “T-DNA Express,” and the cor-responding T-DNA insertion seed stock was obtained from theArabidopsis Biological Resource Center, Ohio State University.Both alleles were characterized genomically by standardprocedures.RT-PCR—To assess the steady state levels of AtMRP1 tran-

scripts and therefore the severity of the knock-outs or knock-downs for the atmrp1 mutants, RNA was extracted from14-day-old atmrp1-1 and atmrp1-2 mutant and wild-type WSandCol-0 plants usingTRIzol reagent (Invitrogen) according tothe manufacturer’s recommendations. For RT-PCR ofAtMRP1, 1-�g aliquots of the RNA samples were reverse-tran-scribed using Superscript II RNaseH� RT (Invitrogen), and thefirst-strand cDNA products were PCR-amplified using theAtMRP1-specific primer pair AtMRP1-RT-F/AtMRP1-RT-R(5�-CGCAGAAATCCTCTTGGTCTTGA-3� and 5�-CCGTT-AGCTTCTCTGGTACGTTTG-3�, respectively). PCR amplifi-cation was for 3 min at 94 °C followed by 27 cycles of 30 s at94 °C, 30 s at 62 °C, and 1 min at 72 °C. To assess the efficacy ofRNA extraction, aliquots of the same RNA samples were alsosubjected to RT-PCR using the Arabidopsis Actin-8 geneprimer pair Actin-F/Actin-R (5�-CCTGCTATGTATGTG-GCTATT-3� and 5�-CTGTGGTGGTGAAAGAGTAAC-3�,respectively) using the same thermal profile except that theannealing step was done at 58 °C.Phenotypic Characterization of atmrp1-1 and atmrp1-2

Mutants—For the preliminary phenotypic screens, seeds ofatmrp1-1 mutant and wild-type WS plants and of atmrp1-2mutant and wild-type Col-0 plants were surface-sterilized with0.05% (w/v) sodiumhypochlorite/0.1% (w/v) Tween 20, washedexhaustively with sterile water, and germinated for 48 h at 4 °Con solid MS medium (pH 5.7) containing 1% (w/v) sucrose.Xenobiotics, elicitors of oxidative stress, and phytohormoneswere incorporated into the medium at the concentrations indi-cated. Thereafter, the plates were grown vertically under con-trolled environmental conditions (24 � 2 °C; continuous coolfluorescent illumination; 70% relative humidity) for 12 daysbefore measuring primary root length. For subsequent detailedscreens of the sensitivity of growth to MTX at the whole plantlevel, surface-sterilized mutant and wild-type seeds were ger-minated for 48 h at 4 °C on solid MS medium containing 0.5g/liter Mes, 0.9% (w/v) Difco-Bacto agar, 1% (w/v) sucrose, andthe indicated concentrations of MTX in plant tissue culturevessels before transfer to a plant growth room for growth at22 � 2 °C and 70% relative humidity under a 16/8-h photope-riod for a further 14 days. In all of the phenotypic screens, pre-cautions were taken to ensure that several independent mutantand wild-type seed batches were treated and screened in paral-lel under identical conditions.Affinity Purification of [3H]PteGlu1—To remove breakdown

products and/or contaminants that were suspected to interferewith transport, the radiolabeled stocks of [3H]PteGlu1 receivedfrom the suppliers were further purified by affinity chromatog-



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raphy before use. For this purpose, 20-�l aliquots of the[3H]PteGlu1 stocks (40–45 Ci/mmol) were added to 300 �l of25 mM potassium phosphate buffer (pH 7.4) and applied to acolumn packed with folate-binding protein (FBP)-agarose (bedvolume 0.5 ml) that had been equilibrated with the same buffer(26). After three successive washes with 2.5 ml of potassiumphosphate buffer (pH 7.4) containing 1 MNaCl, 2.5ml of potas-sium phosphate buffer alone, and 300 �l of 0.1 M HCl, the col-umn was eluted with 1 ml of 0.1 M HCl. The final eluate con-taining purified [3H]PteGlu1 was combined with 0.1 ml of 10mM 2-mercaptoethanol dissolved in 1 M Tris-base to yield a pH7.4 solution that was used immediately or stored at �20 °C.Heterologous Expression of AtMRP1 in Yeast and Purification

of Vacuolar Membrane-enriched Vesicles—For studies of thetransport capabilities of heterologously expressed AtMRP1, S.cerevisiae ycf1� strain DTY168 (MAT� his6 leu2-3,-112 ura3–52 ycf1::hisG) (29) was transformed with pYES3-AtMRP1 orempty pYES3 vector by the LiOAc/polyethylene glycol method(30) and selected for uracil prototrophy by plating on AHCmedium containing tryptophan (31, 32). Vacuolar membrane-enriched vesicles were purified from the transformants asdescribed (31, 32).Preparation of Red Beet Vacuolar Membrane Vesicles—Vac-

uolar membrane vesicles were purified from fresh red beet (B.vulgaris) storage roots by a combination of differential and den-sity gradient centrifugation (10, 22). The final membrane pel-letswere resuspended in suspension buffer (5mMTris/Mes (pH7.5), 1 mM Tris/EDTA, 0.5 mM dithiothreitol, 100 �g/ml buty-lated hydroxytoluene, and 10% (v/v) glycerol) to a final proteinconcentration of 1–2 mg/ml, frozen in liquid nitrogen, andstored at �80 °C.Preparation of Vacuolar Membrane-enriched Vesicles from

atmrp1-1Mutant andWild-typeArabidopsisWSRootCultures—After germination and growth on solid MS medium supple-mented with 1% (w/v) sucrose (pH 5.7) for 7 days, batches of50–75 atmrp1-1 mutant or wild-type Arabidopsis WS seed-lings were transferred to 125-ml culture flasks containing 50mlof half-strength MS medium and cultured for 14 days withshaking at 130 rpm. Vacuolar membrane-enriched vesicleswere purified from the root cultures by a combination of differ-ential and density gradient centrifugation on 10/23% (w/w)sucrose step gradients as described (10).Measurements of Uptake by Yeast, Red Beet, and Arabidopsis

Vacuolar Membrane-enriched Vesicles—Measurements of theMgATP-dependent uptake of [3H]PteGlu1 (1.5 mCi/mmol),[3H]PteGlun (3.5 mCi/mmol), [3H]MTX (7.5 mCi/mmol), or[3H]NEM-GS (6 mCi/mmol) by vacuolar membrane-enrichedmembrane vesicles purified from pYES3-AtMRP1 or emptypYES3 vector-transformed yeastDTY168 cells, red beet storageroot, orArabidopsis root cultures were performed as described(10, 32). Uptake was initiated by the addition of 20 �g of yeastvacuolar membrane-enriched vesicles, 30 �g of red beet vacu-olarmembrane vesicles, or 50�g ofArabidopsis vacuolarmem-brane-enriched vesicles to the uptake media and brief agitationon a vortex mixer. In all of the experiments described here,uptake was measured at 25 °C in 200-�l reaction volumes con-taining 3mMATP, 3mMMgSO4, 10mM creatine phosphate, 16units/ml creatine kinase, 50 mM KCl, 400 mM sorbitol, 25 mM

Tris/Mes buffer (pH 8.0), and the indicated concentrations ofradiolabeled transport substrate. At the times indicated, uptakewas terminated by the addition of 1ml of ice-coldwashmedium(400 mM sorbitol, 3 mM Tris/Mes (pH 8.0)) and vacuum filtra-tion of the suspension through prewettedmembrane filters. Forthe measurements of [3H]PteGlu1, [3H]PteGlun, or [3H]MTXuptake GV Durapore polyvinylidene difluoride (GVWP) filters(0.22-�m pore diameter) were used; for the measurements of[3H[]NEM-GS uptake mixed cellulose ester (HAWP) filters(0.45-�m pore diameter) were used. After two washes with1-ml volumes of ice-cold wash medium, the filters were air-dried, and radioactivity was determined by liquid scintillationcounting in 5-ml volumes of BCS liquid scintillation mixture(Amersham Biosciences).Measurements of Total Cellular and Vacuolar [3H]MTX

Uptake by Protoplasts Isolated from atmrp1-1 Mutant andWild-type Arabidopsis WS Plants—Wild-type WS andatmrp1-1 mutant plants were greenhouse-grown (24 � 2 °C;10/14 hphotoperiod; 80% relative humidity) for 4–5weeks, andprotoplasts were prepared from rosette leaves as described (33).MTX uptake was estimated by incubating equal amounts of thewild-type and atmrp1-1 protoplast suspensions in uptakemedium (10 mM CaCl2, 1 mM MgCl2, 0.5 M sorbitol and 10 mMTris/Mes (pH5.5)) containing 19nM [3H]MTXat 25 °C for 2.5 hwith gentle agitation on an orbital shaker. For the estimation oftotal protoplast (“cellular”) [3H]MTX content, the protoplastswere pelleted by centrifugation at 60 � g for 5 min and washedthree times with fresh uptake medium before the removal ofaliquots for liquid scintillation counting. For the estimation ofvacuolar [3H]MTX content, intact vacuoles were isolated fromthe protoplasts by differential osmolysis and Ficoll flotationcentrifugation through 10% (w/v) and 4% (w/v) Ficoll cushionsprepared in vacuolar resuspension buffer containing 0.45M sor-bitol (34). Bovine serum albumin (5 mg/ml) was added to the10% Ficoll solution to diminish adherence of membranes fromother sources to the surfaces of the vacuoles during fraction-ation (35). Intact vacuoles were collected from the 0/4% Ficollinterface for liquid scintillation counting.Protein Estimations—Protein was estimated by a modifica-

tion of the method of Bradford (36)

RESULTS

MgATP-dependent Transport of Folate Monoglutamate(Pteroyl-L-glutamate, PteGlu1)—Both heterologously expressedAtMRP1 and vacuolar membrane vesicles purified from redbeet storage root are competent in the transport of PteGlu1.The concentration dependence of uptake of affinity-purified[3H]PteGlu1 by vacuolar membrane-enriched vesicles purifiedfrom pYES3-AtMRP1-transformed S. cerevisiae ycf1� strainDTY168 approximates Michaelis-Menten kinetics to yield Kmand Vmax values of 188 � 68 �M and 10.1 � 1.8 nmol/mg/20min, respectively (Fig. 2A). The corresponding values for nativevacuolarmembrane vesicles purified from red beet storage rootare 195 � 14 �M and 6.4 � 0.2 nmol/mg/20 min, respectively(Fig. 2B). Crucial, however, if transport is to be measured reli-ably, is the purity of the [3H]PteGlu1 employed as transportsubstrate. For the experiments shown in Fig. 2 and all of theother experiments employing [3H]PteGlu1, the radiolabeled



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transport substrate as supplied was subjected to FBP-agaroseaffinity purification. If this precaution was not taken, the ratesand extents of uptake were often underestimated. As exempli-fied by the results obtained with red beet vacuolar membranevesicles, the rates and extents of MgATP-dependent uptake of

FBP-agarose-purified [3H]PteGlu1 exceeded those of the unpu-rified compound by severalfold such that purification of the[3H]PteGlu1 stock was accompanied by a decrease in the Kmfrom403� 112 to 195� 15�Mconcomitantwith an increase intheVmax from 2.3 � 0.4 to 6.4 � 0.2 nmol/mg/20min (data notshown). The reason for this difference is not known, but it issuspected to result fromcontamination of the stockwith break-down products that interfere with uptake of the parent com-pound, [3H]PteGlu1, but which lack radiolabel and/or do notundergo appreciable MgATP-dependent uptake themselves.HPLC analysis of the [3H]PteGlu1 stock before and after affinitypurification discloses additional species in the former that areabsent from the latter.5MgATP-dependent Transport of MTX—The kinetics of

uptake of the antifolate MTX by yeast vacuolar membrane-en-riched vesicles containing heterologously expressed AtMRP1 orpurified red beet vacuolar membrane vesicles are fundamentallyequivalent to those of [3H]PteGlu1. In both membrane prepara-tions, MgATP-dependent [3H]MTX uptake approximatesMichaelis-Menten kinetics to yield Km and Vmax values of 243 �55 �M and 8.6 � 0.9 nmol/mg/20 min, respectively, for heterolo-gously expressedAtMRP1 (Fig. 3A) and223�37�Mand3.3�0.2nmol/mg/20 min, respectively, for red beet vacuolar membranevesicles (Fig. 3B).Vanadate Inhibitability of [3H]PteGlu1 and [3H]MTXTrans-

port—All four of the transport processes examined, MgATP-dependent uptake of [3H]PteGlu1 or [3H]MTX into yeast vac-uolar membrane-enriched vesicles containing heterologouslyexpressed AtMRP1 or into red beet vacuolar membrane vesi-cles, are susceptible to inhibition by vanadate (Table 1). On thisbasis and the insensitivity of these processes to the V-ATPaseinhibitor bafilomycin A1 or the protonophore gramicidin-D(data not shown), the transport measured appears to be largelyattributable to the primary energization of ABC transporters byMgATP rather than secondaryH�-coupled transport. The onlyqualitative difference between the kinetics of [3H]PteGlu1 and[3H]MTX transport by heterologously expressed AtMRP1 andthe red beet membrane preparations is that �20% of the trans-port measured in the latter is insensitive to inhibition by vana-date. This necessitates subtraction of the uninhibitable compo-nent from the total uptake measured when enumerating theconcentration of vanadate required to inhibit the inhibitablecomponent by 50%. When this correction is applied, the I50values for the inhibition of [3H]PteGlu1 and [3H]MTX uptakeby vacuolar membrane-enriched vesicles containing heterolo-gously expressed AtMRP1 and red beet vacuolar membranevesicles fall in the same range: 3.3 � 0.5 and 6.7 � 1.9 �M for[3H]PteGlu1 and [3H]MTX uptake by AtMRP1; 3.2 � 1.0 and9.6� 4.6 �M for [3H]PteGlu1; and [3H]MTX uptake by red beet(Table 1).Transport of PteGlu1 andGS-conjugates by a CommonMRP-

type Functionality—AtMRP1was first characterized in terms ofits capacity to catalyze the vanadate-inhibitable, MgATP-de-pendent transport of GS-conjugates (32), a property it shareswith many other MRP-type ABC transporters (13). Accord-

5 V. Naponelli, A. D. Hanson, and J. Gregory, unpublished data.

FIGURE 2. Concentration dependence of MgATP-dependent [3H]PteGlu1uptake by vacuolar membrane-enriched vesicles purified from pYES3-AtMRP1-transformed S. cerevisiae ycf1� strain DTY168 (DTY168/pYES3-AtMRP1) cells (A) and by vacuolar membrane vesicles purified from redbeet storage root (B). MgATP-dependent [3H]PteGlu1 uptake by AtMRP1 (A)was calculated by subtracting the radioactivity taken up by vacuolar mem-brane-enriched vesicles purified from empty pYES3 vector-transformedstrain DTY168 (DTY168/pYES3) cells from that taken up by the equivalentmembrane fraction from DTY168/pYES3-AtMRP1 cells. The data were fittedto a single Michaelis-Menten function by nonlinear least squares analysis toyield Km and Vmax values of 188 � 68 �M and 10.1 � 1.8 nmol/mg/20 min,respectively, for heterologously expressed AtMRP1 (A) and 195 � 15 �M and6.4 � 0.2 nmol/mg/20 min, respectively, for red beet vacuolar membranevesicles (B). Values shown are means � S.E. (n � 3).



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ingly, heterologously expressed AtMRP1 and, as would beexpected if similar transporters are to be found in native plantvacuolarmembranes, red beet vacuolarmembrane vesicles cat-alyze the vanadate-inhibitable uptake of [3H]NEM-GS. TheKmand Vmax values for [3H]NEM-GS uptake by AtMRP1 and redbeet are 292 � 144 �M and 8.8 � 2.2 nmol/mg/20 min and

258 � 46 �M and 10.6 � 0.9 nmol/mg/20 min (Fig. 4, A and B);the corresponding I50 values for inhibition by vanadate are2.3 � 0.5 and 4.4 � 0.5 �M (after subtraction of the uninhibit-able component in the latter case) (Table 1).An intriguing finding, however, is that although NEM-GS

interacts with the same MRP-type functionality as PteGlu1, itdoes so noncompetitively. Although inclusion of 100 �MNEM-GS in the uptake medium decreases the Vmax value for[3H]PteGlu1 uptake by vacuolar membrane-enriched vesiclespurified from yeast heterologously expressing AtMRP1 from10.1� 1.8 nmol/mg/20min to 5.7� 0.8 nmol/mg/20min, it haslittle or no effect on the Km value, which has values of 181 � 52and 188 � 68 �M, respectively, in the presence and absence ofNEM-GS (Fig. 5A). Qualitatively similar results are obtainedwhen the same experiment is performed on red beet vacuolarmembrane vesicles; the Vmax value for [3H]PteGlu1 uptake isdecreased from 6.4 � 0.2 nmol/mg/20 min to 2.3 � 0.1 nmol/mg/20 min while leaving Km relatively unaffected at values of195 � 15 and 181 � 12 �M (Fig. 5B) when NEM-GS is includedin the uptake medium. Because under all four conditions[3H]PteGlu1 uptake closely approximates Michaelis-Mentenkinetics to yield strictly linear Hanes-Woolf plots, the implica-tion is that PteGlu1 andNEM-GS interactwith a common func-tionality in both membrane preparations but at different sites.The interaction of heterologously expressed AtMRP1 or itsequivalents in red beet with NEM-GS interferes with [3H]Pte-Glu1 transport without interferingwith the binding of the latterto the transporter.Polyglutamylated Folate as a Poor Transport Substrate—By

comparison with the folyl monoglutamates, PteGlu1 andMTX,folyl polyglutamates (PteGlun) undergo only very low rates ofMgATP-dependent uptake. Net uptake of 50 �M [3H]PteGluninto yeast vacuolar membrane-enriched vesicles containingheterologously expressed AtMRP1 or red beet vacuolar mem-brane vesicles is 7–10- and�3-fold lower than the net uptake of100 �M [3H]PteGlu1 measured after 10 or 20 min under thesame conditions (Fig. 6,A and B). Moreover, whereas the inclu-sion of 50 �M PteGlun in the [3H]PteGlu1 uptake mediumweakly but consistently inhibits uptake mediated by heterolo-gously expressed AtMRP1, suggesting that PteGlu1 and Pte-Glun compete for common binding sites (Fig. 6A), the converse

FIGURE 3. Concentration dependence of MgATP-dependent [3H]MTXuptake by vacuolar membrane-enriched vesicles purified from DTY168/pYES3-AtMRP1 cells (A) and by vacuolar membrane vesicles purifiedfrom red beet storage root (B). The data were fitted to a Michaelis-Mentenfunction to yield Km and Vmax values of 243 � 55 �M and 8.6 � 0.9 nmol/mg/20 min, respectively, for heterologously expressed AtMRP1 and 223 � 37�M and 3.3 � 0.2 nmol/mg/20 min, respectively, for red beet vacuolar mem-brane vesicles. Values shown are means � S.E. (n � 3).

TABLE 1Sensitivity of MgATP-dependent 3HPteGlu1, 3HMTX, or 3HNEM-GSuptake by yeast vacuolar membrane-enriched vesicles containingheterologously expressed AtMRP1 and vacuolar membrane vesiclespurified from red beet storage root to inhibition by vanadateThe rates of MgATP-dependent uptake of 100 �M concentrations of 3HPteGlu1,3HMTX, or 3HNEM-GS from assay media containing 0–80 �M vanadate wereestimated as described in Figs. 2 and 4. Each data set was fitted to a single negativeexponential function by nonlinear least squares analysis to yield estimates of theconcentrations of vanadate required for 50% inhibition of net uptake (I50 values).Note that the I50 values calculated for red beet vacuolarmembrane vesicles are thosefor the inhibitable component obtained by subtracting the uninhibitable compo-nent, which accounted for approximately 20% of total uptake, from total uptake.Values shown are means � S.E. (n � 3).

Transportsubstrate

I50 (�M) for inhibition by vanadateHeterologously expressed

AtMRP1Red beet vacuolarmembrane vesicles

PteGlu1 3.3 � 0.5 3.2 � 1.0MTX 6.7 � 1.9 9.6 � 4.6NEM-GS 2.3 � 0.5 4.4 � 0.5



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is seen in the red beet system. Addition of 50 �M PteGlunincreases the net uptake of 100 �M [3H]PteGlu1 by red beetvacuolar membrane vesicles by factors of 1.3 and 2.2 after 10and 20min, respectively (Fig. 6B). The significance of this effectis not known, but the increase in [3H]PteGlu1 uptake seenwhenPteGlun is added to the assay medium is at least partially inhib-ited by vanadate (Fig. 6B), which is consistent with the partici-pation of an ABC transporter.Isolation and Genomic Characterization of AtMRP1 Knock-

outMutants—With the aim of gaining insight into the functionof AtMRP1 in the intact plant, or at least processes for which itsloss of function has discernible phenotypic consequences, twoT-DNA insertion mutant alleles of AtMRP1, atmrp1-1 andatmrp1-2 from ecotypes Wassilewskia (WS) and Columbia

(Col-0), respectively, were obtained. The former was isolated inin-house screens; the latter was obtained from the ArabidopsisBiological Resource Center Salk collection.As confirmed by the results shown in Fig. 7, both T-DNA

insertions, atmrp1-1 that maps to exon 23 of the genomicsequence of AtMRP1 and atmrp1-2 that maps to exon 25 (Fig.7A), are associated with a knock-out or severe knockdown ofexpression (Fig. 7B). RT-PCR of total RNA extracted fromatmrp1-1 or atmrp1-2 mutants and the corresponding wildtypes using the AtMRP1-specific primer pair AtMRP1-RT-F/AtMRP1-RT (see “Materials andMethods”) yields a strong sig-nal that matches the 512-bp amplification product expected

FIGURE 4. Concentration dependence of MgATP-dependent [3H]NEM-GSuptake by vacuolar membrane-enriched vesicles purified from DTY168/pYES3-AtMRP1 cells (A) and by red beet vacuolar membrane vesicles (B).The data were fitted to a single Michaelis-Menten function to yield Km andVmax values of 292 � 144 �M and 8.8 � 2.2 nmol/mg/20 min, respectively, forheterologously expressed AtMRP1, and 258 � 46 �M and 10.6 � 0.9 nmol/mg/20 min, respectively, for red beet vacuolar membrane vesicles. Valuesshown are means � S.E. (n � 3).

FIGURE 5. Kinetics of inhibition of MgATP-dependent [3H]PteGlu1 uptakeby vacuolar membrane-enriched vesicles purified from DTY168/pYES3-AtMRP1 cells (A) and red beet vacuolar membrane vesicles (B) by NEM-GS. Shown are Hanes-Woolf plots of [PteGlu1] (�M)/uptake (nmol/mg/20 min)versus [PteGlu1] (�M) in the absence (E) and presence (F) of 100 �M NEM-GS.The Km and Vmax values for AtMRP1-mediated [3H]PteGlu1 uptake (A) in theabsence (E) and presence (F) of 100 �M NEM-GS were 188 � 68 �M and10.1 � 1.8 nmol/mg/20 min, and 181 � 52 �M and 5.7 � 0.8 nmol/mg/20 min,respectively. The corresponding values for [3H]PteGlu1 uptake by red beetvacuolar membrane vesicles (B) were 195 � 15 �M and 6.4 � 0.2 nmol/mg/20min, and 181 � 12 �M and 2.3 � 0.1 nmol/mg/20 min, respectively. Valuesshown are means � S.E. (n � 3).



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from the mature transcript from the wild-type RNA extractsbut no signal from the mutant extracts (Fig. 7B).MTX Hypersensitivity of atmrp1-1 and atmrp1-2 Knock-

out Mutants—Having established that both atmrp1-1 andatmrp1-2 are null mutants and that heterologously expressedAtMRP1 is competent in the MgATP-dependent transport oftoxic amphipathic anions, such as MTX, as well as some GS-conjugable xenobiotics, screenswere initiated to assess the sen-sitivity of thesemutants by comparisonwithwild-type plants tothe toxic effects of these agents.In the first instance, seeds from atmrp1-1 and atmrp1-2

plants and their correspondingWS and Col-0 wild-type equiv-alents were germinated and grown in the light on sterile platescontaining a broad range of concentrations of the antifolateMTX, CDNB (a cytotoxic generic glutathione S-transferase(GST) substrate), the GS-conjugable herbicides atrazine (a tri-azine derivative) or metolachlor (a chloroacetanilide), menadi-one (an elicitor of oxidative stress), or abscisic acid (ABA, asesquiterpenoid stress hormone). In this way it was determinedthat although neither of thesemutants is more susceptible thantheir corresponding wild types to any of the GS-conjugable

xenobiotics tested or menadione or ABA, both are more sensi-tive to MTX in the growth medium. When germinated andgrown vertically on MS medium supplemented with 1–10 �Matrazine or metolachlor, 5–60 �M CDNB, 5–100 �M menadi-one or 1–5 �M ABA, no difference between the mutant andwild-type seedlings was discernable; all showed similar degreesof root growth inhibition (data not shown). However, when thesame experiments were conducted with MTX, differencesbetween the atmrp1-1 and atmrp1-2 mutants and their corre-sponding wild types were evident in the nanomolar range.Expansion of the screens to the effects of MTX at the level of

the intact seedling reinforced these findings. When grown onsolid MS medium containing 0–40 nM MTX in plant tissueculture vessels under a 16/8-h photoperiod for a further 14 daysafter germination, significant, albeit small to moderate, differ-ences between the atmrp1 mutants and their correspondingwild types are evident at both the root and shoot levels (Fig. 8).The wild-type and atmrp1mutant seedlings are indistinguish-able when grown on solid medium lacking MTX. However,starting at MTX concentrations of 10 nM in the case of ecotypeWS and 15 nM in the case of ecotype Col-0, differences betweenthe mutant and wild-type seedlings in terms of both root andshoot growth become evident such that although both themutants and wild types are subject to growth retardation, theretardations are greater in both the atmrp1-1 and atmrp1-2mutants at all MTX concentrations greater than 25 nM (Fig. 8).At the highest MTX concentrations examined, 30 and 40 nM,the mutants are more prone than the wild types to chlorosis,anthocyanin accumulation, and eventual necrosis (Fig. 8).Protoplasts from atmrp1-1 Mutants Are Defective in Vacuo-

lar [3H]MTX Accumulation—In view of the capacity of heter-ologously expressed AtMRP1 for MgATP-dependent MTXtransport in vitro and localization of this transport protein tothe vacuolar membrane in planta, the most straightforwardexplanation for the increased susceptibility of atmrp1-1 andatmrp1-2 mutants to the toxic action of this antifolate is thatby comparison with their wild-type counterparts they areimpaired in the vacuolar sequestration of this compound. Totest this proposal, two approaches were adopted. In the first,total cellular [3H]MTX uptake and the vacuolar levels achievedby protoplasts isolated from the leaves of atmrp1-1mutant andwild-type WS plants were compared. In the second, the capac-ities of vacuolar membrane-enriched vesicles purified fromatmrp1-1 mutant and wild-type WS liquid root cultures forMgATP-dependent [3H]MTX uptake in vitro were compared.The results obtained from both approaches were consistent

with a deficiency in vacuolar MTX sequestration in atmrp1-1mutants.When incubated in culturemedium containing 19 nM[3H]MTX for 2.5 h, protoplasts isolated from atmrp1-1mutants and wild-type WS plants achieve similar total cellularlevels of the antifolate (61.7 � 3.1 and 63.8 � 2.6 fmol/mgprotein, respectively) (Fig. 9A). However, if aliquots of the samesamples of protoplasts are gently disrupted by differentialosmolysis to release intact vacuoles and the [3H]MTX contentsof these are estimated, there is a marked difference dependingon whether the vacuoles are fractionated from atmrp1-1mutant or wild-type WS protoplasts. Vacuoles prepared fromwild-type WS protoplasts achieve [3H]MTX contents of 4.4 �

FIGURE 6. Net MgATP-dependent uptake of folate monoglutamate andfolate polyglutamates by vacuolar membrane vesicles purified fromDTY168/pYES3-AtMRP1 cells (A) and purified red beet vacuolar mem-brane vesicles (B). Net MgATP-dependent [3H]PteGlu1 (100 �M) (�) or[3H]PteGlun (50 �M) uptake (u), or [3H]PteGlu1 (100 �M) uptake from assaymedia containing unlabeled PteGlun (50 �M) (f) were measured after 10 and20 min. In one set of experiments on red beet vacuolar membrane vesicles,the vanadate inhibitabilities of [3H]PteGlu1 and [3H]PteGlun uptake and of[3H]PteGlu1 uptake in the presence of unlabeled PteGlun were determinedafter the addition of 100 �M vanadate to the assay medium. Values shown aremeans � S.E. (n � 3).



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0.1 pmol/mg protein, whereas the[3H]MTX content of the corre-sponding fraction from atmrp1-1mutant protoplasts is only 2.1 � 0.2pmol/mg protein (Fig. 9B). An�50% diminution of the vacuolar[3H]MTX content of atmrp1-1mutant versus wild-type WS proto-plasts despite comparable levels oftotal protoplast uptake indicatesthat the atmrp1-1 mutants are notimpaired in total cellular MTXuptake or extrusion but are insteadimpaired in delivery of this com-pound into the vacuole.In agreement with this interpre-

tation, the rates and extents ofMgATP-dependent [3H]MTX up-take by vacuolar membrane-en-riched vesicles purified fromatmrp1-1 mutant root cultureswhenmeasured at an initial concen-tration of 50 �M are diminished by�60 and 40%, respectively, by com-parison with the correspondingmembrane fraction from wild-typeWS root cultures (Fig. 10). More-over, regardless of the source ofthemembrane vesicles,MgATP-de-pendent [3H]MTX uptake is morethan 85% inhibited by the inclusionof 100 �M vanadate in the uptakemedium (data not shown), whichimplies that not only theAtMRP1-dependent componentbut also the AtMRP1-independentcomponent of uptake is largelyattributable to ABC transportersand not to secondary H�-coupled,V-ATPase-energized uptake.

DISCUSSION

The findings presented establishthat heterologously expressedAtMRP1 is not only competent intheMgATP-dependent transport ofGS-conjugates but also monoglu-tamylated folates, as exemplified byPteGlu1 and MTX, and that this is aproperty shared by vesicles derivedfrom the vacuolar membrane of redbeet storage root. Moreover, it isshown that whereas both of theknock-out mutant alleles ofAtMRP1, atmrp1-1 and atmrp1-2from Arabidopsis ecotypes WS andCol-0, respectively, are indistin-guishable from their wild-type

FIGURE 7. Schematic diagram depicting positions of T-DNA insertions in genome of Arabidopsis WSatmrp1-1 and Col-0 atmrp1-2 knock-out mutants (A), and results of RT-PCR analyses of AtMRP1 expres-sion in wild-type and atmrp1-1 mutant WS plants and wild-type and atmrp1-2 mutant Col-0 plants (B).The DNA encompassing AtMRP1 starting at position 9384 and the left border of the T-DNA insertion of homozy-gous WS atmrp1-1 mutants was amplified using the primer pair AtMRP1-R1/T-DNA-LB (5�-TGCAAGTAGTGGTC-GAATATTTTG-3� and 5�-GATGCACTCGAAATCAGCCAATTTTAGAC-3�) to map the insertion in this allele to exon23. The DNA encompassing AtMRP1 starting at position 9705 and the left border of the T-DNA insertion ofhomozygous Col-0 atmrp1-2 mutants was amplified using the primer pair AtMRP1–2 RP/T-DNA-LB1 (5�-GATT-TCAAGGCTTTGGAGGTC-3� and 5�-GCCTTTTCAGAAATGGATAAATAGCCTTGCTTCC-3�) to map this insertion toexon 25. RT-PCR of total RNA extracted from wild-type and atmrp1-1 mutant WS plants and from wild-type andatpmrp1-2 mutant Col-0 plants was performed using the primer pair AtMRP1-RT-F/AtMRP1-RT-R (see under“Materials and Methods”), which should yield a 512-bp amplification product from reverse-transcribed matureAtMRP1 transcript. To ensure equivalence between the extractions and sample loads, the same RNA sampleswere subjected to RT-PCR using the actin-F/actin-R primer pair (see under “Materials and Methods”), whichyields a 207-bp amplification product.

FIGURE 8. Differential sensitivities of the growth of Arabidopsis WS atmrp1-1 mutant and wild-typeplants (A) and Arabidopsis Col-0 atmrp1-2 and wild-type plants (B) to the inclusion of MTX in the growthmedium. Seedlings were grown for 14 days on solid MS medium or MS medium supplemented with theconcentrations of MTX indicated at 22 � 2 °C and 70% relative humidity under a 16/8-h photoperiod.



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counterparts when grown under standard conditions or onsolid media containing GS-conjugable xenobiotics, they aremore sensitive to nanomolar concentrations of MTX in thegrowth medium. In the case of the mutant allele that was char-acterized further,atmrp1-1,MTXhypersensitivity is associatedwith impaired vacuolar transport and sequestration of this anti-folate. Evidently, AtMRP1 and its functional equivalent in thevacuolar membrane of red beet translocate folate monogluta-mates and antifolates in vitro and the former contributes toantifolate tolerance in vivo.Estimates of the internal volume of red beet vacuolar mem-

brane vesicles prepared in the same manner as in this studyyield a value of 10 �l/mgmembrane protein (10, 37). Assuminga 1:1mixture of right-side-out and inside-out vesicles, uptake of�5 nmol/mg protein after 60 min from a medium containing100 �M [3H]PteGlu1 amounts to an accumulation ratio of 10.The corresponding value for the uptake of [3H]NEM-GS by the

same preparation is 16. Given that the transport capabilities ofyeast vacuolar membrane-enriched vesicles containing heter-ologously expressed AtMRP1 are similar to and often greaterthan those of red beet vacuolar membrane vesicles, it is appar-ent that transport is against a moderately steep concentrationgradient in both preparations.The substrate selectivity of AtMRP1 strictly parallels those of

the MRPs from mammalian sources that have been implicatedin the transport of folates. Human MRPs 1 and 3, for instance,are high capacity, low affinity MTX and folate transportersexhibiting little or no affinity toward polyglutamates (7). MRP3and several other human MRPs confer resistance to MTX intransfected cultured cells (8). Equivalently, AtMRP1 is a highcapacity, intermediate affinity folate and MTX transporterexhibiting little or no activity toward polyglutamates; it medi-ates very low rates of [3H]PteGlun transport, and the inclusionof PteGlun in the uptake medium only weakly inhibits [3H]Pte-Glu1 uptake. Knock-out mutants for AtMRP1 are hypersensi-tive toMTX in the growthmedium and deficient in its vacuolarsequestration.AtMRP1 and its functional equivalents in other plants have

the characteristics of MgATP-dependent vacuolar pumpscapable of contributing to the detoxification of non-glutathio-nylated amphipathic anions. That this should be the case, how-ever, raises the following question: why does AtMRP1 appearnot to confer tolerance toward CDNB ormetolachlor, a genericcytotoxin and herbicide, respectively, whose GS-conjugates arealso in vitro transport substrates (12, 32) if it confers tolerancetoward the transport substrate MTX?The answer to this question is not known, but functional

redundancy or a lack thereof may be an important consider-ation. Because only a subset of plant MRPs have appreciablefolate and antifolate transport activity, the problems of func-tional redundancy associated with screens involving GS-conju-gable xenobiotics, namely the ability ofmostMRPs to transportGS-conjugates, apply less toMTX. An alternate or supplemen-tary explanation is that, unlike the screens deploying GS-con-jugable xenobiotics, the efficacy of MTX as a screening agent isprobably not rate-limited by upstream enzymes, such as GSTs,whose activity in the case of GS-conjugable xenobiotics wouldset an upper limit on the rate of formation of transport-activederivatives. In lieu of findings to the contrary, one other possi-bility that cannot be ruled out is that the importance of vacuolarsequestration has simply been overstated in that conjugationof GS-conjugable xenobiotics, alone, might be sufficient forthe detoxification of many xenobiotics regardless of whether theconjugates remain in the cytosol or are transported into thevacuole (38–40).The MTX hypersensitivity profiles of atmrp1 mutants are

reminiscent of the properties of atmrp2 knock-out mutants.Despite the capacity of heterologously expressed AtMRP2 forhigh rates of GS-conjugate transport, the highest reported todate for anyMRP regardless of source (16, 41), themost strikingphenotype of atmrp2mutants is not associated with GS-conju-gate transport but instead with the vacuolar sequestration ofchlorophyll catabolites (40), amphipathic anions whose trans-port, as exemplified by the linearized tetrapyrrole Brassica

FIGURE 9. Comparison of the MTX contents of protoplasts (A) and intactvacuoles (B) isolated from atmrp1-1 mutant and wild-type (WT) Arabi-dopsis WS plants after incubation in media containing [3H]MTX. Equiva-lent amounts of protoplasts, estimated as total protein, isolated from rosetteleaves of atmrp1-1 mutant and wild-type WS plants were incubated at 25 °Cwith gentle shaking in uptake medium containing 19 nM [3H]MTX. After 2.5 h,the protoplasts were sedimented by centrifugation at low speed and washedthree times in the same medium lacking [3H]MTX. After removing aliquots ofthe suspended protoplasts for the estimation of total protoplast [3H]MTXcontent (A), samples of the suspensions were subjected to gentle disruptionby differential osmolysis and Ficoll floatation centrifugation for the purifica-tion of intact vacuoles and estimation of their [3H]MTX content (B). Valuesshown are means � S.E. (n � 3).

FIGURE 10. Time course of MgATP-dependent [3H]MTX uptake by vacuo-lar membrane-enriched vesicles purified from liquid root cultures ofatmrp1-1 (F) and wild-type Arabidopsis WS plants (E). MgATP-dependentMTX uptake from media containing 50 �M [3H]MTX was estimated asdescribed in Fig. 3. Values shown are means � S.E. (n � 3).



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napus nonfluorescent chlorophyll catabolite 1 (Bn-NCC-1),does not depend on upstream GST action (41).Another striking similarity betweenAtMRP1 andAtMRP2 is

that both interact with both GS-conjugates and non-glutathio-nylated amphipathic anions but not in a simple competitivemanner. Heterologously expressed AtMRP2 catalyzes thetransport of GS-conjugates, Bn-NCC-1 and glucuronides suchas 17�-estradiol 17-(�-D-glucuronide) (E217�G), but not in amanner consistent with the interaction of these three classes ofsubstrate with a common binding or transport site (41). Forinstance, DNP-GS and Bn-NCC-1 exert little or no effect oneach other’s transport via AtMRP2; both are transported atsimilar rates regardless ofwhether the other transport substrateis in the uptakemedium (41). Rather than competing with eachother for uptake by AtMRP2, DNP-GS stimulates the uptake ofE217�G and vice versa (16). The kinetics of AtMRP2-mediatedtransport are consistent with a scheme in which DNP-GS,E217�G, and Bn-NCC-1 undergo transport through differentAtMRP2-dependent pathways and that DNP-GS and E217�Gpromote each other’s transport by binding sites distinct frombut tightly coupled to the other’s transport pathway (13, 16).The behavior of AtMRP1 is not as pronounced as that ofAtMRP2 in this regard. Nonetheless, the binding and/or trans-port sites for NEM-GS and PteGlu1 behave as if distinct, butcoupled.NEM-GSnoncompetitively inhibits PteGlu1 transportand vice versa, implying that the two substrates interact withnonidentical binding sites on the same functionality. This is nota property peculiar to AtMRP1 because the equivalent trans-port processes in red beet vacuolar membrane vesicles behavesimilarly.If MRP-type transporters contribute to the accumulation of

vacuolar folates in planta, they likely do so by catalyzing uptakeof the monoglutamylated rather than the polyglutamylatedcomponent. Because polyglutamylated folates represent such asizeable fraction of the total vacuolar folate pool, a pressingquestion that remains to be answered is how polyglutamylatedfolatesmake their appearance in this compartment?Must alter-nate mechanisms for the delivery of polyglutamates into thevacuole be invoked if neither heterologously expressedAtMRP1 nor red beet vacuolar membrane vesicles catalyzeappreciable PteGlun uptake and the vacuolar lumen is devoid ofFPGS and ATP (3), so precluding intravacuolar polyglutamyla-tion of the incoming monoglutamates?A subsidiary consideration that has a bearing on whether

AtMRP1 and its equivalents in other plant vacuolarmembranesactually transport folate monoglutamates in vivo are their rela-tively low affinities for these compounds in vitro. TheKm valuesfor PteGlu1 transport by heterologously expressed AtMRP1and red beet vacuolar membrane vesicles (188 and 195 �M,respectively) are far higher than the probable steady state cyto-solic concentrations of these compounds in plant cells. Assum-ing a typical folate content of 2 nmol/g fresh weight (42) ofwhich 5% or less is monoglutamylated (3), it is likely that thesteady state cytosolic concentration of PteGlu1 seldom exceeds1 �M if the cytosol amounts to �34 �l/g fresh weight (43, 44),and about 20%of the total pool is allocated to this compartment(3). In short, if AtMRP1 and its equivalents are to participate invacuolar folate sequestration, the conditions for their assay in

vitro are far from optimal and/or the transporters in questionoperate far from their maximal capacity in vivo.

Notwithstanding these uncertainties, there can be littledoubt of the relevance of AtMRP1 for the detoxification of anti-folates despite its correspondingly low in vitro Km value forMTX (243 �M) in that atmrp1 knock-out mutants are hyper-sensitive to nanomolar concentrations of this compound.Moreover, even when exposed to only nanomolar concentra-tions ofMTX, there is amarked difference between the capacityof wild-type and atmrp1 knock-outmutant protoplasts for vac-uolar sequestration of this antifolate, which might imply thateven when operating suboptimally MRP-type transporters canmake a significant contribution to the intracellular distributionof these and related compounds.That said, there is still appreciable MTX uptake into the

vacuoles of atmrp1-1mutant protoplasts, and in vitromeasure-ments of the uptake of [3H]MTX by vacuolar membrane-en-riched vesicles purified fromatmrp1-1 root cultures reveal con-siderable residual vanadate-inhibitable MgATP-dependentuptake. Knowing that heterologously expressed AtMRP4 iscapable of high rates of PteGlu1 and MTX transport (17) andthat at least a fraction of total cellular AtMRP4 may localize tothe vacuolar membrane (19), there is a possibility that thistransporter also contributes to the vacuolar uptake of folylmonoglutamates.It is instructive to note that because physiological folates

compete with antifolates for binding to their target enzymes,dihydrofolate reductase and FPGS, any factor that influencesphysiological folate pool size could affect antifolate efficacy. It istherefore conceivable that a diminution of vacuolar folateuptake, as might be the case for atmrp1 knock-out mutants,would confer MTX hypersensitivity by comparison with wildtypes not only because of a decrease in the capacity for vacuolarMTX sequestration but also because of a decrease in cellularfolate pool size.Of the many facets of vacuolar folate uptake that have yet to

be resolved, one that is especially perplexing is the seeminglocalization of folate polyglutamates and �-glutamyl hydrolase(GGH) to the same compartment (3). If this is correct and theactivity of GGH in vitro approximates its activity in vivo, thevacuolar folate polyglutamate pools ofArabidopsis and red beetwould be predicted to have half-lives of only �7 s and 5 min,respectively, under steady state conditions (3). This paradoxhas yet to be reconciled, but a possibility that cannot be ignoredis that it is notmonoglutamates but instead polyglutamates thatare transported into the vacuole in vivo by a non-MRP-typefunctionality, and that the former are derived from the latterintravacuolarly through the action of a vacuolar GGH, i.e. theremay be parallels betweenwhat happens in plants andwhat hap-pens in mammals, where lysosomes do not store folates butinstead import folate polyglutamates, hydrolyze them, andexport monoglutamates (45, 46). Alternatively, vacuolar folatepolyglutamates are protected from intravacuolar hydrolysisthrough their interaction with FBPs or by the presence of apotent GGH inhibitor in this compartment. Although there isno experimental evidence for or against either possibility, thelatter is the less likely in that the inhibition of vacuolar GGHwould not only diminish the deglutamylation of folate polyglu-



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tamates but also the deglutamylation of p-aminobenzoate poly-glutamates, which would disrupt folate recycling (3). Anotherpossibility, perhaps the most straightforward one, is that folatepolyglutamates, or at least a sizeable fraction of this pool, andGGH do no reside in precisely the same vacuolar compartmentbut instead localize to different intravacuolar compartments ordifferent subpopulations of vacuoles in the same cells or differ-ent cells within the same or different tissues.

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

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