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
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ABSTRACT |
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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 17 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 ycf1 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 17 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 E217 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 ycf1
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
ycf1 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
Measurement of Transport--
Uptake of
[3H]E217 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]E217 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.
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 ycf1
From these experiments it was determined that AtMRP2 catalyzed the low
affinity, high capacity MgATP-energized transport of [3H]E217 DNP-GS-promoted E217
The results of a screen of the effects of a broad range of GS
conjugates demonstrated that at a sub-Km
concentration of [3H]E217
The stimulatory action of DNP-GS on AtMRP2-mediated
E217 E217
Some of the GS derivatives, as exemplified by decyl-GS, that did not
promote AtMRP2-mediated [3H]E217
In close agreement with what had been determined previously for
[3H]DNP-GS transport by AtMRP2 (17), Bn-NCC-1
only marginally inhibited [3H]E217 Simultaneous Parallel Transport of E217
The simple additivity of the activated rates of uptake of
[3H]E217
Several mechanisms, including trans-activation,
counter-transport, cis-activation, or cotransport, might be
envisaged for how E217 Nonreciprocal GSH-promoted E217
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]E217
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]E2
The finding that the concentrations of DNP-GS required to promote
[3H]E217 Other MRPs--
The capacity of DNP-GS and E217 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 E217 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 E217 Of the various models that might be proposed for how
E217 A tentative scheme capable of accounting for the properties of AtMRP2
is depicted in Fig. 11. According to
this scheme: (i) E217-estradiol 17-(
-D-glucuronide)
(E217
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 E217
G
uptake by AtMRP2. Reduced glutathione, although subject to transport by
AtMRP2 and able to markedly promote E217
G uptake, neither competes with DNP-GS for uptake nor is subject to
E217
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.
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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
-estradiol 17-(
-D-glucuronide)
(E217
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.
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 E217
G transport, the enhancements are not attributable
to GSH-E217
G cotransport but instead to nonreciprocal
GSH-mediated cis-activation of glucuronide uptake.
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strain DTY168 (MAT
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).
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).
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).
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]E217
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]E217
G. After incubation for a further
10 min, uptake was terminated, and the radioactivity retained after
filtration was estimated as described above.
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.
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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.
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 E217
G.
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]E217
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]E217
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 E217
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]E217 G by
vacuolar membrane-enriched vesicles purified from
pYES3-AtMRP2-transformed (
) and pYES3-transformed (
) S. cerevisiae ycf1
strain DTY168.
MgATP-dependent uptake was measured in standard uptake
medium containing 100 µM
[3H]E217
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]E217 G uptake by
vacuolar membrane-enriched vesicles purified from pYES3-AtMRP2/DTY168
cells. Uptake of the indicated concentrations of
[3H]E217
G in the presence (
) or absence
of 100 µM unlabeled DNP-GS (
) was measured as
described in Fig. 1. The data for MgATP-dependent
[3H]E217
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).
G Uptake--
DNP-GS promoted
AtMRP2-mediated E217
G uptake. Having established that
AtMRP2 was competent in the transport of E217
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 E217
G uptake were examined. To preclude
attenuation of any of the effects exerted through carrier saturation,
all of the GS derivatives and E217
G were added at
concentrations equivalent to or lower than their respective
Km values.
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]E217
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
E217
G (Table I), the facility of a particular GS
conjugate for transport by AtMRP2 was not correlated with its effect on
E217
G uptake. DNP-GS underwent moderate rates of
transport but markedly stimulated E217
G uptake, C3G-GS
underwent high rates of transport but only weakly stimulated
E217
G uptake, and metolachlor-GS underwent high rates of
transport but weakly inhibited E217
G uptake (Table I).
Rates of uptake and effects of different GS conjugates on
MgATP- dependent AtMRP2-mediated [3H]E217G
uptake
G uptake, pYES3-AtMRP2/DTY168 membranes
were incubated in uptake medium containing 100 µM
[3H]E217
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 E217
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).
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]E217
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).
G-promoted DNP-GS Uptake--
The interactions
of DNP-GS and E217
G with AtMRP2 were mutual. Not only
did DNP-GS promote the uptake of
[3H]E217
G, but E217
G
promoted the uptake of [3H]DNP-GS. Inclusion of 100 µM E217
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
E217 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 E217
G (
) 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 E217
G and values
of 20.1 ± 6.7 µM and 18.0 ± 1.1 nmol/mg/10
min in media containing E217
G, respectively.
Individual data points are the means ± S.E. (n = 3-6).
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]E217
G uptake with an
I50 of 3.8 ± 1.5 µM (Fig.
5).
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Fig. 5.
Effects of GSSG ( ),
Bn-NCC-1 (
), and decyl-GS (
) on
MgATP-dependent
[3H]E217
G uptake by
vacuolar membrane-enriched vesicles purified from pYES3-AtMRP2/DTY168
cells. Uptake of 100 µM
[3H]E217
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]E217
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).
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 E217
G or DNP-GS, it was not investigated further here.
G and
DNP-GS--
E217
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]E217
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]E217
G uptake medium and 50 µM unlabeled E217
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]
E217
G and 50 µM [3H]DNP-GS
(Fig. 6). Thus, the rate of uptake when
50 µM concentrations of
[3H]E217
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]E217 G
and [3H]DNP-GS into vacuolar membrane-enriched vesicles
purified from pYES3-AtMRP2/DTY168 cells. Uptake of the indicated
concentrations of [3H]E217
G and/or
[3H]DNP-GS in media lacking or containing
unlabeled E217
G ([1H]E217
G)
or unlabeled DNP-GS ([1H]DNP-GS) was measured as
described in the legend to Fig. 1.
[3H]E217
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.
G and [3H]DNP-GS was
instructive in two respects. First, it implied that E217
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.
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 E217
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]E217
G showed no evidence of enhanced
[3H]E217
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.
Effect of preloading vacuolar membrane-enriched vesicles purified from
pYES3-AtMRP2/DTY168 cells with DNP-GS on MgATP- dependent uptake of
[3H]E217G
G. Uptake of
[3H]E217
G was terminated after incubation for a
further 10 min. Values shown are the means ± S.E.
(n = 3).
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
E217
G uptake, it was decided to determine whether this
GS conjugate might exert its effects by stereochemically simulating
GSH.
G
uptake; (iii) by determining whether E217
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.
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]E217
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]E217
G uptake, but the
redox-active thiol dithiothreitol did not (Fig. 8A). None of
the other precursors of DNP-GS and E217
G, 1-chloro-2,4-dinitrobenzene, glucuronate, or
-estradiol,
promoted [3H]E217
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 E217
G were subject to cotransport, the uptake of [3H]GSH was not promoted by E217
G but
instead inhibited (Fig. 7), and GSH did not compete with
[3H]DNP-GS for uptake. Inclusion of 100 µM
E217
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
) or pYES3/DTY168 cells (
) measured in the presence (
) or
absence of E217
G (100 µM) (
and
). 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 (
), and dithiothreitol (
) on
MgATP-dependent uptake of
[3H]E217
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]E217
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).
Effects of precursors of E217G and DNP-GS on rates of
MgATP- dependent AtMRP2-mediated uptake of
[3H]E217
G or [3H]DNP-GS
G or 100 µM
[3H]DNP-GS plus 100 µM or 5 mM
concentrations of unlabeled 1-chloro-2,4-dinitrobenzene (CDNB),
-estradiol, or glucuronate, and MgATP-dependent uptake
of the radiolabeled compounds was measured. Values shown are the
means ± S.E. (n = 3).
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]E217
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]E217
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 E217
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]E217 G uptake
into pYES3-AtMRP2/DTY168 vacuolar membrane-enriched vesicles by
DNP-GS. Uptake of 50 (
), 100 (
), or 200 µM
[3H]E217
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]E217
G, estimated graphically, were 7, 2, and 1 µM, respectively.
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 E217
G
individually. MgATP-dependent uptake of a Km (386 µM) concentration of
[3H]E217
G by wild type DTY7 yeast vacuolar
membrane vesicles was 4.3-fold greater than that mediated by membranes
purified from ycf1
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 E217
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]E217
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]E217
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 E217
G. Although the MgATP-dependent
uptake of a Km (25 µM) concentration
of [3H]E217
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]E217 G
by YCF1 (A), AtMRP1 (B), and HmMRP3
(C). MgATP-dependent uptake of
Km concentrations of
[3H]E217
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 ycf1
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]E217
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
G. E217
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.
G transport nor the observation that GSH promotes E217
G transport but
E217
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).
G and DNP-GS interact, those involving
counter-transport and/or trans-activation are the least
plausible inasmuch as stimulation of E217
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 E217
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 E217
G uptake; GS conjugates, other than DNP-GS, are
subject to high rates of transport but do not promote
E217
G transport. Transport of DNP-GS per se
does not appear to promote E217
G uptake; although DNP-GS
and E217
G undergo parallel simultaneous transport by
AtMRP2, the concentrations of DNP-GS required for the half-maximal
promotion of E217
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
E217
G-DNP-GS cotransport model, namely that DNP-GS
promotes E217
G transport by simulating GSH as a
cotransported species is also excluded by the nonreciprocal nature of
the interactions between GSH and E217
G. GSH and its
S-methylated, redox-inactive derivative,
S-methyl-GS, promote AtMRP2-mediated E217
G
transport but E217
G, rather than promoting GSH
transport, inhibits it. Evidently, GSH, like DNP-GS, promotes
AtMRP2-mediated E217
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.
G, DNP-GS, GSH, and by implication
Bn-NCC-1 undergo transport via different
AtMRP2-dependent pathways; (ii) E217
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 E217
G
transport, do so at a site distinct from that responsible for the
promotion of E217
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 E217
G
transport are themselves not able to promote E217
G
transport.
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Fig. 11.
Schematic diagram depicting the interactions
of E217 G and different GS
derivatives with AtMRP2. DNP-GS, GSH, Bn-NCC-1, and
E217
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. E217
G
promotes DNP-GS transport and DNP-GS promotes E217
G
transport by interacting with sites distinct from but coupled to the
DNP-GS and E217
G transport pathways, respectively. GSH
and S-methyl-GS promote E217
G transport by
interacting with different sites. Decyl-GS and GSSG compete with DNP-GS
for binding to the site responsible for promoting E217
G
transport. E217
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 E217G and DNP-GS. All of the other
MRPs examined, regardless of source, catalyze both E217
G
and DNP-GS transport, but none catalyzes E217
G transport
in a DNP-GS-stimulated manner. On the contrary, E217
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.
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;
E217G, 17
-estradiol
17-(
-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 |
---|
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---|
1. | Higgins, C. F. (1992) Annu. Rev. Cell Biol. 8, 67-113[CrossRef] |
2. |
Dudler, R.,
and Hertig, C.
(1992)
J. Biol. Chem.
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