From the University of Cologne, Botanical Institute,
Gyrhofstrasse 15, D-50931 Cologne, Federal Republic of Germany and
the § University of Neuchatel, Institut de Botanique,
Laboratoire de Physiologie végétales, Rue Emile Argand 13, CH-2007 Neuchatel, Switzerland
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ABSTRACT |
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A directly energized vacuolar pump for
glutathione (GS) conjugates has been described for several plant
species. Since glucuronate conjugates also occur in plants, we
addressed the question whether plant vacuoles take up the abiotic
glucuronate conjugate estradiol 17-(-glucuronide)
(E217G) via a GS conjugate pump, which in some cases
has been reported to accept various organic anions as substrates, or
via a distinct glucuronate transporter. Uptake studies into vacuoles from rye and barley were performed with E217G and
metolachlor-GS (MOC-GS), a substrate of the GS conjugate ATPase, to
compare glucuronate conjugate transport into vacuoles containing
endogenous flavone glucuronides with those lacking specific glucuronate
conjugates, respectively. Our results indicate that E217G
and MOC-GS are taken up into vacuoles of both plants via a directly
energized mechanism since transport was (i) strictly
ATP-dependent; (ii) inhibited by vanadate but not by
bafilomycin A1, azide, verapamil, nor by dissipation of the vacuolar
pH or
; (iii) E217G uptake into rye vacuoles was
partially driven by other nucleotides in the following order of
efficiency: ATP > GTP > UTP
CTP, whereas the
non-hydrolyzable ATP analogue 5
-adenylyl-
,
-imidodiphosphate, ADP, or PPi did not energize uptake. E217G
transport into rye vacuoles was saturable (Km
0.2 mM). The rye-specific luteolin glucuronides
decreased uptake rates of E217G and MOC-GS into rye and
barley vacuoles to comparable degrees with the mono- and
diglucuronidated derivatives (40-60% inhibition) being more effective
than the triglucuronide. Inhibition of E217G uptake by
luteolin 7-O-diglucuronide was competitive
(Ki = 120 µM). Taurocholate had no
effect on E217G transport, and uptake of MOC-GS was not
inhibited by E217G. Although GS conjugates and oxidized GS
decreased MOC-GS transport, E217G uptake into rye and
barley vacuoles was stimulated up to 7-fold in a
concentration-dependent manner by these substances, with
dinitrobenzene-GS being most effective. The stimulation of the GS
conjugates was not due to detergent or redox effects and was specific
for the E217G pump. GS conjugate stimulation of glucuronate
uptake was unique for plants as E217G uptake into yeast
microsomal vesicles was not affected. By comparison with a
YCF1
yeast mutant, defective in vacuolar transport of GS conjugates mediated
by YCF1, it was shown that E217G was taken up into yeast
vesicles via a YCF1-independent directly energized pump. These results
indicate that E217G as a glucuronate conjugate is
transported across the vacuolar membranes of plants and yeast by a
carrier distinct from the GS conjugate ATPase.
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INTRODUCTION |
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In plants, many secondary compounds such as phenolic acids, coumarins, tannins, and flavonoids are detoxified and stored in the large central vacuole, a compartment with low metabolic activity (1). Enzymatic conjugation of these substances with one or more hydrophilic ligands, primarily sugars, catalyzed by respective transferases is an integral part of the detoxification process and leads to higher water solubility. However, in several cases conjugation with glucuronate is also observed. In addition, conjugation of an anthocyanin with glutathione is apparently necessary for its vacuolar deposition in maize (2). Another type of reaction is the esterification of phenolic compounds with polar substituents (3).
Glucuronate conjugates are mainly produced in animals, where they play
an important role in the degradation of heme resulting in bilirubin
diglucuronide which is secreted into bile. Although a series of
phenolic glucuronates have been identified in different plant taxa (4),
glucuronyltransferases and the subsequent vacuolar deposition are
poorly understood. In rye, two glucuronidated flavones, luteolin
7-O-diglucuronyl-4-O-glucuronide
(R1)1 and luteolin
7-O-diglucuronide (R2), are specifically localized in the
mesophyll tissue of primary leaves, whereas the epidermal layers
contain glycosylated flavonoids (5). The metabolism of these flavone
glucuronates starting from luteolin is sequentially catalyzed by three
anabolic cytosolic UDP-glucuronate:flavone-glucuronosyltransferases (6)
followed by vacuolar storage via an unknown mechanism and may also
involve subsequent degradation of the luteolin triglucuronide initiated
by a specific
-glucuronidase located in the cell wall (7).
Accordingly, vacuolar steady-state concentrations of the flavone
glucuronides show a maximum in the young rye leaf followed by a rapid
decline during later development (8). In animals, glucuronate transport
across the liver canalicular membrane has been investigated in detail
and was shown to be mediated by a directly energized transporter
belonging to the ABC family recognizing several anionic substances
called MOAT (multiple organic anion transporter; see Ref. 9).
In plants, final deposition of glutathione conjugates within the plant
vacuole is mediated by a directly energized transport process also
involving an ABC transporter (10, 11). It has recently been shown that
a human (MRP1; Ref. 12) and a yeast (YCF1; Ref. 13) transporter
belonging to the ABC family are able to catalyze glutathione (GS)
conjugate transport (Refs. 14 and 15 and references therein).
Complementation of the cadmium hypersensitive ycf1 deletion
mutant with MRP1 restores cadmium tolerance as well as glutathione
conjugate transport activity (15). Cadmium tolerance is due to the
transport of bis(glutathionato)cadmium complexes in yeast (16). The
human and rat liver canalicular MOAT represents a liver-specific
isoform of the human multidrug resistance associated protein MRP1
(therefore denoted MRP2), and the transport function of the
heterologously expressed mrp2 gene could be shown for a GS
conjugate and leukotriene C4 (17-19). As shown for MOAT,
MRP1 is capable of transporting not only glutathione but also other
negatively charged conjugates (20) including estradiol
17-(-D-glucuronide) (E217G; Ref. 21).
In the case of glucose conjugates in plants, at least two mechanisms coexist as follows: species-specific plant flavonoid glucosides apparently use the proton gradient established by the vacuolar proton pumps, a vacuolar H+-ATPase and a H+-pyrophosphatase, and are transported by a species-specific proton antiport mechanism. In contrast, uptake of an abiotic herbicide glucoside is directly energized by MgATP (22). This difference may indicate that some glucosides are definitively stored within the vacuole, and others may be re-metabolized at a later stage of development.
As outlined above, rye mesophyll cells accumulate specific flavone glucuronides. Therefore we started 14C and 3H labeling experiments with these substances to study the mechanism of their vacuolar deposition. However, so far different procedures have failed to specifically label the rye flavone glucuronides. To find evidence for a possible general mechanism of glucuronate transport into plant vacuoles, we investigated the transport of the steroid conjugate E217G, presumably not occurring in plants, and compared the uptake characteristics into rye vacuoles (containing endogenous glucuronates) and barley vacuoles (obviously lacking these compounds). Furthermore, we were interested in comparing the transport of metolachlor conjuated to glutathione (MOC-GS) with E217G, to elucidate whether the vacuolar uptake system for GS conjugates corresponds to a transporter with broad substrate specificity for anionic conjugates as shown for the canalicular MOAT/MRP2 (9) or the human multidrug resistance associated protein MRP1 (20).
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EXPERIMENTAL PROCEDURES |
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Chemicals and Isolation of Flavonoid Glucuronides from
Rye--
-Estradiol 17-(
-D-glucuronide)
[estradiol-6,7-3H] (1.8 × 109 Bq/mol)
and [14C]sucrose (23.6 × 1012 Bq/mol)
were obtained from NEN Life Science Products (Bad Homburg, Germany) and
Amersham-Buchler (Braunschweig, Germany), respectively. [phenyl-U-14C]Metolachlor-glutathione
(1.7 × 106 Bq/mol, gift of Dr. K. Kreuz, Novartis,
Basel, Switzerland) was synthesized as described (23). Percoll and
Ficoll 400 were supplied by Pharmacia (Freiburg, Germany). Luteolin was
from Roth (Karlsruhe, Germany). The flavonoid glucuronides luteolin
7-O-diglucuronide (R2) and luteolin
7-O-diglucuronyl-4
-O-glucuronide (R1; see Ref. 5) were isolated from Secale cereale L. cv. Kustro primary leaves grown in the field for 10 days as described (24) with the
following modification: the TLC step was omitted, and separation and
final purification were done with a Sephadex LH-20 column (Pharmacia,
Freiburg, Germany) using water as eluant.
Luteolin-7-O-glucuronide was isolated from Antirrhinum
majus L. petals (25). Purity of flavonoid glucuronides was higher
than 95% as checked by HPLC. Dinitrobenzene-glutathione (DNB-GS) was
prepared chemically by stirring 5 mM
1-chloro-2,4-dinitrobenzene and 10 mM reduced glutathione in water adjusted to pH 8.5 for 24 h at room temperature. Excess of 1-chloro-2,4-dinitrobenzene was removed by 3-fold shaking the mixture out with tert-butyl methyl ether. The aqueous phase
was evaporated to dryness, and the concentration of the redissolved DNB-GS was determined photometrically (
340 nm = 10 mM
1·cm
1). All other chemicals
and solvents were of highest purity available and were mainly supplied
by Sigma (Deisenhofen, Germany) and Fluka (Buchs, Switzerland).
Plant Material and Growth Conditions--
Barley (Hordeum
vulgare L. cv. Bakara) and rye (S. cereale L. cv.
Kustro; Lochow-Petkus, Bergen, Germany) were grown on vermiculite for 8 days in a growth cabinet with 13 h of fluorescent light (approximately 100 µmol × m2 s
1) at
20 °C and 70% relative humidity and were watered daily with Hoagland's solution (22).
Yeast Strains--
The isogenic Saccharomyces
cerevisiae strains DTY7 (Mat, ura3-52, leu2-3,-112,
his6) and DTY168 (Mat
ycf1
::hisG,
ura3-52, leu2-3,-112, his6) were used for transport experiments
into isolated microsomes.
Preparation of Mesophyll Vacuoles from Primary Leaves of Barley
and Rye--
Protoplasts of both plants and barley vacuoles were
isolated as described earlier (26). Contamination of barley vacuoles with other cell constituents was less than 3% as measured by
marker-enzyme activities (26). As mechanical shearing forces using
different needles and lysis conditions did not result in an acceptable
yield of rye vacuoles, a different strategy was established. After
digestion, rye protoplasts were washed twice with 0.5 M
sorbitol, 1 mM CaCl2, 10 mM
MES-KOH, pH 5.8, with a layer of Percoll containing 0.5 M
sorbitol and 20 mM MES, pH 6, underneath and sedimented on
this layer for 5 min at 200 × g (Rotixa K., Hettich,
Tuttlingen, Germany). Either this crude preparation or protoplasts
purified on a step gradient as described (26) were used to lyse rye
protoplasts by a combination of pH and osmotic shock. One part of
concentrated protoplasts was mixed gently with four parts of lysis
medium (10% (w/v) Ficoll 400, 20 mM MES-BTP, pH 8.5, 5 mM EGTA, 1 mg·ml1 BSA, 5 mM DTT
and sorbitol, total osmolarity of 0.22 osmol·kg
1) at
20 °C. After 15-20 min of gentle shaking a gradient was prepared consisting of 20 ml of the lysis mixture (bottom), an equal volume of
medium A (0.4 M sorbitol, 5.5% (v/v) Percoll, 30 mM KCl, 1 mM DTT, 1 mg·ml
1 BSA,
20 mM MES-BTP, pH 7.2) and 5 ml of medium B (0.4 M glycine betaine, 30 mM KCl, 1 mM
DTT, 1 mg·ml
1 BSA, 20 mM MES-BTP, pH 7.2;
top) in glass centrifugation tubes. Vacuoles were separated from
unlysed protoplasts by centrifugation (5 min at 1000 × g, Rotixa K) and were collected at the interphase of medium
A and B. To concentrate the vacuoles, Percoll was added to a final
concentration of 20% (v/v), and the gradient and centrifugation steps
were repeated using 10-ml glass centrifugation tubes. All steps except
for the lysis step were performed on ice and surveyed microscopically.
Rye vacuoles were contaminated with less than 6% of nonvacuolar marker
enzymes and retained almost 100% of soluble vacuolar hydrolases (data
not shown).
Preparation of Yeast Microsomes--
Yeast microsomal fractions
were isolated from DTY7 and DTY168 strains grown overnight in YPD
medium (1% (w/v) Bacto-yeast extract (Difco), 2% (w/v) Bacto-peptone
(Difco), 2% (w/v) glucose) at 30 °C to an
A600 of approximately 10 by disruption of
enzymatically prepared spheroblasts in a glass/Teflon potter and
pelleting membranes at 100,000 × g for 45 min as
described in detail (15). Microsomal pellets were resuspended at an
A600 of 4 in 1.1 M glycerol, 50 mM Tris-MES, pH 7.4, 1 mM EDTA, 1 mM DTT, 2 mg × ml1 BSA, 1 mM phenylmethylsulfonyl fluoride, 1 µg × ml
1 leupeptin. For transport experiments, yeast membranes
were either used immediately after isolation or stored in liquid
nitrogen.
Uptake Experiments with Plant Vacuoles--
Vacuolar uptake
experiments with [3H]-estradiol
17-(
-D-glucuronide) (E217G) or
[14C]metolachlor-glutathione (MOC-GS) were performed as
described earlier (26). Unless indicated otherwise, for each time point and condition polyethylene microcentrifugation tubes (0.4-ml capacity) were prepared as follows: 70 µl of medium C (23% (v/v) Percoll, 0.4 M sorbitol, 30 mM KCl, 20 mM
MES-BTP, pH 7.2, 0.12% (w/v) BSA, 1 mM DTT, and 1.5 kBq of
[3H]E217G, 0.2 kBq of
[14C]MOC-GS, or 1.8 kBq of [14C]sucrose,
and further solutes as indicated in figures and tables) were placed on
the bottom of the tube. Uptake was started by adding 30 µl of
concentrated vacuole suspension. The samples were rapidly overlayered
with 200 µl of silicone oil AR 200 and 60 µl of water. The
incubation was terminated by flotation of the vacuoles (10,000 × g for 15 s, Beckman Microfuge 11, München,
Germany). 50 µl of the aqueous phase was used to determine the
radioactivity. Vacuolar volume was calculated by the addition of 3.7 kBq of 3H2O which equilibrates rapidly between
the medium and the vacuolar lumen. 3H2O was
added directly to medium C in experiments with
[14C]MOC-GS and in separate assays of identical
composition for the experiments with
[3H]E217G. Experiments in the presence of GSH
and its conjugates were performed without DTT which had no effect on
the uptake of either substrate. Unless stated otherwise, uptake rates
of [3H]E217G or [14C]MOC-GS
into barley and rye vacuoles were calculated by subtracting the
radioactivity measured after 2 min of incubation from corresponding 18-min values. Km, Vmax, and
Ki data were calculated using a computer program
(Enzfitter, Elsevier Biosoft, Cambridge, UK).
Identification of [3H]E217G after
Uptake into Rye Vacuoles--
After incubation of rye vacuoles in
medium C supplied with 7.4 kBq [3H]E217G and
further solutes as indicated in Fig. 1, 2 × 50 µl of the
aqueous supernatants following silicone oil centrifugation containing
about 6 µl of vacuolar volume were pooled and subjected to HPLC (22)
under the following conditions: Nucleosil RP-8 column (125 × 4.6 mm; 5-µm grain size; CS Chromatographie Langerwehe, Germany); flow
rate 1 ml × min1; solvent A, H2O
containing 1% (v/v) H3PO4; solvent B, MeOH;
linear gradient from 50 to 70% B in 23 min, sample volume 50 µl.
0.5-ml fractions were collected, and the radioactivity was determined by liquid scintillation counting. The radioactive peak of the vacuolar
content was compared with that of the authentic substrate [3H]E217G and with unlabeled
E217G and
-estradiol, both detected at 260 nm.
Uptake Experiments with Yeast Microsomal Vesicles-- Uptake of [3H]E217G was measured by the rapid filtration technique using nitrocellulose filters of 0.45-µm pore size (Schleicher & Schuell, Dassel, Germany) as described (15). In short, 1 part of microsomes was mixed with 6 parts of 0.4 M glycerol, 0.1 M KCl, 20 mM Tris-MES, pH 7.4, 1 mM DTT, 10 µM [3H]E217G, and additional compounds as indicated in figures and tables. Incubation was at 25 °C. At times indicated, samples of 100 µl were removed from the incubation medium and filtered through nitrocellulose filters premoistened with medium D (0.4 M glycerol, 0.1 M KCl, 20 mM Tris-MES, pH 7.4). Filters were washed with 5 ml of ice-cold medium D. Radioactivity was determined by liquid scintillation counting after addition of 5 ml Readysafe (Beckman) and shaking until filters were dissolved. Unless stated otherwise, uptake rates were calculated by subtracting the radioactivity measured after 0.5 min of incubation from corresponding 10-min values. Conditions were repeated with at least three independent microsome preparations, each performed in triplicate.
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RESULTS |
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It has been shown previously that plants have the ability to
deposit xenobiotics conjugated to glutathione in their vacuoles by a
directly energized, ATP-driven glutathione (GS) conjugate pump (10, 11,
27). In animals, a directly energized transport system named MOAT or
MRP2 has been described which is responsible for the removal of
conjugates of lipophilic substrates with glutathione but also
with glucuronate or sulfate across the canalicular membrane of liver
cells into bile (for review, see Ref. 28). The present work was
performed to address the following questions: (i) do plant vacuolar
membranes possess an active transport system that is able to transfer
other negatively charged conjugates like -estradiol 17-(
-D-glucuronide) (E217G) apart from GS
conjugates into the vacuole, and (ii) are these conjugate substrates of
the transporter described for GS conjugates, thus corresponding to a
"plant MOAT-like" pump?
Intact rye vacuoles incubated in the presence of 36 nM
[3H]E217G contained a single radioactive
compound after HPLC separation that coeluted with authentic labeled or
unlabeled E217G at identical retention times (Fig.
1). Up to 60 min of incubation, no
further radioactive peak was detected in vacuolar supernatants,
excluding a possible modification of the transported compound,
especially deglucuronidation to -estradiol (Fig. 1). Rye as well as
barley mesophyll vacuoles took up the abiotic glucuronate conjugate, [3H]E217G, in an ATP-dependent
manner (Figs. 1C and 2A and Table I). ATP-stimulated uptake of the
glucuronate into barley and rye vacuoles is linear for at least 22 min
(Fig. 2A). As shown earlier
for barley (10, 29), vacuolar uptake of [14C]MOC-GS into
rye vacuoles was ATP-dependent and linear for at least 20 min (Fig. 2B). In the presence of ATP and E217G
at a concentration of 8.2 nM, vacuolar transport rates for
E217G were comparable for both plant species (8.70 ± 1.69 and 8.48 ± 1.35 pmol E217G × (liter
vacuolar volume × s)
1 for rye and barley vacuoles,
respectively) suggesting the existence of an E217G carrier
located in plant vacuolar membranes irrespective of the capacity to
synthesize and store endogenous glucuronides. After 18 min of
incubation in the presence of 8.2 nM E217G and ATP, vacuolar concentrations of the glucuronate ranged between 6.61 and
11.75 nM and between 6.24 and 10.16 nM in
vacuoles isolated from rye and barley leaves, respectively. Thus, these
results do not clearly demonstrate accumulation against the
concentration gradient. Extending the incubation time up to 45 min
resulted in only a weak further increase in vacuolar concentration
(data not shown).
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In contrast, MOC-GS clearly accumulated in rye and barley vacuoles
against the concentration gradient in the presence of ATP (Fig.
2B and Table I). After 18 min vacuoles of rye contained between 97 and 182 µM (4.8-9.1-fold accumulation) of the
MOC-GS (20 µM), and in barley vacuoles concentrations
ranged between 89 and 127 µM (4.4-6.3-fold
accumulation). The corresponding uptake rates of 131.5 ± 36.5 and
108.6 ± 19.1 nmol of MOC-GS × (liter vacuolar volume × s)1 for rye and barley vacuoles, respectively, suggest
again that both plant species possess comparable transport activities
for the deposition of xenobiotic compounds conjugated to
glutathione. Uptake rates are in accordance with data
published for barley (e.g. Ref. 29).
In the presence of ATP, uptake of E217G into rye vacuoles
is a saturable process with an apparent Km of
208 ± 58 µM and a Vmax of
188 ± 53 nmol of E217G × (s × liter)1 (means of three independent experiments; Fig.
3). For barley, vacuolar uptake of MOC-
and N-ethylmaleimide-glutathione was shown to be saturable
with apparent Km values of 40-60 and 500 µM, respectively, in a previous publication (10).
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Inhibitors of different transport ATPases and reagents affecting the
vacuolar proton gradient or membrane potential showed comparable
effects on the uptake of E217G into vacuoles of both plants
(Table I). Transport of the E217G into vacuoles of barley and rye was not affected by bafilomycin A1 (30). The proton pumping
activity of the rye vacuolar ATPase into intact vacuoles was completely
abolished in the presence of 20 nM inhibitor (data not
shown). In contrast, vanadate, an inhibitor acting as a phosphate analogue on P-type ATPases, strongly decreased the uptake rate of the
glucuronate into rye and barley vacuoles (Table I). Vacuolar transport
was not sensitive toward azide. In addition, verapamil, a
calcium-channel blocker known to (i) reverse multidrug resistance (31),
(ii) to be a potent inhibitor of P-glycoprotein-mediated drug transport
(32, 33), and (iii) to stimulate ATP hydrolysis activity of
P-glycoprotein (34) did not affect vacuolar uptake of the
E217G. Neither dissipation of the pH gradient across the vacuolar membrane by the addition of NH4Cl nor dissipation
of the membrane potential (inside positive) by valinomycin in the presence of K+ ions had an effect on E217G
uptake into vacuoles of either plant species (Table I). In the absence
of ATP, the addition of NH4Cl did not further reduce
E217G uptake compared with uptake rates without ATP. Thus,
a pH-dependent, secondary energized glucuronate uptake
activity distinct from the vanadate-sensitive system could not be
detected, as it has been suggested for abiotic glucosides (22).
Essentially, uptake of MOC-GS into barley and rye vacuoles showed an
identical sensitivity toward the different effectors compared with
E217G as the substrate (Table I). Transport of the GS
conjugate was insensitive toward bafilomycin A1, azide, verapamil, and
dissipation of pH or
, whereas vanadate reduced uptake to
10-20% of the control rate.
Other nucleoside triphosphates could substitute for
MgATP-dependent uptake of E217G into rye
vacuoles: MgATP > MgGTP > MgUTP MgCTP (Table
II). The non-hydrolyzable ATP analogue
AMP-PNP could not drive glucuronate uptake. ADP and PPi
(the substrate of the vacuolar H+-pumping pyrophosphatase)
were not able to drive glucuronate transport (Table II).
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To obtain further information on the substrate specificities of the
transporters responsible for vacuolar uptake of the glucuronate and GS
conjugate in both plant species, competition experiments with (i)
endogenous rye-specific flavone glucuronides, (ii) abiotic glucuronates
(Table III), and (iii) glutathione
S-conjugates (Table IV) were performed.
Uptake rates of both E217G and MOC-GS into vacuoles of
barley and rye were decreased in the presence of the natural rye
flavone glucuronides luteolin
7-O-diglucuronyl-4-O-glucuronide (R1), luteolin
7-O-diglucuronide (R2), and
luteolin-7-O-glucuronide, all at 0.2 mM, to a
comparable extent (Table III). R1, bearing three glucuronate residues,
inhibited transport of E217G and metolachlor-GS about 30%,
whereas R2, carrying two glucuronates, was more effective, inhibiting
E217G uptake to 35% of the control rate and metolachlor-GS uptake to 43%, irrespective of the plant investigated. The
monoglucuronidated flavone luteolin 7-O-glucuronide
inhibited E217G transport into rye and barley vacuoles to
43 and 32%, respectively. MOC-GS as a substrate was differentially
affected by luteolin 7-O-glucuronide in barley and rye;
uptake into rye vacuoles was decreased only to 66% of the control
rate, whereas luteolin 7-O-glucuronide was almost as
effective as R2 when barley vacuoles were studied (43% of the control
activity, Table III). The flavone aglycone luteolin decreased
E217G transport rate into rye vacuoles about 55%. R2 acted
as a competitive inhibitor on transport of E217G into rye vacuoles with a Ki of 121 ± 16 µM (Fig. 4).
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The abiotic glucuronate conjugates p-nitrophenol
-D-glucuronide and 4-methylumbelliferyl-glucuronide were
much less inhibitory than the flavone glucuronides (Table III). The
unconjugated steroid estradiol inhibited vacuolar transport of
E217G about 60%, whereas uptake of metolachlor-GS was not
affected. Estradiol conjugated with glucuronate and sulfate residues
(
-estradiol 3-sulfate-17-(
-D-glucuronide)) as anionic
substituents strongly reduced glucuronate uptake into rye vacuoles.
Interestingly, E217G added to MOC-GS uptake assays did not
affect transport of the GS conjugate into rye vacuoles (Table III).
The bile acid taurocholate which is a substrate of a directly energized vacuolar bile acid transporter recently characterized in barley (29) had no effect on vacuolar uptake of E217G into rye vacuoles (Table III).
As shown earlier for barley, various substances conjugated to glutathione inhibited vacuolar uptake of glutathione S-conjugates competitively (10, 35). Here we show that decyl-GS and dinitrobenzene-GS (DNB-GS) present in a 10-fold excess reduced transport of MOC-GS into rye vacuoles by about 85 and 63%, respectively (Table IV). Oxidized glutathione (GSSG) inhibited transport of MOC-GS about 45%. Reduced glutathione (GSH) had only a very low effect on MOC-GS uptake into rye vacuoles.
Surprisingly, GS conjugates did not inhibit but strongly stimulated vacuolar uptake of E217G into rye and barley vacuoles (Table IV). The glucuronate conjugate transport rates increased 5-7-fold in the presence of decyl-GS or DNB-GS and 2.5-5.4-fold in the presence of GSSG, whereas GSH did not affect the transport rate. The HPLC analysis of vacuolar contents after uptake of [3H]E217G for 60 min in the presence of DNB-GS and ATP confirmed that the stimulation by DNB-GS was exclusively due to a rise of the [3H]E217G peak (Fig. 1C). Thus, the addition of DNB-GS led to an increase in the transport rate of [3H]E217G, and no modification of the transported glucuronate occurred. It can be excluded that the stimulation of glucuronate conjugate transport by oxidized glutathione was due to its redox potential, as NADP added at the same concentration did not enhance but rather reduced E217G uptake in barley and rye vacuoles (Table IV). The unconjugated fatty acid caprinic acid had no effect on glucuronate uptake. The 5-fold increase of the E217G uptake rate into rye vacuoles induced by decyl-GS was even retained in the presence of bafilomycin A1 and NH4Cl. The stimulation of E217G transport was dependent on the presence of ATP; decyl-GS (0.2 mM) added in the absence of MgATP resulted in E217G uptake rates of about 2% the values in the presence of MgATP which means that decyl-GS did not increase the uptake observed in the absence of ATP (Table I). Finally, the stimulatory effect of GS conjugates was not unspecific; vacuolar uptake of [14C]sucrose into vacuoles of barley and rye showed the typical behavior of an ATP-independent sucrose permease described for green mesophyll tissues (36) without any influence of GS conjugates, oxidized or reduced glutathione, caprinic acid, or NADP (Table IV).
The stimulation of E217G uptake into rye vacuoles was dependent on the concentration of GS conjugates or GSSG (Fig. 5). Half-maximal increase in E217G uptake rate required 9 ± 2 and 138 ± 19 µM decyl-GS and GSSG, respectively.
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It has recently been shown that the gene product encoded by the ycf1 gene contributes to cadmium tolerance in yeast (13) and that YCF1 is a GS conjugate transporter located in the vacuolar membrane (14-16). To compare vacuolar uptake characteristics of glucuronate conjugates in plants with yeast, transport of E217G into microsomal vesicles isolated from a wild type strain (DTY7) and a ycf1 deletion mutant (DTY168) was studied. As in plant vacuoles, transport of the glucuronate conjugate was dependent on the presence of ATP and strongly inhibited by vanadate (Table V and Fig. 6). Up to 10 min, ATP-dependent uptake into vesicles isolated from DTY7 and DTY168 was linear with time (Fig. 6). Microsomal uptake of E217G into DTY168 vesicles was not significantly reduced when compared with vesicles isolated from the wild type strain DTY7 (Table V), indicating that YCF1 may not be the major transport protein for glucuronate conjugates or it does not accept these conjugates as substrates. In contrast to plant vacuoles, uptake of E217G (10 µM) into DTY7 microsomes was not stimulated in the presence of decyl-GS or DNB-GS, both at 20 µM (Table V) or 200 µM decyl-GS (data not shown).
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DISCUSSION |
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Apart from different glutathione (GS) conjugates tested so far
(10, 11), the substrate specificity of the plant vacuolar GS conjugate
pump has not been investigated in detail. Two possibilities exist: (i)
the GS conjugate pump accepts a variety of different organic anions
different from GS conjugates and functions in analogy to the animal
liver-specific MOAT/MRP2, or (ii) glutathione and glucuronate
conjugates are transported by distinct uptake mechanisms. Here we
compared the vacuolar transport of the steroid conjugate -estradiol
17-(
-D-glucuronide) (E217G) with that of
metolachlor-glutathione (MOC-GS) to be able to distinguish between both
possibilities.
Principally, uptake of MOC-GS into rye vacuoles showed the typical basic characteristics of a directly energized transport system as already shown for barley mesophyll vacuoles (10), vacuolar membrane vesicles from Vigna radiata hypocotyl, and other plant sources (11). Vacuolar uptake was dependent on the presence of MgATP, was insensitive toward inhibitors of the vacuolar proton pumping ATPase, and was strongly inhibited by vanadate (Table I). Dissipation of either the proton gradient (inside acidic) or the membrane potential (inside positive) across the vacuolar membrane had no effect on MOC-GS uptake, ruling out the possibility of a secondary energized mechanism, e.g. via [H+] antiport (Table I). The transport rate for MOC-GS entry into rye vacuoles is comparable to that of barley vacuoles (Ref. 29 and this paper). Furthermore, vacuolar accumulation against the concentration gradient could be clearly demonstrated for rye and barley vacuoles, suggesting that xenobiotic glutathione conjugates are deposited in the plant vacuole by a ubiquitous uptake system directly using MgATP as an energy source which is thermodynamically more effective than a secondary active mechanism (27). In contrast to results obtained with V. radiata hypocotyl vesicles (11), verapamil, an agent known to revert multidrug resistance caused by P-glycoprotein, had no inhibitory effect on MOC-GS uptake into rye vacuoles added at 0.1 mM (Table I). It has been shown that vacuolar uptake of GS conjugates and oxidized glutathione (GSSG) is inhibited competitively by other GS conjugates (11, 35, this report, and Table IV) but not by corresponding molecules lacking the glutathione residue (11), suggesting that this residue serves as a tag for vacuolar deposition of potentially toxic substances.
Plant vacuolar membranes possess an uptake system for E217G
representing a glucuronate conjugate. The transport characteristics of
E217G into rye and barley vacuoles were similar to those
described for glutathione conjugates (10, 11), chlorophyll catabolites (37), bile acids (29), and an abiotic glucoside (22). It is therefore
likely that E217G is recognized by an ABC transporter. (i)
Vacuolar uptake of E217G was strongly
MgATP-dependent, and in the absence of ATP the glucuronide
transport amounted to only about 5 (rye) or 8% (barley) of the
transport rates obtained in the presence of MgATP (Table I and Fig. 2).
(ii) Transport into barley and rye vacuoles was sensitive toward
vanadate, whereas bafilomycin A1 and azide did not affect
E217G uptake. Dissipation of pH or
did not
decrease the glucuronate transport rates (Table I). In addition,
inorganic pyrophosphate, which is the substrate of the proton pumping
pyrophosphatase present in plant vacuolar membranes (38), was not able
to stimulate E217G uptake. Taken together these data
suggest that E217G transport does not depend on a secondary
active energization mechanism utilizing
pH or
as driving
forces but is energized by a vanadate-sensitive ATPase. (iii) As in
other cases where directly energized transport has been observed in
plants (10, 22, 29, 37), other nucleoside triphosphates but not ADP and
AMP-PNP could partially substitute for ATP (Table II). The latter fact
indicates that ATP hydrolysis is necessary for the transport
activity.
Compared with vacuolar transport of GS conjugates, bile acids, or
chlorophyll catabolites we could not clearly obtain an accumulation of
E217G against a concentration gradient within 45 min.
However, in the presence of DNB-GS, a 7.7-fold vacuolar accumulation
could be observed within 18 min. There are two lines of evidence that vacuolar transport depended on the presence of the glucuronate residue
in E217G. (i) The uptake rate decreased about 70% after treatment of E217G with a commercial -glucuronidase
(data not shown). (ii) More significantly, after incubation of rye
vacuoles with [3H]E217G the analysis of the
vacuolar contents confirmed that the substrate was taken up as such
without any structural changes (Fig. 1). Accordingly, uptake of free
-estradiol eventually liberated from E217G by an
unspecific
-glucuronidase could be excluded. A comparison of the
transport rate of the GS conjugate (see above) to that of
E217G showed that vacuoles of both species exhibited about
2-fold higher potential to transport the GS compared with the
glucuronate conjugate, both at 20 µM. Compared with rat
canalicular membrane vesicles (39), the apparent Km
value for E217G uptake into rye vacuoles indicates
that the vacuolar transporter may have a lower affinity for the
glucuronate than the animal MOAT (200 µM
versus 75 µM).
Based on the inhibition studies performed (Table III), we propose that
E217G is not transported by the GS conjugate pump but by a
separate, novel glucuronate conjugate transporter. Although MOC-GS and
E217G uptake was inhibited by the rye-specific flavone glucuronides to comparable degrees and was largely unaffected by the
monoglucuronated derivatives of methylumbelliferone and p-nitrophenol, E217G did not inhibit MOC-GS
uptake into rye vacuoles when the competitor E217G was
present in a 10-fold excess. Vacuolar uptake of E217G also
was not decreased in the presence of GS conjugates but strongly
stimulated (Table IV). Methylumbelliferyl- and p-nitrophenol -D-glucuronide, which did not inhibit E217G
uptake into barley and rye vacuoles, were in contrast taken up by
isolated canalicular plasma membrane vesicles in an
ATP-dependent manner (9). Therefore, the plant vacuolar
ATPase for E217G obviously does not transport glucuronates
in general as proposed for the animal MOAT. However, our results do not
allow us to draw clear-cut conclusions on the chemical structure of the
residual part of the molecule bound to glucuronate to serve as a
substrate for the E217G transporter. Furthermore, our
results indicate that the principal vacuolar GS conjugate transporter
does not correspond to a MOAT/MRP2-like pump accepting different
anionic substrates. In addition, the transport rate of
E217G into yeast microsomal vesicles isolated from a
ycf1 deletion mutant is comparable to vesicles isolated from
the corresponding wild type (Table V and Fig. 6). Thus, glucuronate and
GS conjugates may be transported by distinct membrane proteins in yeast
with YCF1 being responsible for yeast GS transport (14-16). Strict ATP
dependence and the strong inhibition by vanadate suggest that the yeast
E217G transporter may be a further directly energized pump.
For plant vacuoles, it can also be excluded that E217G is
transported via the bile acid carrier described recently (29), as
taurocholate did not inhibit the uptake of E217G into rye
vacuoles significantly, although taurocholate is a steroidal derivative. E217G uptake into rye vacuoles was decreased in
the presence of the flavone aglycone luteolin and
-estradiol (Table III). Plant phenolics including flavonoid aglycones were shown to
inhibit the efflux of dinitrophenol-GS from human colon adenocarcinoma cells (40). Furthermore, unspecific interactions with enzymes and
proteins are well known for unconjugated phenolic compounds in plant
biochemistry. Thus, we cannot judge from these data whether the
inhibition by luteolin and estradiol was specific or caused by
unspecific hydrophobic interactions. One may speculate that estradiol
and luteolin bind to the E217G transporter due to
similarities in the core structure of E217G but are not
transported due to the absence of a negative charge.
The striking fact that vacuolar uptake rates and inhibition characteristics of E217G transport were comparable in barley and rye and thus independent of the presence of endogenous glucuronate conjugates argues against a specific role of the E217G transporter in vacuolar deposition of the rye-specific flavone glucuronides. These flavone glucuronides inhibited uptake of E217G to comparable degrees into vacuoles of both plant species (Table III) with the mono- (luteolin 7-O-glucuronide) and diglucuronated (R2) luteolin derivatives being more effective than the triglucuronated one (R1), although R1 is the major component in rye mesophyll vacuoles in young primary leaves (6). The inhibition of E217G uptake into rye vacuoles by R2 was competitive (Fig. 4), although with the relatively high Ki of about 120 µM. Therefore, we cannot exclude the existence of a further high affinity (secondary activated ?) carrier specific for flavone glucuronides in rye in addition to the glucuronate conjugate pump described for the abiotic E217G as shown for glucoside conjugates in barley (22).
Uptake of E217G into rye and barley vacuoles was strongly
stimulated by GS conjugates and GSSG (Table IV and Fig. 5), and the
resulting transport activity for E217G in the presence of decyl-GS was comparable to the rates observed for glutathione conjugates as substrates. The stimulation increased in the following order: GSSG < decyl-GS < DNB-GS and the concentrations
required for half-maximal stimulation indicated that substrates with a high affinity toward the GS conjugate pump (10, 35) also have a high
"affinity" for stimulation. First, we assumed the stimulation by
decyl-GS or DNB-GS to be due to detergent effects of the lipophilic residues of these molecules. However, stimulation was also seen with
GSSG which is lacking an alkyl chain or an aromatic residue, and the
amphipolar C10-fatty acid caprinic acid corresponding to
the hydrophobic part of decyl-GS had no effect on E217G
uptake. In addition, GSH did not reduce vacuolar E217G
transport. Uptake of [14C]sucrose into rye and barley
vacuoles was not affected by any of the GS compounds tested showing the
typical characteristics of a sucrose permease (36) suggesting that GS
stimulation was specific for the glucuronate pump. The fact that
stimulation of E217G uptake by decyl-GS was not decreased
in the presence of agents blocking the vacuolar H+-ATPase
or dissipating the pH suggests that no secondary active mechanism
was involved in the activation but rather the stimulation of a directly
energized pump. On the basis of the results shown here, two
explanations for the stimulatory effect of glutathione conjugates on
vacuolar E217G uptake are possible. (i) Different binding
sites are present at the same transporter for GS conjugates as a
modulator and E217G as the substrate. In that case, the
activating action of GS conjugates on a glucuronate conjugate pump may
be a unique feature of plants as (a) E217G
uptake into yeast microsomal vesicles was not stimulated by GS
conjugates (Table V) and (b) MRP1-dependent
E217G transport across plasma membrane vesicles isolated
from MRP-transfected HeLa cells is not stimulated but strongly
inhibited by leukotriene C4 (21). (ii) The vacuolar glucuronate transporter described here interacts with another protein
activated by bound GS conjugates (the GS conjugate pump ?). Again,
there is no evidence for this possibility in yeast. On the other hand,
the interaction between K+ channels and an ABC transporter
has been reported (41).
Although detoxification reactions of xenobiotics via conjugation to glucuronate are well known in animal systems (42), to our knowledge no abiotic glucuronates have been described in plants. Due to the occurrence of endogenous glucuronates in rye together with corresponding UDP-glucuronyltransferases and the evidence presented here on the existence of a directly energized glucuronate conjugate transporter for E217G, this species may be a suitable model system to study herbicide detoxification via possible glucuronidation.
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ACKNOWLEDGEMENTS |
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We thank P. Burchard, B. Kammerer, A. Krämer, G. Sachs, and R. Schmitz for technical help; R. Tommasini, ETH Zürich, CH, for some suggestions, Dr. K. Kreuz, Novartis, Basel, CH, for the gift of [14C]metolachlor-GS; and Dr. J. F. Bornman, Lund University, for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by the Deutsche Forschungsgemeinschaft Bonn Grant Az. We 630/12-2 and by the Freunde und Förderer der Universität zu Köln.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.
¶ To whom correspondence should be addressed: Universität zu Köln, Botanisches Institut, Lehrstuhl I, Gyrhofstrasse 15, D-50931 Köln, Germany. Tel.: 49 221 470 3859; Fax 49 221 470 5181; E-mail: agweiss{at}novell.biolan.uni-koeln.de.
1
The abbreviations used are: R1, luteolin
7-O-diglucuronyl-4-O-glucuronide; R2, luteolin
7-O-diglucuronide; ABC, ATP-binding cassette; AMP-PNP,
5
-adenylyl
,
-imidodiphosphate; DNB, dinitrobenzene; E217G,
-estradiol 17-(
-D-glucuronide);
GS, glutathione; GSH, reduced glutathione; GSSG, oxidized glutathione;
MOC-GS, metolachlor-glutathione; MOAT, multiple organic anion
transporter; MRP, multidrug resistance associated protein; YCF1, yeast
cadmium factor 1; DTT, dithiothreitol; BSA, bovine serum albumin; MES,
4-morpholineethanesulfonic acid; BTP, bis-Tris propane; HPLC, high
performance liquid chromatography.
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REFERENCES |
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