Enhanced Multispecificity of Arabidopsis Vacuolar Multidrug Resistance-associated Protein-type ATP-binding Cassette Transporter, AtMRP2*

Guosheng Liu, Rocío Sánchez-FernándezDagger, Ze-Sheng Li§, and Philip A. Rea

From the Plant Science Institute, Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6018

Received for publication, October 23, 2000, and in revised form, December 13, 2000


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Recent investigations have established that Arabidopsis thaliana contains a family of genes encoding ATP-binding cassette transporters belonging to the multidrug resistance-associated protein (MRP) family. So named because of the phenotypes conferred by their animal prototypes, many MRPs are MgATP-energized pumps active in the transport of glutathione (GS) conjugates and other bulky amphipathic anions across membranes. Here we show that Arabidopsis MRP2 (AtMRP2) localizes to the vacuolar membrane fraction from seedlings and is not only competent in the transport of GS conjugates but also glucuronate conjugates after heterologous expression in yeast. Based on the stimulatory action of the model GS conjugate 2,4-dinitrophenyl-GS (DNP-GS) on uptake of the model glucuronide 17beta -estradiol 17-(beta -D-glucuronide) (E217beta G) and vice versa, double-label experiments demonstrating that the two substrates are subject to simultaneous transport by AtMRP2 and preloading experiments suggesting that the effects seen result from cis, not trans, interactions, it is inferred that some GS conjugates and some glucuronides reciprocally activate each other's transport via distinct but coupled binding sites. The results of parallel experiments on AtMRP1 and representative yeast and mammalian MRPs indicate that these properties are specific to AtMRP2. The effects exerted by DNP-GS on AtMRP2 are not, however, common to all GS conjugates and not simulated by oxidized glutathione or reduced glutathione. Decyl-GS, metolachlor-GS, and oxidized glutathione, although competitive with DNP-GS, do not promote E217beta G uptake by AtMRP2. Reduced glutathione, although subject to transport by AtMRP2 and able to markedly promote E217beta G uptake, neither competes with DNP-GS for uptake nor is subject to E217beta G-promoted uptake. A multisite model comprising three or four semi-autonomous transport pathways plus distinct but tightly coupled binding sites is invoked for AtMRP2.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

ATP-binding cassette (ABC)1 transporters are starting to assume prominence in considerations of energy-dependent transport in plants. Constituted of one or two copies each of two core structural elements: a hydrophobic, membrane spanning domain (MSD) containing multiple (usually four or six) transmembrane spans and a cytosolically oriented ATP-binding domain (nucleotide binding fold, NBF) containing Walker A, Walker B, and ABC signature sequence motifs, ABC transporters are MgATP-energized pumps that as a superfamily are active in the transport of a broad range of substances including alkaloids, amino acids, sugars and sugar conjugates, peptides and peptide conjugates, heavy metal chelates, and lipids across membranes (1).

Two classes of findings were instrumental in prompting studies of ABC transporters in plants. The first was the molecular cloning of a multidrug resistance (MDR)-like gene from Arabidopsis thaliana (2) and the subsequent independent isolation of other MDR homologs from the same and other plant species (3, 4). Because all of these genes encode ABC transporters bearing strong sequence similarities to one another and to the animal MDR gene products, it seemed likely that ATP-dependent, primary active transport functions, analogous to those catalyzed by the MDRs from other organisms, were also operative in plants. The second class of finding was that intact vacuoles and vacuolar membrane vesicles isolated from plants mediate the MgATP-dependent, H+ gradient-independent accumulation of glutathione (GS) conjugates (5, 6).

Of these two classes of finding, investigations of vacuolar GS conjugate transport have provided the most definitive elucidation of the molecular identity and biochemical function of a plant ABC transporter. Analyses of vacuolar GS conjugate uptake, a process implicated in herbicide detoxification, cell pigmentation, the alleviation of oxidative stress, and the storage of antimicrobial compounds, have established that the transporters responsible belong to the multidrug resistance-associated (MRP) family of ABC transporters (7, 8). Although several plant MDRs and their close relatives the pleiotropic drug resistance proteins have been cloned in their entirety (2-4, 9), the transport capabilities of the proteins encoded by these genes has eluded definition. AtPGP1, for instance, is the most thoroughly characterized MDR from a plant source, and elegant investigations of transgenic plants have shown this ABC transporter to be a plasma membrane protein involved in light-dependent hypocotyl elongation (10, 11), but its mode of action remains obscure.

To date, 15 MRP coding sequences have been identified in Arabidopsis2 of which five have been isolated as full-length cDNAs and shown to encode functional GS conjugate pumps. Transformation of Saccharomyces cerevisiae ycf1Delta strains from which more than 95% of the coding sequence of the yeast MRP gene YCF1 has been deleted (12), and high affinity MgATP-energized vacuolar GS conjugate transport is grossly impaired (13-15) with expression vectors containing the entire open reading frame of AtMRP1, AtMRP2, AtMRP3, AtMRP4, or AtMRP5 restores GS conjugate transport (16-18).3,4

Despite this basic conformity of core function there are nevertheless strong indications of functional differences among the AtMRPs. Specifically, AtMRP1 and AtMRP2, the only two AtMRPs for which quantitative data are available, exhibit marked differences in transport capacity and substrate preference (17). AtMRP2 but not AtMRP1 is not only able to transport GS conjugates but also other bulky amphipathic anions, such as the linearized tetrapyrrole Brassica napus nonfluorescent chlorophyll catabolite 1 (Bn-NCC-1), a product of chlorophyll breakdown (17).

The facility of AtMRP2 for the transport of Bn-NCC-1 is of particular interest in three respects. First, Bn-NCC-1, although not glutathionated, is the most efficacious known substrate for a plant GS conjugate pump (Vmax/Km = 4.2 nmol/mg/10 min/µM versus 1.8 nmol/mg/10 min/µM for the next most efficacious AtMRP2 transport substrate, metolachlor-GS; Ref. 17). Second, AtMRP2-dependent uptake of Bn-NCC-1 is nearly 3-fold less sensitive to inhibition by DNP-GS than would be predicted if Bn-NCC-1 and DNP-GS competed for a common binding site. Reciprocally, AtMRP2-dependent DNP-GS uptake is not inhibited appreciably by the inclusion of Bn-NCC-1 in the uptake medium (17). Third, double-label experiments demonstrate simultaneous parallel transport of [14C]Bn-NCC-1 and [3H]DNP-GS by AtMRP2 (17). Although it was initially surprising to find that AtMRP2 can transport both Bn-NCC-1 and GS conjugates, because the functionality, and by implication the pump, responsible for MgATP-energized Bn-NCC-1 uptake by barley vacuoles had earlier been concluded to be different from that responsible for GS conjugate uptake in that GS conjugates did not compete with Bn-NCC-1 for uptake (19), it is now clear, at least in the case of some AtMRPs, that lack of competition between candidate substrates does not automatically preclude their transport by the same transporter.

The general applicability of these findings is not known but a phenomenon that may be related is the capacity of DNP-GS for stimulating the uptake of flavone glucuronides and the model glucuronide 17beta -estradiol 17-(beta -D-glucuronide) (E217beta G) into barley and rye vacuoles via an MRP-like transporter or transporters (20, 21). DNP-GS consistently promotes the uptake of glucuronides by both membrane preparations, whereas other GS derivatives, such as GSSG and decyl-GS, act as activators or inhibitors depending on the membrane source or glucuronide under investigation. A key question with regard to these findings and the known multispecificity of MRPs, such as AtMRP2, is whether the substrate interactions seen in isolated plant vacuolar membranes are a reflection of the properties of individual MRP species or instead reflect interactions between different MRPs or between MRPs and other vacuolar transporters.

In this paper we address this question in two steps. First, by determining whether AtMRP2 localizes to the vacuolar membrane fraction from seedlings of Arabidopsis. Second, by defining the transport characteristics of vacuolar membrane-enriched vesicles purified from AtMRP2-transformed yeast YCF1 disruptants. In so doing, we demonstrate that AtMRP2 is a vacuolar transporter that has a number of unusual properties that distinguish it from other MRPs and confer on it the capacity to account for the complex transport characteristics of native plant vacuolar membranes. It is shown that the multispecificity of heterologously expressed AtMRP2 is not limited to GS conjugates and linearized tetrapyrroles but also extends to glucuronides and that DNP-GS and E217beta G mutually promote each other's uptake by interacting with distinct but coupled binding sites. Moreover, it is determined that although AtMRP2 is competent in the MgATP-energized transport of GSH and GSH promotes E217beta G transport, the enhancements are not attributable to GSH-E217beta G cotransport but instead to nonreciprocal GSH-mediated cis-activation of glucuronide uptake.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Heterologous Expression of AtMRP2 or AtMRP1 in S. cerevisiae and Spodoptera frugiperda Cells-- For the preparation of transport-competent vacuolar membrane-enriched vesicles containing heterologously expressed AtMRP2 or AtMRP1, plasmids pYES3-AtMRP2 and pYES3-AMRP1 were constructed as described (16, 17) and S. cerevisiae ycf1Delta strain DTY168 (MATalpha his6 leu2-3,-112 ura3-52 ycf1::hisG) (12) was transformed with these or empty vector lacking AtMRP2 or AtMRP1 insert (pYES3) by the LiOAc/polyethylene glycol method (22). Transformants were selected for uracil prototrophy as described previously (23).

For the preparation of membranes containing high levels of heterologously expressed AtMRP2 or AtMRP1 for tests of the efficacy and specificity of antibodies raised against synthetic peptides, insect cell line Sf9, derived from S. frugiperda, was transfected with bacmids pVL1392-AtMRP2 or pVL1392-AtMRP1, containing the coding sequences of AtMRP2 or AtMRP1, respectively. The transfections were performed using a BaculogoldTM Transfection Kit (PharMingen Co., San Diego, CA) according to the manufacturer's recommendations, and amplified recombinant virus was prepared by harvesting and pooling the culture supernatants from Sf9 cells 3 days post-infection.

Preparation of Membrane Vesicles-- For the Western analyses shown in Fig. 1, the vacuolar membrane-enriched fraction from Arabidopsis (Col-0) seedlings after growth on Murashige-Skoog medium for 18 days under standard conditions was prepared by a modification of the procedure of Rea et al. (24). For the transport measurements on AtMRP2 and AtMRP1, pYES3-AMRP2/DTY168 or pYES3-AtMRP1/DTY168 cells were grown and vacuolar membrane-enriched vesicles were prepared as described (16, 17). For the transport measurements on membranes from S. cerevisiae ycf1Delta strain DTY168 and the isogenic wild type strain DTY7 (12), vacuoles were isolated and vesiculated as described (13). For the transport measurements on heterologously expressed HmMRP3, membrane vesicles were purified from HmMRP3-transfected human embryonic kidney cell line 293 (HEK293/MRP3-5 cells) and from empty vector control transfectants (HEK293 cells) as described (25). For the routine preparation of insect cell membranes, Sf9 cells infected with amplified pVL1392-AtMRP2 or pVL1392-AtMRP1 were harvested 4 days post-infection, rinsed in phosphate-buffered saline, and subjected to homogenization, differential centrifugation, and density gradient centrifugation as described (26).

Preparation of Peptide-specific Antibody for Immunodetection of AtMRP2-- The AtMRP2-specific rabbit polyclonal antibody used in these investigations (PABAtMRP2) was raised against synthetic peptide with the sequence AESLEEHNISR, corresponding to positions 1569-1579 of the deduced amino acid sequence of AtMRP2 (17). The peptide was synthesized to contain a C-terminal cysteine residue and was coupled to keyhole limpet hemocyanin as described (27). To maximize monospecificity, the antibody was affinity purified against the Mr 176,000 band of the membrane fraction from pVL1392-AtMRP2-infected Sf9 cells. Membranes from pVL1392-AtMRP2-infected Sf9 cells (1.6 mg of protein) were subjected to preparative SDS-PAGE on a single-well, 1.5 mm, 7% (w/v) acrylamide slab gel and electrotransferred to nitrocellulose, and two narrow strips were cut from each of the filters and subjected to immunoreaction with crude PABAtMRP2 serum as described below. After visualization of the immunoreactive Mr 176,000 band, the strips were aligned with the remainder of the blot, and a narrow horizontal band corresponding to AtMRP2 was excised. The excised band was blocked in phosphate-buffered saline containing 3% (w/v) bovine serum albumin for 16 h, after which time it was incubated in crude serum for 16 h at 4 °C. After three brief rinses in Tris-buffered saline containing 0.1% (w/v) bovine serum albumin and 0.1% (w/v) Nonidet P-40, bound antibody was eluted with 150 µl 0.2 M glycine-HCl (pH 2.5) and neutralized immediately by the addition of 75 µl of 1.0 M potassium phosphate (pH 9.0). The final eluate was desalted by two cycles of centrifugal ultrafiltration through CentriconTM 30 membrane filters (Millipore Corp., Bedford, MA) and resuspended in 250 µl of phosphate-buffered saline.

SDS-PAGE and Western Analyses-- Membrane samples were denatured in denaturation buffer for 10 min at 25 °C and subjected to one-dimensional SDS-PAGE on 7% (w/v) acrylamide slab gels in a Bio-Rad mini-gel apparatus (28). In the case of the vacuolar membrane-enriched fraction from pYES3-AtMRP2/DTY168 cells, the samples were delipidated in a 1:1 (v/v) mixture of acetone:ethanol at -20 °C as described (29) before denaturation and SDS-PAGE to improve resolution in the high molecular weight range. For immunodetection of AtMRP2, the separated samples were electrotransferred to nitrocellulose membranes using a wet transfer system for 16 h at 4 °C at a current density of 3 mA/cm2 in Towbin buffer (20% (v/v) methanol, 25 mM Tris, 192 mM glycine, 0.025% (w/v) SDS) (30). The nitrocellulose blots were probed with purified PABAtMRP2 (1:150) and with secondary horseradish peroxidase-conjugated anti-rabbit antibody (1:50,000) by standard procedures. To assess the enrichment of the membrane fractions from Arabidopsis seedlings, total microsomes and the vacuolar membrane fraction were probed with rabbit polyclonal antibody PABTK specific for the Arabidopsis vacuolar H+-pyrophosphatase (AVP), as described (31). Immunoreactive bands were visualized by ECL using the SuperSignalTM system (Pierce).

Measurement of Transport-- Uptake of [3H]E217beta G, [3H]DNP-GS, [3H]GSH, [3H]GSSG, [14C]metolachlor-GS, and [3H]C3G-GS by vacuolar membrane-enriched vesicles purified from pYES3-AtMRP2/DTY168 or pYES3-AtMRP1/DTY168 cells and by vacuolar membrane vesicles purified from untransformed DTY168 or DTY7 cells was measured at 25 °C in 200-µl reaction volumes containing 3 mM ATP, 3 mM MgSO4, 5 µM gramicidin-D, 10 mM creatine phosphate, 16 units/ml creatine phosphate kinase, 50 mM KCl, 1 mg/ml bovine serum albumin, 400 mM sorbitol, 25 mM Tris-Mes buffer (pH 8.0) and the indicated concentration(s) of radiolabeled transport substrate(s) (13, 16, 17). In the case of [3H]GSH, oxidation and the formation of [3H]GSSG was minimized by degassing all solutions before the dissolution of GSH and by purging the freshly prepared GSH stock solutions and uptake media with nitrogen gas immediately before use. In all cases, uptake was initiated by the addition of membrane vesicles and terminated by the addition of 1 ml of ice-cold wash medium (400 mM sorbitol in 3 mM Tris-Mes buffer, pH 8.0) and vacuum filtration of the suspension through prewetted Millipore Durapore hydrophilic polyvinylidene fluoride filters (pore size, 0.22 µM). The filters were rinsed twice with wash medium, and the radioactivity retained was determined by liquid scintillation counting. Nonenergized (MgATP-independent) uptake was estimated by the same procedure except that ATP was omitted from the uptake medium. Uptake of [3H]E217beta G or [3H]DNP-GS by membrane vesicles purified from HEK/MRP3-5 cells or from HEK293 cells was measured by a similar procedure except that the uptake buffer contained 4 mM ATP, 10 mM MgCl2, 10 mM creatine phosphate, 16 units/ml creatine phosphate kinase, 250 mM sucrose, and 10 mM Tris-HCl (pH 7.4), and the wash medium contained 250 mM sucrose and 10 mM Tris-HCl (pH 7.4) (25). Nonenergized uptake was estimated by the same procedure except that AMP (4 mM) replaced ATP in the uptake medium. For the preloading experiments (see Table II), vacuolar membrane-enriched vesicles purified from pYES-AtMRP2/DTY168 cells were preincubated at 25 °C for 30 min in standard uptake medium lacking or containing 200 µM unlabeled DNP-GS before the addition of 200 µM [3H]E217beta G. After incubation for a further 10 min, uptake was terminated, and the radioactivity retained after filtration was estimated as described above.

Measurement of Protein-- Protein was estimated by a modification of the method of Peterson (32).

Computations-- Lines of best fit and kinetic parameters were estimated by nonlinear least squares analysis (33) using the Ultrafit nonlinear curve fitting package from BioSoft (Ferguson, MO).

Chemicals-- [3H]DNP-GS (418.7 mCi/mmol), [3H]GSSG (44.0 mCi/mmol), [3H]C3G-GS (17.4 mCi/mmol), and [14C]metolachlor-GS (8.3 mCi/mmol) were synthesized and purified as described (6, 16, 17). [3H]GSH (44 Ci/mmol) and [3H]E217beta G (55.0 mCi/mmol) were purchased from PerkinElmer Life Sciences. [14C]Metolachlor was a gift from Novartis (Greensboro, NC). All of the general reagents were obtained from Fisher, Research Organics, Inc. (Cleveland, OH), or Sigma.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Vacuolar Membrane Localization of AtMRP2-- AtMRP2 was associated with the vacuolar membrane fraction from Arabidopsis. Regardless of whether Western blots of membranes from recombinant baculovirus pVL1392-AtMRP2-infected Sf9 cells or vacuolar membrane-enriched vesicles from yeast pYES3-AtMRP2/DTY168 cells (Fig. 1A) or from 18 day-old Arabidopsis seedlings were probed (Fig. 1B), polyclonal antibody PABAtMRP2, raised against the AtMRP2-specific peptide AESLEEHNISR (Fig. 1C), reacted with an Mr 176,000 polypeptide species. In all cases the Mr 176,000 polypeptide was the only species that reacted with PABAtMRP2, and in the case of membranes from Sf9 cells and yeast, this species was absent from uninfected and vector control cells, respectively (Fig. 1A). On this basis and because it did not react with membranes from Sf9 cells expressing the one other AtMRP, AtMRP1, known to contain the C2 domain encompassing the AtMRP2 sequence AESLEEHNISR (8) (data not shown), PABAtMRP2 was inferred to be monospecific. As would be expected if AtMRP2 is preferentially associated with the vacuolar membrane fraction from Arabidopsis, parallel Western analyses demonstrated a proportionate enrichment of AtMRP2 and the vacuolar H+-pyrophosphatase, AVP (28), in the vacuolar membrane fraction versus the total microsome fraction from seedlings (Fig. 1B). Moreover, in view of the proximity of the sequence recognized by PABAtMRP2 to the C terminus of AtMRP2 (Fig. 1C), the equivalence of the mobility of the AtMRP2 translation product in all three of the systems examined and, the greater than 97% correspondence between the measured Mr of AtMRP2 (176,400 ± 5,400) and the calculated mass of the polypeptide encoded by the open reading frame of AtMRP2 (181 kDa) (17), AtMRP2 appeared to undergo incorporation into the vacuolar membrane fraction with little or no proteolytic processing.


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Fig. 1.   Western analyses of PABAtMRP2-reactive polypeptides in the membrane fractions from Sf9 cells, yeast, and Arabidopsis. A, membrane fractions from uninfected (Control) and pVL1392-AtMRP2-infected (AtMRP2) Sf9 cells (1 µg/lane), and vacuolar membrane-enriched fractions from pYES3-transformed (Control) and pYES3-AtMRP2-transformed (AtMRP2) yeast DTY168 cells (30 µg/lane). B, total microsome fraction (TMF) and vacuolar membrane fraction (VMF) from 18-day-old Arabidopsis seedlings. The protein loads for the immunodetection of AtMRP2 and the vacuolar H+-pyrophosphatase, AVP, were 30 and 1 µg/lane, respectively. Membrane protein was subjected to SDS-PAGE on 7% (w/v) gels, electrotransferred, and probed with purified PABAtMRP2 or with AVP antibody PABTK as described under "Experimental Procedures." The bands shown are the only immunoreactive species detected. C, putative domain organization of AtMRP2. The amino acid sequence against which PABAtMRP2 was raised (AESLEEHNISR), corresponding to positions 1569-1579 of AtMRP2, is located in the second half of the C-terminal (C) domain (C2). The C2 domain is specific to AtMRPs 1 and 2. The other putative domains shown are: TM0, TM1, and TM2, transmembranous sector of the N-terminal extension, and transmembrane domains 1 and 2, respectively; L0 and L1, N-terminal linker and linker 1, respectively; and NBF1 and NBF2, nucleotide-binding folds 1 and 2, respectively. Numbers delimit the amino acid residues encompassed by these domains.

AtMRP2-mediated Glucuronide Transport-- AtMRP2 catalyzed glucuronide transport. The capacity of heterologously expressed AtMRP2 for glucuronide transport was assayed as described previously for GS conjugates and Bn-NCC-1 (17) except that the rapid filtration assays were performed using Durapore (hydrophilic polyvinylidene fluoride) membrane filters instead of cellulose nitrate membrane filters to minimize background binding of this class of compound. S. cerevisiae ycf1Delta strain DTY168 was transformed with empty vector (pYES3) or vector containing the entire open reading frame of AtMRP2 (pYES3-AtMRP2) under the control of the constitutive yeast phosphoglycerate kinase gene (PGK) promoter (17). After growth on selective media, vacuolar membrane-enriched vesicles (23) were prepared from these and untransformed DTY168 cells and assayed for transport of the model glucuronide E217beta G.

From these experiments it was determined that AtMRP2 catalyzed the low affinity, high capacity MgATP-energized transport of [3H]E217beta G. When assayed at a concentration of 100 µM, MgATP-dependent uptake by vesicles purified from pYES3/DTY168 cells was approximately linear for the first 10 min and proceeded at a rate of 1.3 nmol/mg/min (Fig. 2). By contrast, MgATP-dependent [3H]E217beta G uptake by vesicles purified from pYES3/DTY168 cells or untransformed DTY168 cells (data not shown) was negligible (Fig. 2). As would be expected if the bulk of the transport measured were attributable to AtMRP2, the rate of MgATP-dependent [3H]E217beta G uptake by membrane vesicles from pYES3/DTY168 cells was consistently less than 2.5 nmol/mg/10 min and increased as a simple linear function of E217beta G concentration (data not shown), whereas the concentration dependence of uptake by the corresponding membrane fraction from pYES3-AtMRP2/DTY168 cells approximated Michaelis-Menten kinetics to yield Km and Vmax values of 752.3 ± 219.8 µM and 88.1 ± 17.2 nmol/mg/10 min, respectively (Fig. 3).


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Fig. 2.   Time dependence of MgATP-dependent uptake of [3H]E217beta G by vacuolar membrane-enriched vesicles purified from pYES3-AtMRP2-transformed () and pYES3-transformed (open circle ) S. cerevisiae ycf1Delta strain DTY168. MgATP-dependent uptake was measured in standard uptake medium containing 100 µM [3H]E217beta G and was enumerated as the increase in uptake consequent on the provision of 3 mM ATP. Values shown are the means ± S.E. (n = 3-6).


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Fig. 3.   Effect of DNP-GS on concentration dependence of MgATP-dependent [3H]E217beta G uptake by vacuolar membrane-enriched vesicles purified from pYES3-AtMRP2/DTY168 cells. Uptake of the indicated concentrations of [3H]E217beta G in the presence () or absence of 100 µM unlabeled DNP-GS (open circle ) was measured as described in Fig. 1. The data for MgATP-dependent [3H]E217beta G uptake were fitted to the Michaelis-Menten equation by nonlinear least squares analysis to yield Km and Vmax values of 752.3 ± 219.8 µM and 88.1 ± 7.2 nmol/mg/10 min in media lacking DNP-GS and values of 238.2 ± 79.2 µM and 115.2 ± 18.1 nmol/mg/10 min in media containing DNP-GS, respectively. Individual data points are the means ± S.E. (n = 3-6).

DNP-GS-promoted E217beta G Uptake-- DNP-GS promoted AtMRP2-mediated E217beta G uptake. Having established that AtMRP2 was competent in the transport of E217beta G, albeit at low affinity, and knowing from studies of native plant vacuolar membranes that some GS conjugates promote MgATP-energized glucuronide uptake (20, 21), the effects of several GS conjugates on AtMRP2-mediated E217beta G uptake were examined. To preclude attenuation of any of the effects exerted through carrier saturation, all of the GS derivatives and E217beta G were added at concentrations equivalent to or lower than their respective Km values.

The results of a screen of the effects of a broad range of GS conjugates demonstrated that at a sub-Km concentration of [3H]E217beta G (100 µM) DNP-GS was the only GS conjugate that stimulated glucuronide uptake (Table I). Whereas 100 µM concentrations of GSSG and C3G-GS exerted little or no stimulatory effect on [3H]E217beta G uptake while metolachlor-GS and decyl-GS were inhibitory, DNP-GS increased uptake by 3.8-fold versus controls assayed in media lacking GS conjugates (Table I). As confirmed by the finding that 100 µM concentrations of DNP-GS, GSSG, C3G-GS, and metolachlor-GS were transported at rates of 10.5, 21.3, 96.8, and 72.4 nmol/mg/10 min, respectively, when assayed in the same medium minus E217beta G (Table I), the facility of a particular GS conjugate for transport by AtMRP2 was not correlated with its effect on E217beta G uptake. DNP-GS underwent moderate rates of transport but markedly stimulated E217beta G uptake, C3G-GS underwent high rates of transport but only weakly stimulated E217beta G uptake, and metolachlor-GS underwent high rates of transport but weakly inhibited E217beta G uptake (Table I).

                              
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Table I
Rates of uptake and effects of different GS conjugates on MgATP- dependent AtMRP2-mediated [3H]E217beta G uptake
For the measurements of AtMRP2-dependent GS conjugate uptake, vacuolar membrane-enriched vesicles purified from pYES3-AtMRP2/DTY168 cells were incubated in standard uptake medium containing 100 µM concentrations of [3H]DNP-GS, [3H]GSSG, [3H]C3G-GS, or [14C]metolachlor-GS. For the measurements of [3H]E217beta G uptake, pYES3-AtMRP2/DTY168 membranes were incubated in uptake medium containing 100 µM [3H]E217beta G plus or minus 100 µM concentrations of unlabeled DNP-GS, C3G-GS, GSSG, metolachlor-GS, or decyl-GS. MgATP-dependent uptake was measured as described in the legend to Fig. 1. Uptake rates for E217beta G are the means ± S.E. (n = 3). Rates for AtMRP2-dependent uptake of GS-conjugates are the mean values taken from Lu et al. (17).

The stimulatory action of DNP-GS on AtMRP2-mediated E217beta G uptake was primarily exerted at the Km level. Inclusion of a Km (100 µM) concentration of DNP-GS in the uptake medium decreased the Km for [3H]E217beta G uptake by 3.2-fold, from 752.3 ± 219.8 to 238.2 ± 79.2 µM, while increasing Vmax by only 1.3-fold, from 88.1 ± 17.2 to 115.2 ± 18.1 nmol/mg/10 min (Fig. 3).

E217beta G-promoted DNP-GS Uptake-- The interactions of DNP-GS and E217beta G with AtMRP2 were mutual. Not only did DNP-GS promote the uptake of [3H]E217beta G, but E217beta G promoted the uptake of [3H]DNP-GS. Inclusion of 100 µM E217beta G in the uptake medium decreased the Km for [3H]DNP-GS uptake by vacuolar membrane-enriched vesicles purified from pYES3-AtMRP2/DTY168 cells by 5.3-fold from 126.0 ± 52.6 µM to 20.1 ± 6.7 µM while decreasing Vmax by only 1.3-fold from 23.6 ± 3.5 nmol/mg/10 min to 18.0 ± 1.1 nmol/mg/10 min (Fig. 4).


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Fig. 4.   Effect of E217beta G on concentration dependence of MgATP-dependent [3H]DNP-GS uptake by vacuolar membrane-enriched vesicles purified from pYES3-AtMRP2/DTY168 cells. Uptake of the indicated concentrations of [3H]DNP-GS in the presence () or absence of 100 µM unlabeled E217beta G (open circle ) was measured as described in Fig. 1. The data were fitted to the Michaelis-Menten equation to yield Km and Vmax values of 126.0 ± 52.6 µM and 23.6 ± 3.5 nmol/mg/10 min in media lacking E217beta G and values of 20.1 ± 6.7 µM and 18.0 ± 1.1 nmol/mg/10 min in media containing E217beta G, respectively. Individual data points are the means ± S.E. (n = 3-6).

Some of the GS derivatives, as exemplified by decyl-GS, that did not promote AtMRP2-mediated [3H]E217beta G uptake instead inhibited it. By comparison with GSSG, for instance, which was a weak inhibitor and exerted only 20% inhibition at a concentration of 200 µM, decyl-GS inhibited [3H]E217beta G uptake with an I50 of 3.8 ± 1.5 µM (Fig. 5).


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Fig. 5.   Effects of GSSG (open circle ), Bn-NCC-1 (black-square), and decyl-GS () on MgATP-dependent [3H]E217beta G uptake by vacuolar membrane-enriched vesicles purified from pYES3-AtMRP2/DTY168 cells. Uptake of 100 µM [3H]E217beta G was measured in standard uptake medium containing the indicated concentrations of unlabeled GSSG, Bn-NCC-1 or decyl-GS. The I50 value for the inhibition of [3H]E217beta G uptake by decyl-GS was estimated to be 3.8 ± 1.5 µM by nonlinear least squares analysis. Data points are the means ± S.E. (n = 3).

In close agreement with what had been determined previously for [3H]DNP-GS transport by AtMRP2 (17), Bn-NCC-1 only marginally inhibited [3H]E217beta G uptake (Fig. 5). At concentrations of less than 50 µM, Bn-NCC-1 was ~4-fold less inhibitory than would be expected from its Km for transport by AtMRP2 (15.2 ± 2.3 µM) (17). At concentrations of 50 µM or more, the inhibitions exerted by Bn-NCC-1 did not exceed 35% (Fig. 5). Because Bn-NCC-1 did not appreciably modulate the transport of either E217beta G or DNP-GS, it was not investigated further here.

Simultaneous Parallel Transport of E217beta G and DNP-GS-- E217beta G and DNP-GS not only stimulated each other's uptake but underwent simultaneous parallel AtMRP2-dependent transport. Sub-Km (50 µM) concentrations of [3H]E217beta G and [3H]DNP-GS were transported into vacuolar membrane-enriched vesicles purified from pYES3-AtMRP2/DTY168 cells at rates of 4.5 ± 1.1 and 8.9 ± 0.5 nmol/mg/10 min, respectively, when added singly to the uptake medium, at rates of 16.2 ± 1.4 nmol/mg/min and 13.6 ± 1.1 nmol/mg/10 min when 50 µM unlabeled DNP-GS was added to the [3H]E217beta G uptake medium and 50 µM unlabeled E217beta G was added to the [3H]DNP-GS uptake medium, respectively, and at an aggregate rate of 32.3 ± 1.8 nmol/mg/10 min when the uptake medium contained both 50 µM [3H] E217beta G and 50 µM [3H]DNP-GS (Fig. 6). Thus, the rate of uptake when 50 µM concentrations of [3H]E217beta G and [3H]DNP-GS were provided simultaneously was the sum of the activated rates of uptake (i.e. 32.3 = 16.2 + 13.6 = 29.8 nmol/mg/10 min), not the sum of the individual nonactivated rates of uptake (4.5 + 8.9 = 13.4 nmol/mg/10 min) (Fig. 6).


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Fig. 6.   Simultaneous MgATP-dependent uptake of [3H]E217beta G and [3H]DNP-GS into vacuolar membrane-enriched vesicles purified from pYES3-AtMRP2/DTY168 cells. Uptake of the indicated concentrations of [3H]E217beta G and/or [3H]DNP-GS in media lacking or containing unlabeled E217beta G ([1H]E217beta G) or unlabeled DNP-GS ([1H]DNP-GS) was measured as described in the legend to Fig. 1. [3H]E217beta G and [3H]DNP-GS were added to the uptake medium at equivalent radiospecific activities. Values shown (uptake rates, nmol/mg/10 min) are the means ± S.E.

The simple additivity of the activated rates of uptake of [3H]E217beta G and [3H]DNP-GS was instructive in two respects. First, it implied that E217beta G and DNP-GS are subject to simultaneous transport, possibly via different pathways. Second, it implied that the substrate for one pathway promotes transport of the other substrate via another pathway while at the same time undergoing transport itself.

Several mechanisms, including trans-activation, counter-transport, cis-activation, or cotransport, might be envisaged for how E217beta G and DNP-GS directly or indirectly promote each other's uptake. Of these, the first two were the least likely. Counter-transport was incompatible with the mutual activations of E217beta G and DNP-GS uptake measured. Both counter-transport and trans-activation were refuted by the finding that pYES3-AtMRP2/DTY168 membrane vesicles that had been preloaded by preincubation with 200 µM DNP-GS in medium containing MgATP for 30 min before transfer to uptake medium containing [3H]E217beta G showed no evidence of enhanced [3H]E217beta G uptake versus controls incubated for the same length of time in the same medium lacking DNP-GS (Table II). Two potential mechanisms, cis-activation and/or cotransport, however, remained.

                              
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Table II
Effect of preloading vacuolar membrane-enriched vesicles purified from pYES3-AtMRP2/DTY168 cells with DNP-GS on MgATP- dependent uptake of [3H]E217beta G
Vesicles were preincubated for 30 min in standard uptake medium plus or minus 200 µM unlabeled DNP-GS before the addition of 200 µM [3H]E217beta G. Uptake of [3H]E217beta G was terminated after incubation for a further 10 min. Values shown are the means ± S.E. (n = 3).

Nonreciprocal GSH-promoted E217beta G Uptake-- The results of cellular and in vitro transport measurements indicate that the natural products etoposide and vincristine are cotransported with free GSH by human MRP1 (HmMRP1) (34, 35). Analogous experiments on the canalicular multispecific organic anion transporter, cMOAT (alias MRP2), imply a similar GSH dependence for the transport of vinblastine by this MRP (36). It has therefore been proposed that HmMRP1, and by implication cMOAT, has a bipartite substrate-binding pocket consisting of a binding site for GSH and another for hydrophobic molecules. Against this background, and in view of the finding that, of the five GS derivatives tested here, only DNP-GS markedly promoted E217beta G uptake, it was decided to determine whether this GS conjugate might exert its effects by stereochemically simulating GSH.

The capacity of GSH to substitute for DNP-GS was examined at four levels: (i) by determining whether [3H]GSH was subject to MgATP-dependent, AtMRP2-mediated uptake; (ii) by determining whether GSH promoted [3H]E217beta G uptake; (iii) by determining whether E217beta G promoted [3H]GSH uptake; and (iv) by determining whether GSH competed with [3H]DNP-GS for uptake. If the effects of DNP-GS were explicable in terms of its capacity to substitute for GSH in cotransport processes analogous to those inferred for HmMRP1 and cMOAT, criteria i-iv would be expected to apply.

Direct tests of the applicability of these criteria showed that GSH was indeed amenable to MgATP-dependent transport by AtMRP2 and able to promote [3H]E2beta G uptake. Vacuolar membrane-enriched vesicles purified from pYES3-AtMRP2/DTY168 cells catalyzed the MgATP-dependent uptake of 1 mM [3H]GSH at a rate of 2.2 nmol/mg/min, whereas uptake by the equivalent membrane fraction from pYES3/DTY168 cells was negligible (Fig. 7). Addition of 0.5-10 mM concentrations of GSH to the standard uptake medium promoted the MgATP-dependent uptake of 100 µM [3H]E217beta G by pYES3-AtMRP2/DTY168 membranes with a Michaelian concentration dependence consistent with Km and Vmax values of 4.7 ± 1.3 mM and 32.8 ± 10.1 nmol/mg/10 min, respectively (Fig. 8A). Moreover, none of these effects were either attributable to the redox action of GSH or could be simulated by other precursors of the conjugates concerned. The redox-inactive GSH derivative S-methyl-glutathione (S-methyl-GS) more than 50% substituted for GSH in promoting [3H]E217beta G uptake, but the redox-active thiol dithiothreitol did not (Fig. 8A). None of the other precursors of DNP-GS and E217beta G, 1-chloro-2,4-dinitrobenzene, glucuronate, or beta -estradiol, promoted [3H]E217beta G or [3H]DNP-GS uptake (Table III), implying that the effects seen with GSH were specific to this compound. However, in direct contradiction to what would be predicted if GSH and DNP-GS were interchangeable and GSH and E217beta G were subject to cotransport, the uptake of [3H]GSH was not promoted by E217beta G but instead inhibited (Fig. 7), and GSH did not compete with [3H]DNP-GS for uptake. Inclusion of 100 µM E217beta G in the uptake medium almost totally abolished the MgATP-dependent uptake of 1 mM [3H]GSH (Fig. 7), and neither GSH nor S-methyl-GS competed with 100 µM [3H]DNP-GS for uptake (Fig. 8B).


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Fig. 7.   Time dependence of MgATP-dependent uptake of [3H]GSH by vacuolar membrane-enriched vesicles purified from pYES3-AtMRP2/DTY168 ( and black-square) or pYES3/DTY168 cells (open circle ) measured in the presence (black-square) or absence of E217beta G (100 µM) ( and open circle ). Uptake of [3H]GSH (1 mM) from standard uptake medium was measured as described in the legend to Fig. 1. Values shown are the means ± S.E. (n = 3).


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Fig. 8.   Effects of GSH (), S-methyl-GS (open circle ), and dithiothreitol (black-square) on MgATP-dependent uptake of [3H]E217beta G (100 µM) (A) and [3H]DNP-GS (100 µM) (B) by vacuolar membrane-enriched vesicles purified from pYES3-AtMRP2/DTY168 cells. The data for GSH-promoted [3H]E217beta G uptake were fitted to the Michaelis-Menten equation to yield Km and Vmax values of 4.7 ± 1.3 mM and 32.8 ± 11.1 nmol/mg/10 min, respectively. Individual data points are the means ± S.E. (n = 3).

                              
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Table III
Effects of precursors of E217beta G and DNP-GS on rates of MgATP- dependent AtMRP2-mediated uptake of [3H]E217beta G or [3H]DNP-GS
Vacuolar membrane-enriched vesicles purified from pYES3-AtMRP2/DTY168 cells were incubated in standard uptake medium containing 100 µM [3H]E217beta G or 100 µM [3H]DNP-GS plus 100 µM or 5 mM concentrations of unlabeled 1-chloro-2,4-dinitrobenzene (CDNB), beta -estradiol, or glucuronate, and MgATP-dependent uptake of the radiolabeled compounds was measured. Values shown are the means ± S.E. (n = 3).

The finding that the concentrations of DNP-GS required to promote [3H]E217beta G uptake were 1-2 orders of magnitude lower than those required for the transport of DNP-GS itself further confirmed that the promotion of [3H]E217beta G uptake by GSH or S-methyl-GS did not have the properties expected of symport. The concentrations of DNP-GS sufficient for half-maximal stimulation of the uptake of 50, 100, and 200 µM [3H]E217beta G were only 7, 2, and 1 µM, respectively (Fig. 9); the corresponding values for half-maximal MgATP-dependent [3H]DNP-GS uptake by pYES3-AtMRP2/DTY168 membranes in the presence or absence of 100 µM E217beta G were 20 and 125 µM or greater, respectively (Fig. 4).


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Fig. 9.   Concentration dependence of promotion of MgATP- dependent [3H]E217beta G uptake into pYES3-AtMRP2/DTY168 vacuolar membrane-enriched vesicles by DNP-GS. Uptake of 50 (black-square), 100 (open circle ), or 200 µM [3H]E217beta G () was measured as described in the legend to Fig. 1 in media containing the concentrations of unlabeled DNP-GS indicated. The concentrations of DNP-GS required for half-maximal uptake of 50, 100, or 200 µM [3H]E217beta G, estimated graphically, were 7, 2, and 1 µM, respectively.

Other MRPs-- The capacity of DNP-GS and E217beta G to promote each other's uptake was a feature peculiar to AtMRP2. None of the other MRPs examined, Arabidopsis AtMRP1, yeast YCF1, and human MRP3 (HmMRP3), exhibited the same behavior despite their capacities for the transport of DNP-GS and E217beta G individually. MgATP-dependent uptake of a Km (386 µM) concentration of [3H]E217beta G by wild type DTY7 yeast vacuolar membrane vesicles was 4.3-fold greater than that mediated by membranes purified from ycf1Delta strain DTY168 (14.9 ± 2.6 versus 3.5 ± 0.7 nmol/mg/10 min) but insignificantly affected by the addition of a Km (5.9 µM) concentration of DNP-GS to the uptake medium (Fig. 10A), and addition of a Km concentration of E217beta G simply diminished [3H]DNP-GS uptake by wild type membranes from 16.5 ± 1.2 to 9.2 ± 0.5 nmol/mg/10 min (Fig. 10A). MgATP-dependent uptake of 100 µM [3H]E217beta G by vacuolar membrane-enriched vesicles purified from pYES3-AMRP1/DTY168 cells was appreciable by comparison with the near-zero values measured for the corresponding membrane fraction from pYES3/DTY168 cells (0.7 ± 0.1 versus 0.0 ± 0.1 nmol/mg/10 min), yet the addition of 100 µM DNP-GS to the uptake medium largely abolished [3H]E217beta G uptake (Fig. 10B). MgATP-dependent uptake of [3H]DNP-GS by the same membranes was unaffected or only weakly inhibited by the addition of E217beta G. Although the MgATP-dependent uptake of a Km (25 µM) concentration of [3H]E217beta G (25) by membrane vesicles purified from HmMRP3-transfected HEK293 cells was almost exclusively attributable to HmMRP3 in that the same membrane fraction from empty vector-transfected cells catalyzed little or no uptake (Fig. 10C), addition of a Km concentrations of DNP-GS to the uptake medium did not promote MgATP-dependent uptake Fig. 10C).


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Fig. 10.   Effects of DNP-GS on MgATP-dependent transport of [3H]E217beta G by YCF1 (A), AtMRP1 (B), and HmMRP3 (C). MgATP-dependent uptake of Km concentrations of [3H]E217beta G (386 or 25 µM) in the absence or presence of Km (5.8 or 6.0 µM) concentrations of DNP-GS by vacuolar membrane vesicles purified from yeast ycf1Delta strain DTY168 and the isogenic wild type strain DTY7 (A) and by membrane vesicles isolated from HEK293/MRP3-5 or control HEK293 cells (C) was measured as described under "Experimental Procedures." MgATP-dependent uptake of 100 µM [3H]E217beta G by vacuolar membrane-enriched vesicles purified from pYES3-AtMRP1/DTY168 and pYES3/DTY168 cells in the absence and presence of 100 µM DNP-GS (B) was assayed as described in the legend to Fig. 1. Values shown are the means ± S.E. (n = 3).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The results of the experiments described here show that AtMRP2 has the properties of a vacuolar ABC transporter. AtMRP2 localizes to the vacuolar membrane-enriched fraction from Arabidopsis seedlings. Heterologously expressed AtMRP2, like native plant vacuolar membranes (20, 21), is not only competent in the transport of GS conjugates and linearized tetrapyrroles (17) but also glucuronides as exemplified by E217beta G. E217beta G and DNP-GS promote each other's uptake into both yeast vacuolar membrane-enriched vesicles containing heterologously expressed AtMRP2 and into isolated plant vacuoles.

It is now established that many GS conjugates and other amphipathic anions are accumulated in the vacuolar compartment of protoplasts and intact plant cells. In situ cytosolic glutathionation of monochlorobimane to its fluorescent derivative, bimane-GS, and accumulation of the latter in the vacuoles of protoplasts and cell suspension cultures has been demonstrated (37) as has a greater than 50-fold accumulation of alachor-GS in the vacuoles of intact barley leaves (38). Likewise, it is evident that an MRP-like functionality is responsible for the vacuolar uptake of both glutathionated and nonglutathionated dyes by barley aleurone cells (39). However, except for one isolated report of Mr 170,000 band that reacts with antibody raised against a synthetic peptide corresponding to a wheat MRP homolog that is enriched in the vacuolar membrane fraction from herbicide safener-treated but not untreated plants (40), virtually nothing is known, other than by implication, of the membrane localization of plant MRPs. The findings reported here therefore provide the first direct demonstration of the localization to the vacuolar membrane of a functionally defined MRP from a plant source. Moreover, given how monospecific PABAtMRP2 is, it can be concluded that the immunoreactive Mr 176,000 polypeptide in the vacuolar membrane fraction from Arabidopsis is indeed AtMRP2. AtMRP2 is the only MRP, except for AtMRP1, in the Arabidopsis genome data base that contains a C2 domain, and although AtMRP1 contains a C2 domain motif (1568EDSLNQSDISR1578) resembling the motif against which PABAtMRP2 was raised, this antibody does not react with Sf9 cell-expressed AtMRP1.

Although the capacity of AtMRP2 for the transport of GS conjugates, linearized tetrapyrroles, and glucuronides establishes a basis for the molecular manipulation of three previously identified ABC transporter-like activities in plant vacuoles, it refutes our earlier proposal that AtMRP2 is constituted of two functionally distinguishable semi-autonomous modules residing in different half-molecules (17). A simple two-module model can neither explain how some transport-active GS conjugates (e.g. DNP-GS) but not others (e.g. metolachlor-GS) promote E217beta G transport nor the observation that GSH promotes E217beta G transport but E217beta G does not promote GSH transport. AtMRP2 and other members of the MRP subclass of ABC transporters undoubtedly contain two homologous halves, each of which contains an NBF located on the cytosolic face of the membrane and an MSD containing multiple transmembrane spans. However, it is improbable, as has been proposed to explain the semi-autonomous transport of DNP-GS and Bn-NCC-1 by this transporter (17), that one NBF-MSD pair translocates one class of compounds (e.g. GS conjugates) across the vacuolar membrane, whereas the other NBF-MSD pair translocates another class of compounds (e.g. linearized tetrapyrroles). Instead, it is more likely, now that the extent of its transport capabilities are better defined, that each half-molecule of AtMRP2 contains several substrate transport sites. Analogies may therefore be drawn with Lmr(A), a half-molecule ABC transporter from Lactococcus lactis that transports drugs with kinetics consistent with cooperative interactions between two or more nonidentical sites (41) and with mammalian P-glycoprotein (MDR) whose kinetic properties implicate a multisite drug-binding model (42).

Of the various models that might be proposed for how E217beta G and DNP-GS interact, those involving counter-transport and/or trans-activation are the least plausible inasmuch as stimulation of E217beta G uptake into vacuolar membrane-enriched vesicles containing AMRP2 by the addition of DNP-GS to the uptake medium is instantaneous and not enhanced or simulated by preloading the vesicles with DNP-GS. Similarly, a model based exclusively on the cotransport of E217beta G and DNP-GS is not capable of explaining all of the characteristics of AtMRP2-mediated transport. Transportability is not the sole criterion to be satisfied by a GS conjugate for it to stimulate E217beta G uptake; GS conjugates, other than DNP-GS, are subject to high rates of transport but do not promote E217beta G transport. Transport of DNP-GS per se does not appear to promote E217beta G uptake; although DNP-GS and E217beta G undergo parallel simultaneous transport by AtMRP2, the concentrations of DNP-GS required for the half-maximal promotion of E217beta G transport are 1-2 orders of magnitude lower than those required for half-maximal net transport of DNP-GS, itself. Likewise, an extension of the E217beta G-DNP-GS cotransport model, namely that DNP-GS promotes E217beta G transport by simulating GSH as a cotransported species is also excluded by the nonreciprocal nature of the interactions between GSH and E217beta G. GSH and its S-methylated, redox-inactive derivative, S-methyl-GS, promote AtMRP2-mediated E217beta G transport but E217beta G, rather than promoting GSH transport, inhibits it. Evidently, GSH, like DNP-GS, promotes AtMRP2-mediated E217beta G transport by interacting with a site different from that involved in its own transport. As implied by the inability of both GSH and S-methyl-GS to compete with DNP-GS for uptake, GSH, in turn, appears to be transported via a pathway distinct from that for DNP-GS.

A tentative scheme capable of accounting for the properties of AtMRP2 is depicted in Fig. 11. According to this scheme: (i) E217beta G, DNP-GS, GSH, and by implication Bn-NCC-1 undergo transport via different AtMRP2-dependent pathways; (ii) E217beta G and DNP-GS promote each other's transport by binding sites distinct from but tightly coupled to the other's transport pathway; (iii) GSH and S-methyl-GS, although able to promote E217beta G transport, do so at a site distinct from that responsible for the promotion of E217beta G transport by DNP-GS and (iv) decyl-GS, and to a lesser extent GSSG, although able to compete with DNP-GS for the site responsible for promoting E217beta G transport are themselves not able to promote E217beta G transport.


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Fig. 11.   Schematic diagram depicting the interactions of E217beta G and different GS derivatives with AtMRP2. DNP-GS, GSH, Bn-NCC-1, and E217beta G are considered to be transported via semi-autonomous pathways. Other GS conjugates such as metolachlor-GS and C3G-GS compete with DNP-GS for transport. E217beta G promotes DNP-GS transport and DNP-GS promotes E217beta G transport by interacting with sites distinct from but coupled to the DNP-GS and E217beta G transport pathways, respectively. GSH and S-methyl-GS promote E217beta G transport by interacting with different sites. Decyl-GS and GSSG compete with DNP-GS for binding to the site responsible for promoting E217beta G transport. E217beta G blocks the transport of GSH.

One of the most striking findings to come from these investigations is an appreciation of how unusual AtMRP2 is in its susceptibility to mutual activation by E217beta G and DNP-GS. All of the other MRPs examined, regardless of source, catalyze both E217beta G and DNP-GS transport, but none catalyzes E217beta G transport in a DNP-GS-stimulated manner. On the contrary, E217beta G transport by AtMRP1, YCF1, and HmMRP3 is either insensitive to or only slightly inhibited by DNP-GS. Even by comparison with its 94% sequence similar homolog AMRP1 (17), AtMRP2 is unique among the MRPs in terms of its facility for the transport of some substrates cooperatively and others semi-autonomously.

The association of AtMRP2 with multiple semi-autonomous transport pathways and the facility of some substrates to promote the transport of others extends considerably our appreciation of the processes that may converge on this transporter. In the case of GS conjugate and tetrapyrrole transport, the processes that may depend on AtMRP2 and its equivalents in plant species other than Arabidopsis may include xenobiotic detoxification, chlorophyll catabolism, antimicrobial compound storage, cell pigmentation, and protection from oxidative stress (7, 8). In the case of glucuronides, the processes that may depend on AtMRP2 and AtMRP2-like transporters may be broadened to encompass the vacuolar storage of flavonoid antifeedants, UV screening agents, and animal attractants. Although the biosynthesis and transport of glucuronate conjugates has been most extensively studied in animals, where they participate in the degradation of heme and its MRP2-mediated secretion into bile as bilirubin diglucuronide (43), there is increasing evidence that at least some plants deploy similar mechanisms for the vacuolar sequestration of flavonoids (44). Intriguing, therefore, is the possibility that the properties of AtMRP2 and its equivalents not only have the potential of providing a molecular basis for the vacuolar uptake of GS conjugates and glucuronides but also for cross-talk between the GSH-dependent and glucuronate-dependent detoxification pathways in plants.

    ACKNOWLEDGEMENTS

We thank Dr. Stefan Hörtensteiner (Department of Plant Biology, University of Zurich, Switzerland) for the kind gift of Bn-NCC-1 and Dr. Gary Kruh (Fox Chase Cancer Center, Philadelphia, PA) for construction and provision of the HmMRP3-transfected HEK cell lines.

    FOOTNOTES

* This work was supported by National Research Initiative Competitive Program Grant 99-35304-8094 from the United States Department of Agriculture (to P. A. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Triagency (Department of Energy/National Science Foundation/United States Department of Agriculture) Plant Training Grant Fellow during the initial stages of this project and sponsored by PlantGenix, Inc. during the final stages.

§ Present address: Dow AgroSciences LLC, 9330 Zionsville Rd., Indianapolis, IN 46268-1054.

To whom correspondence should be addressed. Fax: 215-898-8780; E-mail: parea@sas.upenn.edu.

Published, JBC Papers in Press, December 13, 2000, DOI 10.1074/jbc.M009690200

2 R. Sánchez-Fernández, T. G. E. Davies, J. O. D. Coleman, and P. A. Rea, unpublished data.

3 R. Sánchez-Fernández and P. A. Rea, unpublished data.

4 N. Gaedeke, M. Klein, B. Müller-Rober, and E. Martinoia, unpublished data.

    ABBREVIATIONS

The abbreviations used are: ABC, ATP-binding cassette; Bn-NCC-1, Brassica napus nonfluorescent chlorophyll catabolite 1; C3G, cyanidin-3-glucoside; DNP-GS, 2,4-dinitrophenyl-glutathione; E217beta G, 17beta -estradiol 17-(beta -D-glucuronide); GSH, reduced glutathione; GSSG, oxidized glutathione; MRP, multidrug resistance-associated protein; GS, glutathionyl; MSD, membrane spanning domain; NBF, nucleotide binding fold; MDR, multidrug resistance; PAGE, polyacrylamide gel electrophoresis; Mes, 4-morpholineethanesulfonic acid; AVP, Arabidopsis vacuolar H+ pyrophosphatase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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