From the School of Agricultural
Biotechnology, Seoul National University, Suwon 441-744, Korea and the
§ Division of Biochemistry, National Institute of
Agricultural Science and Technology, Suwon 441-100, Korea
Received for publication, October 16, 2000, and in revised form, December 5, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ABP57 is an auxin-binding
protein that possesses receptor function. In this study, a protocol for
ABP57 purification was developed on the basis of
cross-reactivity shown between ABP57 and antisera raised
against bovine serum albumin, which enabled us to purify ABP57 with a high yield and to further characterize it.
ABP57 activates plant plasma membrane H+-ATPase
(PM H+-ATPase) via direct interaction. The binding of
indole-3-acetic acid (IAA) to the primary binding site on
ABP57 caused a marked increase in the affinity of
ABP57 for PM H+-ATPase, which was accompanied
by a change in ABP57 conformation. Meanwhile, additional
IAA binding to the secondary site on ABP57 nullified the
initial effect without inducing further conformational change. When
ABP57 with IAA occupying only the primary site interacted with PM H+-ATPase, no IAA could access the secondary site.
These results suggest that IAA-induced biphasic alteration in the
affinity of ABP57 for PM H+-ATPase correlates
with a bell-shaped dose response of the enzyme to IAA. There is also a
possibility that, whereas the stimulation phase of the response is
associated with a conformational change of ABP57, the
destimulation phase probably results from hindrance arising
directly from the presence of IAA at the secondary site.
Auxin-binding proteins
(ABPs)1 are a class of low
abundance proteins in plants that bind active auxins with high affinity
and specificity. As a result of ABP-auxin binding, ABP might initiate the auxin signal pathways leading to various cellular responses and
thus have a plant hormone receptor function. Extensive studies have led
to the identification of a number of ABPs in both membrane and soluble
fractions, of which the best characterized in terms of cellular
location, biochemical nature, and putative receptor function is ABP1
(for reviews, see Refs. 1-5). The main natural auxin in most plants is
indole-3-acetic acid (IAA), which appears to exert many effects in
plants, including, most importantly, cell elongation. In fact, the term
"auxin" is used to describe chemical substances that stimulate
elongation growth in coleoptiles and many stems.
There is no clear explanation of the mechanism by which auxin regulates
cell growth. The immediate effect of exposure of plant tissues to auxin
is proton excretion, occurring within minutes. The resulting apoplastic
acidification provides a favorable condition for cell wall loosening,
which could be an early part of auxin-induced cell expansion (1, 3, 6).
H+-ATPase activity of the plasma membrane (PM) in plants is
responsible for proton extrusion from the cell. Therefore, the
acidification of the cell wall space is thought to result from
activation of the electrogenic H+-ATPase in some manner.
There is evidence that auxin stimulates plant PM
H+-ATPase, which is correlated with an increased affinity
of the enzyme for ATP (7) but does not involve a direct effect of auxin
on the enzyme itself (8, 9).
We have previously isolated two isoforms of a soluble ABP in rice
plants, one from shoots and the other from roots, and partially characterized them with respect to IAA binding and interaction with the
H+-ATPase (10, 11). Both isoforms, found to be monomeric
proteins with an estimated molecular mass of about 57 kDa on SDS-PAGE
and thus designated ABP57, work together with IAA to elicit
an auxin response of plant PM over a very wide range of IAA
concentrations. That is, the root isoform elicits a response with IAA
concentrations of
10 In the present investigation we found that ABP57 has a
domain in its internal amino acid sequence that is identical to one known to exist in bovine serum albumin (BSA). This finding led us to
conjecture that the domain might provide a motif that is recognized by
anti-BSA antibody, because serum albumin has been known to have
affinity for indoles, such as tryptophan and IAA (12, 13). Consistent
with this hypothesis, significant cross-reactivity between
ABP57 and antisera raised against BSA was observed.
Based on this observation, immunoaffinity chromatography using
IgGanti-BSA as a probe was employed for isolating the ABP
from rice plants. This approach recovered ABP57 purified to
homogeneity with a yield of about 60%. The high yield preparation
allowed us to further characterize the ABP with respect to its role in
IAA signal transduction in plant PM. We focused particularly on whether
the binding of IAA causes a conformational change of the ABP and, if
so, how it is related to regulating the proton-translocating activity of plant PM. An underlying mechanism for the IAA effect on the H+-ATPase via ABP57 is proposed, with the
relevant evidence provided.
Analysis of Internal Amino Acid Sequence--
An
ABP57 preparation, purified from the shoots of dark-grown
rice seedlings as described previously (10), was freeze-dried, redissolved in 70% formic acid, admixed with molar excess cyanogen bromide, and incubated at room temperature for 24 h in the dark. The resulting CNBr-cleaved peptides were purified in a fast protein liquid chromatography system (Amersham Pharmacia Biotech) with a
Superdex 75 HR column. The fractions of peptide with molecular mass of
about 15 kDa were concentrated under vacuum, run on an SDS-PAGE gel,
electroblotted onto a polyvinylidine difluoride membrane in CAPS buffer
at 50 V for 1 h, and then subjected to analysis in an amino acid
sequencer (Applied Biosystems).
Immunoblotting--
BSA and ABP57 were run on an
SDS-PAGE gel and electroblotted onto a polyvinylidine difluoride
membrane as above. The membrane was blocked with Tris-buffered saline
containing 5% (w/v) casein, then incubated with anti-BSA (Sigma),
washed with Tris-buffered saline-Tween 20 and Tris-buffered saline, and
then reincubated with peroxidase-conjugated anti-IgG (Sigma), as
described by Harlow and Lane (14). The membrane was thoroughly washed
in Tris-buffered saline-Tween 20; then sprayed with a peroxidase
substrate (Supersignal West Pico chemiluminescent substrate, Pierce)
consisting of luminol, chemiluminescence enhancer, and stable peroxide
buffer; and immediately used to expose x-ray film for a few seconds.
Purification of ABP57: A New
Protocol--
Soluble protein, prepared by ammonium sulfate (30-60%)
fractionation of crude extracts from etiolated rice seedlings, was solubilized in 20 mM Tris-Mes buffer (pH 8.0) and loaded
onto an immunoaffinity column (1.7 × 3.5 cm) filled with
anti-BSA-agarose (Sigma). Elution was done with a linear gradient of
ethylene glycol (0-20%) in the same buffer with a flow rate of 0.1 ml/min. The fractions showing ABP57 activity were pooled,
concentrated under vacuum, and passed through a DEAE-Sepharose CL-6B
column (2.5 × 5 cm). The flow-through fractions were then retrieved.
H+-ATPase Preparation--
PM vesicles were prepared
from rice roots as in Larsson et al. (15). The
H+-ATPase was isolated from PM vesicles essentially
according to Serrano (16), with a slight modification for the
detergent-solubilizing step; that is, 0.1%
n-dodecyl-D-maltoside was used instead of 0.6%
lysophosphatidylcholine to solubilize the membrane.
Assay of H+-ATPase Activity--
Vanadate-sensitive
ATP hydrolysis catalyzed by PM preparations as well as by the isolated
enzyme was measured as described by Dufour et al. (17).
H+ uptake by the inside-out PM vesicles was assayed by the
procedure of Palmgren (18).
Chemical Cross-linking of H+-ATPase and
ABP57--
Dimethyl suberimidate (DMS), which can
chemically link closely associated amino groups (19), was used for the
cross-linking experiments. A mixture of isolated H+-ATPase
(70 µg/ml) and ABP57 (40 µg/ml)
in 1 M triethanolamine-HCl (pH 9.7) was incubated with 10 mM DMS for 2 h at room temperature. The reaction was
terminated by boiling in Laemmli buffer, and cross-linking was
identified by SDS-PAGE.
Measurement of Trypsin
Digestibility--
ABP57 (86 µg/ml) was
incubated with trypsin (0.75 benzoyl-L-arginine ethyl ester
units/ml) and IAA (0-1 mM) in 50 mM potassium phosphate (pH 7.8) at 30 °C for 8 min. The digestion was stopped by the addition of type II-T trypsin inhibitor (0.1 µg/ml)
immediately followed by boiling in Laemmli buffer. Samples were run on
SDS-PAGE gel and stained, and the remaining intact ABP was measured
with a Gel-Pro analyzer (Media Cybernetics).
Optical Measurements--
Rayleigh scattering by
ABP57 in 0.2 M potassium phosphate buffer
containing IAA (0-1 mM) was measured at 25 °C in a
fluorescence spectrophotometer (Hitachi F-4500), with both excitation
(incident light) and emission (scattered light) wavelengths set at 350 nm. The circular dichroism (CD) spectra of the ABP were measured under the same buffer and temperature conditions as above in a Jasco spectropolarimeter (Jasco J-715).
Cross-reactivity of ABP57 with Anti-BSA Antibody
Provides the Basis for a Protocol for ABP Purification--
Because a
preliminary investigation revealed that the N terminus of the ABP is
blocked, internal sequencing was performed with a peptide purified from
CNBr-treated ABP57. Interestingly enough, the N-terminal
sequence of the peptide with 18 amino acids identified, such as
REKVLTSSARQRLRCASI, was found to show 100% sequence identity to a part
of domain II of BSA.2 Because
this domain could be an antigenic determinant, we tested whether
BSA-specific polyclonal antibody cross-reacts with ABP57 and found a band emitting chemiluminescence in Western blots on a
polyvinylidine difluoride membrane (Fig.
1). The intensity of luminescence from
the ABP band was estimated to be about one-half of that from a BSA band
of the same amount, indicating that there is significant
cross-reactivity between the ABP and anti-BSA antibody. Based on the
immunoblotting result, we developed a simple procedure for purifying
ABP57 with a high yield by the use of
IgGanti-BSA as an immunoadsorbent. Because the new protocol
often needed a final clean-up step, a DEAE-Sepharose CL-6B column was
used to remove small amounts of several protein contaminants that might bind nonspecifically to the antibody, yielding electrophoretically pure
preparations of ABP57 as shown (Fig.
2). The overall scheme of purification is
compiled in Table I. One unit of
activity was defined as the amount of protein that caused a
2-fold increase in the rate of ATP hydrolysis by 1 mg of protein of
plant PM in the presence of the optimal concentration of IAA at
37 °C and pH 7.0.
ABP57 Interacts Directly with the
H+-ATPase--
Because ABP57 functions as an
activator of PM H+-ATPase (11), a relevant question is
whether the ABP interacts directly with the target enzyme or indirectly
via a specific protein, i.e. a docking protein in the
membrane. If they interact directly to form a complex, a certain
functional group, such as a sulfhydryl or primary amine, on the ABP
molecule and on the H+-ATPase molecule might be in close
proximity in the complex; this could be tested by chemical
cross-linking (19). SDS-PAGE analysis of the mixture of isolated
H+-ATPase and ABP57 incubated with DMS (Fig.
3) revealed that the proteins were
covalently linked through reaction with the bifunctional cross-linker,
which has a spacer arm 11 Å long. Under these conditions, diminution
of the H+-ATPase (102 kDa) and ABP57 (57 kDa)
bands is accompanied by the appearance of a new protein band with
molecular mass of ~162 kDa, virtually equal to the combined mass of
the interacting proteins.
In contrast to experiments with isolated H+-ATPase,
H+-ATPase associated with PM was not cross-linked to ABP by
DMS. This led us to infer that the quaternary structure of the complex
formed between the membrane-associated enzyme and ABP57 may
be somewhat different from that of the isolated
enzyme-ABP57 complex. If such is the case, the primary
amines responsible for the cross-linking may be situated beyond the
range of the spacer arm of the cross-linker, being unable to
react concurrently with the two imidoester groups of DMS.
IAA Influences the Interaction between ABP57 and PM
H+-ATPase by Altering Binding Affinity--
The hyperbolic
dependence of PM H+-ATPase activity on the amount of
ABP57 was studied in the presence of IAA at various
concentrations (0-1 mM). The maximal stimulation of the
target enzyme by the ABP as an effector (Smax) and the
concentration of the effector required for one-half Smax
(S0.5) were determined from the respective hyperbolic
curves and plotted as functions of IAA concentration. This revealed
that S0.5 values changed biphasically with increasing concentrations of IAA, as did Smax, with both the lowest
S0.5 value and the highest Smax value appearing
at 5 µM IAA, as illustrated (Fig.
4). The S0.5 value is often
considered to inversely represent the affinity in an effector-enzyme
interaction (21). Therefore, such an effect of IAA on S0.5
may be taken as an indication that auxin binding affects the intrinsic
affinity of ABP57 for the H+-ATPase in a
biphasic mode, maximizing it at about 5 µM
IAA. Our earlier study has shown that there are two different kinds of IAA-binding sites on the ABP molecule, a primary site with a smaller dissociation constant (Kd) value and a secondary
site with a larger Kd value (11). In the case of the
isoform from rice shoots, the primary site is fully occupied by IAA at 5 µM, above which further binding of IAA to
the secondary site takes place. Taking the results together, it may be
concluded that the binding of IAA to the primary site enhances the
affinity of ABP57 for the H+-ATPase, which, in
turn, causes an increase in catalytic efficiency of the enzyme.
Meanwhile, additional IAA binding to the secondary site diminishes the
enhanced affinity of the ABP as well as the increased catalytic
efficiency.
IAA Binding Induces a Conformational Change of
ABP57--
Three different tests were performed to test
whether IAA binding induces changes in the structure of the
ABP57 molecule. In the first test, light scattering of the
ABP in solution was assessed in the presence of 0-1 mM
IAA. Because the angular dependence of light scattering by the solution
was not measured, it was impossible to obtain significant quantitative
information about the shape and size of the molecule. However, plotting
the relative intensity of the scattered light measured at
90o against IAA concentration gave a titration curve that
still appeared informative with respect to overall conformational
alteration, as shown (Fig.
5A). The occurrence of IAA
binding was manifested by a sharp increase in light scattering. This
may indicate that, upon IAA binding, significant changes took place in
some physical parameters involved in the process of light scattering,
such as the polarizability, the refractive index, or the radius of
gyration, that are closely linked to the overall structural property of a protein (22). The results of CD experiments also appeared to conform
to the concept of IAA-induced conformational alteration. CD data in the
short UV region give some information on the secondary structure of
proteins. For instance, a negative band at about 222 nm and a negative
and positive couplet at about 208 and 190 nm are assigned to
IAA Occupying the Secondary Binding Site Causes Interference with
the H+-ATPase-ABP57 Interaction--
An
intriguing feature of the above results was that, despite a significant
difference in the regulatory effect between ABP57 with IAA
bound only to the primary site and ABP57 with IAA
saturation, no difference in overall conformation was detected between
these two states. Therefore, the binding of IAA to the secondary site, without inducing further conformational change, was thought to somehow
influence the interaction of the ABP with PM H+-ATPase,
reducing it to the level of the interaction between IAA-free ABP57 and PM H+-ATPase. To investigate this
effect, an experiment was conducted. The H+-ATPase of PM
vesicles was activated to the highest degree by the optimal
concentration of IAA (5 µM) in the presence of
the ABP, and excess IAA (1 mM) was subsequently added to
the reaction mixture. It was then tested whether the additional, excess
supply of IAA affects the H+-ATPase activity, revealing
that the enzyme remained fully activated for both ATP hydrolysis and
H+ translocation even under conditions of supraoptimal
auxin concentrations. This was a clear contrast to the assay in which
IAA was added first at 1 mM to the assay system (Fig.
7). An incubation experiment was also
conducted in which samples of PM vesicles plus ABP57 were
incubated with IAA, added first at 5 µM and
then at 1 mM, and assayed for ATP hydrolysis at
fixed time intervals for 20 h. As it turned out, the maximum
activation of the H+-ATPase attained by 5 µM IAA via the ABP was not dissipated at all
by the presence of excess IAA during the prolonged incubation (data not
shown). A slight decrease in the H+-ATPase activity, which
started to occur about 8 h after incubation at 4 °C (3 h at
10 °C), was thought to be due probably to thermal denaturation of
the proteins. From these observations the following inference could be
drawn. ABP57 by itself binds only loosely to the
H+-ATPase, and free IAA can thus have easy access to both
the primary and the secondary binding sites. On the other hand, the ABP
with IAA only at the primary site binds tightly to the
H+-ATPase so that the secondary IAA-binding site is
effectively shielded, limiting the accessibility of IAA. The presence
of IAA at the secondary site on the ABP causes interference, either
sterically or electrostatically, with the protein-protein
interaction.
It is apparent that ABP57 shares a common epitope with
BSA. However, it remains to be confirmed whether the identified partial sequence of 18 amino acids shared by both proteins is directly involved
in the binding of IAA. Whatever the structural characteristics of
ABP57 are, as compared with those of BSA, the
cross-reactivity of the ABP with anti-BSA antibody provides the basis
for large scale preparation of the ABP through practically a single
chromatographic step. ABP57 was formerly identified and
isolated based solely on a biochemical functional assay, rather than on
ligand binding assays. For this, at least four chromatographic
separation steps involving anion exchange, cation exchange, size
exclusion, and tryptophan probe binding were needed for purifying to
homogeneity with yields of about 3% for the ABP isoform from rice
roots and about 6% for that from the shoots (10, 11). Compared with this earlier protocol of ours, the new protocol demonstrates a drastic
improvement of purification in terms of overall yield, simplicity, and rapidity.
ABP57 has intriguing biochemical properties related to
regulating plant PM H+-ATPase. It may first be mentioned
that the ABP interacts directly with the H+-ATPase,
enhancing the catalytic efficiency by altering kinetic parameters, such
as decreasing Km for the substrate and increasing
Vmax of ATP hydrolysis (11). Because PM
H+-ATPase has an autoinhibitory domain in its cytoplasmic
C-terminal region that is removed either reversibly or permanently when
the enzyme is stimulated (25, 26), ABP57 binding to the
H+-ATPase is thought to displace the inhibitory domain in
some manner. The effect of the regulatory domain displacement is
typified by the result of tryptic digestion, in which the truncated
enzyme with the C terminus deleted shows an increased activity with
decreased Km and increased
Vmax (25). A protein modulator of plant PM
H+-ATPase may be found in the 14-3-3 protein that
interacts directly and reversibly with the H+-ATPase, in
which a fungal toxin, fusicoccin, plays a role in stabilizing the
complex formed between 14-3-3 and the enzyme (27-30). Recent evidence
also indicates that phosphorylation of the penultimate threonine
residue of the C terminus of the H+-ATPase facilitates
14-3-3 binding regardless of the presence of fusicoccin (31-33). In a
sense, ABP57 is another kind of intrinsic protein modulator
of PM H+-ATPase in plant cells. However, its major
physiological relevance may rather be found in a role as a mediator of
IAA action on plant PM, which has not been identified with the 14-3-3 protein.
Another important property of ABP57 is believed to have
connection with the presence of the primary and secondary binding sites
with different affinities for IAA. Numerous results presented herein as
well as in an earlier report (11) are consistent with the concept that
the ABP can exist in three different states depending on the degree of
IAA binding, in the presence of a wide range of IAA concentrations. The
second state (State II) is the most "activated" in terms of the
ability to stimulate PM H+-ATPase through a direct
protein-protein interaction, and the first and third states (State I
and State III) are activated to a much lesser extent. The isoform of
ABP57 from rice shoots remains in State I at IAA
concentrations up to 0.1 µM, in State III at concentrations greater than 100 µM, and in State II at
~5 µM IAA. In the case of the isoform from
the roots, State I and State III were found to be attained at [IAA] < 0.5 nM and [IAA] > 100 nM, respectively,
and State II was found to be attained at [IAA] = 5 nM
(data not shown).
Evidence is provided here that IAA binding indeed induces a change in
the conformation of ABP57, which appears related to the
ABP-mediated IAA effect in plant PM as far as stimulation of the
H+-ATPase is concerned. A model of the two-step
conformational change caused by stepwise binding of IAA to the primary
and secondary sites would provide a rather feasible explanation as to
the bell-shaped dose response of the enzyme to IAA, as suggested
earlier (11). However, the present study shows that this is not the
case, because conformational change occurs only once as IAA
concentration increases. We therefore propose a mechanism,
schematically represented herein (Fig.
8), involving a putative
ABP57-docking site on the ABP molecule that is able
to interact intramolecularly with the autoinhibitory domain when it is
docked by the ABP. As a result of such communication, the regulatory
domain may be displaced from the catalytic region of the enzyme.
According to this model, the ABP in State II shows the highest affinity
for PM H+-ATPase because its structure fits well with the
docking site. Meanwhile, the ABP in State I and State III may have low
affinity for the docking site due to poor structural compatibility and hindrance arising from the presence of IAA at the secondary binding site, respectively.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
10-10
7
M, and the shoot isoform elicits a response with IAA
concentrations of
10
7-10
4
M. ABP57 is believed to have an unambiguous IAA
receptor function in many respects. The rates of ATP hydrolysis and
proton translocation by the H+-ATPase activity of plant PM
are regulated biphasically by IAA only in the presence of the ABP.
There are two different types of IAA-binding sites on the ABP molecule,
one showing a higher affinity for IAA and the other a lower affinity.
On its own, the ABP functions as a simple activator of PM
H+-ATPase, but the binding of IAA makes the efficiency of
this activation biphasic.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (97K):
[in a new window]
Fig. 1.
Cross-reactivity of ABP57 with
anti-BSA antibody. The ABP was immunoblotted with antisera raised
against BSA. For comparison, BSA purified by native PAGE was
concurrently subjected to immunoblotting. The results are from an equal
amount (0.8 µg) of the proteins.
View larger version (78K):
[in a new window]
Fig. 2.
Isolation of ABP57 from shoots of
dark-grown rice seedlings. The progress of purification by
ammonium sulfate fractionation of crude extracts (lane I),
immunoaffinity chromatography based on the ABP-IgGanti-BSA
interaction (lane II), and removal of protein impurities
using a DEAE-Sepharose column (lane III) is demonstrated by
SDS-PAGE. The marker proteins are shown in lane M.
Purification of ABP57 from rice seedlings
View larger version (112K):
[in a new window]
Fig. 3.
Dimethyl suberimidate cross-linking of
ABP57 and PM H+-ATPase. Three different
samples, the ABP (40 µg/ml), the H+-ATPase (70 µg/ml), and their mixture with the same concentrations in
1 mM triethanolamine-HCl (pH 9.7), were incubated with 10 mM DMS for 2 h at room temperature and then run on
SDS-PAGE. Neither ABP alone (lane I) nor
H+-ATPase alone (lane II) formed homoconjugates
in the presence of DMS, whereas the cross-linked conjugate of the two
proteins was seen in the incubated mixture (lane III). The
molecular mass markers (lane M) are shown on the same
gel.
View larger version (21K):
[in a new window]
Fig. 4.
Stimulation of PM H+-ATPase by
ABP57 in the presence of IAA (0-1 mM).
The maximum stimulation (Smax) and the concentration of the
ABP required for one-half Smax (S0.5) are shown
as functions of IAA concentration in B and
A, respectively. Typical examples of the hyperbolic
dependence of enzyme activation on the concentration of the ABP in the
presence of 5 µM (a) and 1 mM IAA (b) are given in the inset of
A. Smax and S0.5 were determined
graphically by double-reciprocal analysis of the hyperbolic
relationship. The intrinsic H+-ATPase activity of PM
vesicles was 0.233 ± 0.06 µM
ADP·mg 1·min
1,
as measured in the absence of the ABP. Data are the means ± S.D.
of three independent ABP57 preparations.
-helix (23), and the CD for a
-strand has a
negative band at about 215 nm and a positive band at about 198 nm (24).
The spectra of ABP57 were not measured in wavelengths shorter than 200 nm because of limited sensitivity of the
instrument with a limited amount of sample. Yet, the appearance of a
negative band at 208 nm with a shoulder at around 220-225 nm indicates the presence of both
-helix and
-strand in
ABP57. The binding of IAA gave rise to an increase in the
content of these secondary structures, as shown (Fig. 5B),
which should affect the tertiary structure. Virtually the same
pattern of structural response to IAA binding was observed in the other
experiment, where the degree of protein digestion by trypsin was
measured with the incubation of ABP57, IAA, and trypsin.
The binding of IAA apparently made the ABP more susceptible to tryptic
digestion, probably by inducing a conformational change as illustrated
(Fig. 6).
View larger version (22K):
[in a new window]
Fig. 5.
Effects of IAA on light scattering and CD
properties of ABP57 in solution. Monochromatic (350 nm) light was used for the scattering experiment, and the related data
are presented as means ± S.D. (n = 3) in
A. Two distinctive bundles of CD spectra measured with
ABP57 (50 µg/ml) solutions containing IAA in
the ranges of 0-0.1 µM and 0.005-1
mM are shown in B. In each bundle, the spectra
are virtually identical and are thus unable to be discerned from one
another. IAA at concentrations up to 1 mM did not produce
any CD signal, as indicated by the broken tracing.
mdeg, millidegrees.
View larger version (29K):
[in a new window]
Fig. 6.
Tryptic digestion of ABP57
incubated with IAA at various concentrations. Data are presented
as means ± S.D. (n = 3). The inset
shows a typical SDS-PAGE result.
View larger version (21K):
[in a new window]
Fig. 7.
Time courses of H+ translocation
(A) and ATP hydrolysis (B) by PM
H+-ATPase. The reactions were initiated by adding 1.2 mM MgSO4 to the respective basal mixtures and
monitored at 30 °C by fluorescence intensity of acridine orange and
absorbance of NADH, respectively. ABP57 (0.12 µg/ml) and IAA (5 µM and 1 mM) were subsequently added to the reaction systems, as
indicated by arrows. The basal mixture for assaying
H+ translocation consisted of the inside-out PM vesicles
(20 µg of protein/ml), Na-ATP (1.2 mM),
and acridine orange (2 µM) in 10 mM MOPS-bis-Tris propane buffer (pH 7.0) containing
50 mM KBr and 0.14 M KNO3. The
composition of the basal mixture for assaying ATP hydrolysis was as
follows: PM (50 µg of protein/ml), Na-ATP (0.6 mM), NADH (0.2 mM), phosphenolpyruvate (2 mM), pyruvate kinase (2 units), lactate dehydrogenase (2 units), NaN3 (1 mM), sodium molybdate (1 mM), KCl (50 mM), KNO3 (0.2 M), and Triton X-100 (0.01%) in 20 mM Tris-Mes
(pH 7.0).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (43K):
[in a new window]
Fig. 8.
A plausible mechanism of the
ABP57-mediated IAA effect on the activity of PM
H+-ATPase. The ABP undergoes a conformational change
upon IAA binding that occurs involving the high affinity site
(H) and the low affinity site (L). On the
H+-ATPase molecule, there is a putative
ABP57-docking site (DS) that interacts
intramolecularly with the inhibitory domain (ID) when DS is
occupied by the ABP. As a result of such interaction, ID is displaced
from the catalytic site (CS). The extent of ID displacement
depends on the structural fit of the ABP to DS. IAA involved in the
binding is indicated by an I enclosed in a circle.
Kd values are for the complexes formed between the
ABP isoform from shoots and IAA, taken from the previous report
(11).
The proposed mechanism for describing both the stimulation and destimulation phases of the IAA effect on PM H+-ATPase activity is rather simple and straightforward. In this respect, ABP57 is distinct from other proteins claimed to possess auxin receptor function, most importantly including ABP1, which perceives auxin signals on the cell surface but is predominantly localized to the lumen of the endoplasmic reticulum (3, 34). To explain auxin signal transfer between ABP1 and the H+-ATPase in PM, complex mechanistic schemes need to be formulated, involving the export of ABP1 from the endoplasmic reticulum (35), the existence of a hypothetical ABP1-docking protein in plant PM (34, 36), and signaling intermediates, such as phospholipase A2 and protein kinase (37, 38), etc. Furthermore, despite evidence that ABP1 is involved in hyperpolarization of plant PM by auxin (36, 39, 40), it remains unclear whether auxin-induced variations of transmembrane potential in the presence of exogenous ABP1 result from the modulation of the H+-ATPase. The expression and activity of ion channels may also regulate the electrochemical gradient of protons (41).
In conclusion, the present study not only discloses a peculiar
immunological property of ABP57 that provides the basis for developing a simple protocol for purification, but also, more importantly, provides insights into the underlying mechanism for IAA-regulated H+ efflux in plant cells. The biphasic mode
of the ABP-mediated IAA action observed with in vitro
systems may be correlated with the bell-shaped dose-response curves for
in vivo IAA effects that have long been seen in the
elongation growth of the coleoptiles and apical root segments of plants
(20, 42-44). In addition, it is worth mentioning that other active
auxins, notably including synthetic auxins, such as 1-naphthalene
acetic acid and 2,4-dichlorophenoxyacetic acid, fail to elicit the same
response of plant PM H+-ATPase in the presence of
ABP57 as seen with the main natural auxin, IAA (11). This
failure may conversely implicate that there are some other receptors
that, together with various active auxins, play a role in
electrochemically energizing plant PM.
![]() |
FOOTNOTES |
---|
* This work was supported in part by the Brain Korea21 project.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. Tel.: 82-31-290-2406; Fax: 82-31-293-8608; E-mail: jinjung@snu.ac.kr.
Published, JBC Papers in Press, January 11, 2001, DOI 10.1074/jbc.M009416200
2 GenBankTM accession number M73993, submitted by E. W. Holowachuk (1991).
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: ABP, auxin-binding protein; IAA, indole-3-acetic acid; PM, plasma membrane; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; CAPS, 3-(cyclohexylamino)propanesulfonic acid; Mes, 2-(N-morpholino)ethanesulfonic acid; DMS, dimethyl suberimidate; MOPS, 4-morpholinepropanesulfonic acid.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Jones, A. M., and Prasad, P. V. (1992) Bioessays 14, 43-48 |
2. | Jones, A. M. (1994) Annu. Rev. Plant Physiol. Plant Mol. Biol. 45, 393-420[CrossRef] |
3. | Napier, R. M., and Venis, M. A. (1995) New Phytol 129, 167-201 |
4. | Venis, M. A., and Napier, R. M. (1995) Crit. Rev. Plant Sci. 14, 27-47 |
5. | Macdonald, H. (1997) Physiol. Plant. 100, 423-430[CrossRef] |
6. | Rayle, D. L., and Cleland, R. E. (1992) Plant Physiol. (Bethesda) 99, 1271-1274[Medline] [Order article via Infotrieve] |
7. | Gabathuler, R., and Cleland, R. E. (1985) Plant Physiol. (Bethesda) 79, 1080-1085 |
8. | Santoni, V., Vansuyt, G., and Rossignol, M. (1991) Planta 185, 227-232 |
9. | Szpornarski, W., Vansuyt, G., and Rossignol, M. (1991) Phytochemistry 30, 1391-1395[CrossRef] |
10. | Kim, Y.-S., Kim, D., and Jung, J. (1998) FEBS Lett. 438, 241-244[CrossRef][Medline] [Order article via Infotrieve] |
11. | Kim, Y.-S., Kim, D., and Jung, J. (2000) Plant Growth Regul., in press |
12. | Bertuzzi, A., Mingrone, G., Gandolfi, A., Greco, A. V., Ringoir, S., and Vanholder, R. (1997) Clin. Chim. Acta 265, 183-192[CrossRef][Medline] [Order article via Infotrieve] |
13. | Peters, T., Jr. (ed) (1996) All about Albumin: Biochemistry, Genetics, and Medical Applications , pp. 76-132, Academic Press, San Diego, CA |
14. | Harlow, E., and Lane, D. (1988) Antibodies , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
15. | Larsson, C., Widell, S., and Kjellbom, P. (1987) Methods Enzymol. 148, 558-568 |
16. | Serrano, R. (1988) Methods Enzymol. 157, 533-544[Medline] [Order article via Infotrieve] |
17. | Dufour, J. P., Amory, A., and Goffeau, A. (1988) Methods Enzymol. 157, 513-528[Medline] [Order article via Infotrieve] |
18. | Palmgren, M. G. (1990) Plant Physiol. (Bethesda) 94, 882-886 |
19. | Mattson, G., Conklin, E., Desai, S., Nielander, G., Savage, D., and Morgensen, S. (1993) Mol. Biol. Rep. 17, 167-183[Medline] [Order article via Infotrieve] |
20. | Radermacher, E., and Klämbt, D. (1993) J. Plant Physiol. 141, 698-703 |
21. |
Verghese, G. M.,
Johnson, J. D.,
Vasulka, C.,
Haupt, D. M.,
Stumpo, D. J.,
and Blackshear, P. J.
(1994)
J. Biol. Chem.
269,
9361-9367 |
22. | Pittz, E. P., Lee, J. C., Bablouzian, B., Townsend, R., and Timasheff, S. N. (1971) Methods Enzymol. 27, 209-256 |
23. | Johnson, W. C., and Tinoco, I. (1972) J. Am. Chem. Soc. 94, 4389-4390[Medline] [Order article via Infotrieve] |
24. | Brahms, S., Spach, G., and Brack, A. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 3208-3212[Abstract] |
25. | Palmgren, M. G., Askerlund, P., Fredrikson, K., Widell, S., Sommarin, M., and Larsson, C. (1990) Plant Physiol. (Bethesda) 92, 871-880 |
26. |
Palmgren, M. G.,
Sommarin, M.,
Serrano, R.,
and Larsson, C.
(1991)
J. Biol. Chem.
266,
20470-20475 |
27. |
Jahn, T.,
Fuglsang, A. T.,
Olsson, A.,
Bruntrup, I. M.,
Collinge, D. B.,
Volkmann, D.,
Sommarin, M.,
Palmgren, M. G.,
and Larsson, C.
(1997)
Plant Cell
9,
1805-1814 |
28. | Oecking, C., Piotropski, M., Hagemeier, J., and Hagemann, K. (1997) Plant J. 12, 441-453[CrossRef] |
29. |
Fullone, M. R.,
Visconti, S.,
Marra, M.,
Fogriano, V.,
and Aducci, P.
(1998)
J. Biol. Chem.
273,
7698-7702 |
30. | Baunsgaard, L., Fuglsang, A. T., Jahn, T., Korthout, H. A., de Boer, A. H., and Palmgren, M. G. (1998) Plant J. 13, 661-671[CrossRef][Medline] [Order article via Infotrieve] |
31. |
Svennelid, F.,
Olsson, A.,
Piotropski, M.,
Rosenquist, M.,
Ottman, C.,
Larsson, C.,
Oecking, C.,
and Sommarin, M.
(1999)
Plant Cell
11,
2379-2391 |
32. |
Fuglsang, A. T.,
Visconti, S.,
Drumm, K.,
Jahn, T.,
Stensballe, A.,
Mattei, B.,
Jensen, O. N.,
Aducci, P.,
and Palmgren, M. G.
(1999)
J. Biol. Chem.
274,
36774-36780 |
33. |
Maudoux, O.,
Batoko, H.,
Oecking, C.,
Gevaert, K.,
Vadekerckhove, J.,
Boutry, M.,
and Morsomme, P.
(2000)
J. Biol. Chem.
275,
17762-17770 |
34. | Barbier-Brygoo, H. (1995) Crit. Rev. Plant Sci. 14, 1-25 |
35. | Cross, J. W. (1991) New Biol. 8, 813-819 |
36. | Klämbt, D. (1990) Plant Mol. Biol. 14, 1045-1050[Medline] [Order article via Infotrieve] |
37. | Scherer, G. F. E., and André, B. (1993) Planta 191, 515-523 |
38. | Yi, H., Park, D., and Lee, Y. (1996) Physiol. Plant. 96, 359-368[CrossRef] |
39. | Barbier-Brygoo, H., Maurel, C., Shen, W. H., Ephritikhine, G., Delbarre, A., and Guern, J. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 891-895[Abstract] |
40. | Barbier-Brygoo, H., Ephritikhine, G., Klämbt, D., Maurel, C., Palme, K., Schell, J., and Guern, J. (1991) Plant J. 1, 83-94 |
41. |
Sze, H.,
Li, X.,
and Palmgren, M. G.
(1999)
Plant Cell
11,
677-689 |
42. | Cleland, R. (1972) Planta 104, 1-9 |
43. | Shen, W. H., Petit, A., Guern, J., and Tempe, J. (1988) Proc. Natl. Acad. Sci. U. S. A 5, 3417-3421 |
44. | Karz, W., Stolarek, J., Piettruszka, M., and Malkowski, E. (1990) Physiol. Plant. 80, 257-261[CrossRef] |