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Plant Physiol. (1998) 118: 551-555
A Phosphothreonine Residue at the C-Terminal End of the Plasma
Membrane H+-ATPase Is Protected by Fusicoccin-Induced
14-3-3 Binding1
Anne Olsson*,
Fredrik Svennelid,
Bo Ek,
Marianne Sommarin, and
Christer Larsson
Department of Plant Biochemistry, Lund University, P.O. Box 117, SE-221 00 Lund, Sweden (A.O., F.S., M.S., C.L.); and Uppsala Genetic
Center, Department of Plant Biology, Swedish University of Agricultural
Sciences, P.O. Box 7080, SE-750 07 Uppsala, Sweden (B.E.)
 |
ABSTRACT |
We have isolated the plasma membrane
H+ ATPase in a phosphorylated form from spinach
(Spinacia oleracea L.) leaf tissue incubated with
fusicoccin, a fungal toxin that induces irreversible binding of
14-3-3 protein to the C terminus of the H+-ATPase, thus
activating H+ pumping. We have identified threonine-948,
the second residue from the C-terminal end of the
H+-ATPase, as the phosphorylated amino acid. Turnover of
the phosphate group of phosphothreonine-948 was inhibited by 14-3-3
binding, suggesting that this residue may form part of a binding motif for 14-3-3. This is the first identification to our knowledge of an
in vivo phosphorylation site in the plant plasma membrane H+-ATPase.
| |
INTRODUCTION |
The plant plasma membrane H+-ATPase provides the
driving force for secondary active transport, and controls intra- and
extracellular pH, as well as cell turgor. Considering that the
H+-ATPase plays a major role in the control of many cell
processes, it is not surprising that it is regulated by a number of
factors, including hormones, light, and fungal toxins (for review, see Michelet and Boutry, 1995 ).
The only mechanism for regulation so far demonstrated is that ATP
hydrolytic activity and H+ pumping may be controlled via an
autoinhibitory domain located in the C-terminal region of the
H+-ATPase (Palmgren et al., 1990, 1991). Removal of this
domain by proteolysis or deletion at the gene level results in an
activated form of the enzyme with a higher
Vmax, a lower Km
for ATP, a more alkaline pH optimum, and a more efficient coupling
between ATP hydrolysis and H+ pumping (Palmgren et al.,
1990, 1991; Regenberg et al., 1995). Very similar results were obtained
after incubation of intact tissue with the fungal toxin fusicoccin
(Johansson et al., 1993; Rasi-Caldogno et al., 1993; Lanfermeijer
and Prins, 1994), a treatment suggested to cause a displacement of the
C-terminal autoinhibitory domain.
Fusicoccin was initially suggested to activate the plasma membrane
H+-ATPase by direct interaction with the enzyme
(Marrè, 1979). Later, fusicoccin was demonstrated to bind to a
"receptor" belonging to a family of proteins designated 14-3-3
proteins (Korthout and de Boer, 1994; Marra et al., 1994; Oecking et
al., 1994). 14-3-3 proteins constitute a highly conserved family of
eukaryotic proteins with multiple regulatory functions (for review, see
Aitken, 1996). Recently, it was shown that 14-3-3 proteins bind
directly to the C-terminal region of the H+-ATPase and that
fusicoccin stabilizes the H+-ATPase/14-3-3 complex
formed, rendering the association irreversible (Jahn et al., 1997;
Oecking et al., 1997; Baunsgaard et al., 1998). This explains earlier
observations that plasma membranes isolated from fusicoccin-treated
material contained several times more 14-3-3 than plasma membranes
isolated from the corresponding controls (Korthout and de Boer, 1994;
Oecking et al., 1994). The strong interaction induced by fusicoccin
also allowed the H+-ATPase/14-3-3 complex to be
solubilized and purified from isolated plasma membranes (Jahn et al.,
1997; Oecking et al., 1997). Binding of 14-3-3 proteins to the
C-terminal region of the H+-ATPase was also shown to occur
in the absence of fusicoccin, and it was suggested that 14-3-3
proteins are natural ligands of the H+-ATPase, regulating
H+ pumping by displacing the autoinhibitory domain of the
enzyme (Jahn et al., 1997; Oecking et al., 1997; Baunsgaard et al.,
1998).
Binding of 14-3-3 proteins to their target proteins has been shown to
involve a phosphorylated motif in the target (Muslin et al., 1996). Two
optimal motifs have been identified, RSXpSXP, based on the binding
motif in the animal Ser/Thr protein kinase Raf-1 (Muslin et al., 1996),
and RXY/FXpSXP (Yaffe et al., 1997), identified by using
phosphoserine-oriented peptide libraries to probe mammalian and yeast
14-3-3 proteins. There is, however, no amino acid sequence in the
C-terminal region of the H+-ATPase with obvious similarity
to any of the two optimal binding motifs, suggesting that 14-3-3
binding to this region involves a somewhat different motif. In the
present study we identify a Thr at the C-terminal end of the
H+-ATPase, the phosphorylation of which is affected by
fusicoccin-dependent binding of 14-3-3 to this region.
| |
MATERIALS AND METHODS |
Plant Material
Spinach (Spinacia oleracea L.) was grown in a
greenhouse with supplementary light (23 W/m2,
350-800 nm, G/86/2 HPLR 400 W, Philips, Eindhoven, The Netherlands). Expanding leaves of 4- to 5-week-old plants were used.
In Vivo Phosphorylation
Spinach leaves (5-25 g) were cut into small pieces and
infiltrated under a vacuum with 6 volumes of 0.33 M Suc,
10 mM
Mes/1,3-bis(Tris[hydroxymethyl]methylamino)propane, pH 6.0, 0.1 mM EGTA, 0.1 mM EDTA, and 1 to 7 mCi
32P-orthophosphate. After 1 h of incubation at room
temperature, 5 µM fusicoccin was added and incubation
proceeded for another 30 min. Alternatively, fusicoccin was already
present in the infiltration medium and
32P-orthophosphate was added 15 min later; total
incubation was also for 90 min. Controls did not receive fusicoccin.
After removing the incubation buffer, plasma membranes were isolated as
described previously (Larsson et al., 1994).
Trypsin Treatment
Trypsin treatment of plasma membrane vesicles was essentially as
described by Palmgren et al. (1990); 66 µg of plasma membrane protein
in 66 µL of 10 mM
Mops/ 1,3-bis(Tris[hydroxymethyl]methylamino)propane, pH 7.0, 20%
(v/v) glycerol, 5 mM EDTA, 1 mM DTT (buffer A)
containing 6 mM ATP was mixed with an equal volume of Brij
58 (10 mg/mL in buffer A) to turn all plasma membrane vesicles
cytoplasmic-side-out (Johansson et al., 1995). Then, 2 µL of
buffer A containing 1 µg of trypsin was added. After incubation at
20°C for 5 min, the reaction was stopped by the addition of 2 µL of
100 mM Pefablock (catalog no. 1429868, Boehringer Mannheim)
and put on ice.
SDS-PAGE and Western Blotting
Samples were solubilized at room temperature in standard sample
buffer, and polypeptides were separated by SDS-PAGE according to the
method of Laemmli (1970). Gels were either stained with Coomassie
brilliant blue R 250, or polypeptides were electrophoretically transferred to an Immobilon PVDF transfer membrane (Millipore) for
immunostaining. A monoclonal antibody, 46E5B11F6, raised against a
fusion protein containing the N-terminal region of the maize H+-ATPase and an antiserum raised against a
barley 14-3-3 protein (Brandt et al., 1992) were used. Immunoblots
were developed with nitroblue tetrazolium and
5-bromo-4-chloro-3-indolyl phosphate. P-proteins were visualized with a phosphor
imager (Molecular Dynamics, Sunnyvale, CA).
Protein Digestion and Amino Acid Sequencing
The 100-kD H+-ATPase band, obtained by
SDS-PAGE of in vivo-phosphorylated spinach leaf plasma membranes, was
cut from the gel and digested with LysC from Achromobacter
lyticus (Wako, Osaka, Japan), as described by Rosenfeld et
al. (1992). Separation of eluted peptides by HPLC and amino acid
sequencing were as described previously (Johansson et al., 1996).
Determination of the phosphorylated amino acid sequence was by covalent
sequencing of the radioactive peptide on arylactivated membranes
(Millipore). After several washes with neat trifluoroacetic acid in
a sequencer (model 477A, ABI) the AZT amino acid was extracted with
neat trifluoroacetic acid, collected, and concentrated by evaporation.
Each fraction, corresponding to one released amino acid, was spotted on
TLC plates, and radioactive spots were visualized and quantified using
a phosphor imager (Fuji, Tokyo, Japan).
Protein Determination
Protein was measured essentially as described by Bearden (1978),
with BSA as the standard.
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RESULTS AND DISCUSSION |
Fusicoccin-Dependent 32P-Labeling of the
H+-ATPase
To investigate whether binding of 14-3-3 to the plasma membrane
H+-ATPase may involve a phosphorylated amino acid residue,
pieces of spinach leaves were infiltrated with
32P-orthophosphate and with fusicoccin to promote
H+-ATPase/14-3-3 complex formation (Jahn et al., 1997;
Oecking et al., 1997). This was followed by plasma membrane isolation,
separation of polypeptides, and visualization of phosphorylated
polypeptides. A 100-kD band coinciding with the position of the
H+-ATPase was labeled, provided that the plant material was
preincubated for 1 h with 32P-orthophosphate to
equilibrate the ATP pool with 32P before addition of
fusicoccin (Fig. 1, B and D, lanes 2). No P-labeling of the H+-ATPase was observed in
the absence of fusicoccin (lanes 1), or if fusicoccin was added 15 min
before addition of 32P-orthophosphate (lanes 3). Fusicoccin
treatment led to an increase in binding of 14-3-3 to the plasma
membrane (Fig. 1E), in agreement with earlier results (Korthout and de
Boer, 1994; Oecking et al., 1994; Jahn et al., 1997).
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| Figure 1.
In vivo phosphorylation of spinach leaf plasma
membrane polypeptides. Polypeptides were stained with Coomassie
brilliant blue R (A), and a phosphor imager was used to visualize
32P-polypeptides in the dried gel (B). Immunostaining of
the H+-ATPase (C) shows that the 100-kD
H+-ATPase band coincides with the
32P-polypeptide, as visualized with a phosphor imager (D).
E, Immunostaining of 14-3-3 protein. Pieces of spinach leaves were
infiltrated with medium containing 32P-orthophosphate in
the absence (lanes 1) or presence (lanes 2 and 3) of fusicoccin and
incubated for 90 min. In lanes 2, leaves were incubated for 1 h
with 32P-orthophosphate before addition of fusicoccin,
whereas in lanes 3, leaves were incubated with fusicoccin for 15 min
before addition of 32P-orthophosphate. Plasma membranes
were isolated and polypeptides were separated by SDS-PAGE (20 µg of
protein per lane). Polypeptides were either stained with Coomassie
brilliant blue R, or detected by immunoblotting using antibodies raised
against a maize H+-ATPase or a barley 14-3-3 protein.
Arrowheads mark the position of the 100-kD H+-ATPase.
Numbers at left refer to molecular mass standards.
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In vivo activation of spinach leaf H+-ATPase by fusicoccin
is a rapid process and reaches a plateau within minutes (Johansson et
al., 1993). This means that formation of stable
H+-ATPase/14-3-3 complexes (Jahn et al., 1997; Oecking et
al., 1997) is completed within minutes, locking the enzyme in an
activated and phosphorylated (Fig. 1, lanes 2) form. In the absence of
fusicoccin, no phosphorylation of the H+-ATPase was
observed (Fig. 1, B and D, lanes 1). This may indicate that the level
of phosphorylation, as well as the level of
H+-ATPase/14-3-3 complex formation, is normally very low,
and that phosphorylation may be observed only when the equilibrium is
shifted toward complex formation by the action of fusicoccin.
Alternatively, the in vivo level of H+-ATPase/14-3-3
complex formation is high, but the binding is readily reversible and
the complexes are disrupted and the phosphate lost due to endogenous
protein phosphatase activity during homogenization of the tissue.
That 14-3-3 binding to the H+-ATPase protects the
phosphorylated amino acid residue from dephosphorylation was
demonstrated by incubating the leaf tissue with fusicoccin before
addition of 32P-orthophosphate (i.e.
H+-ATPase/14-3-3 complex formation preceded
32P-labeling of the ATP pool). In that experiment, no
32P-labeling of the H+-ATPase was observed,
suggesting that the fusicoccin-induced binding of 14-3-3 somehow
shielded the phosphorylated amino acid residue from protein
phosphatase/kinase activity (Fig. 1, B and D, lanes 3). Thus, taken
together, our results suggest that binding of 14-3-3 to the
H+-ATPase protects a phosphorylated amino acid residue in
the enzyme from being turned over.
Localization of the Phosphorylation Site
Mild trypsin treatment removes a 7- to 10-kD fragment from the
C-terminal end of the H+-ATPase, thereby
activating the enzyme (Palmgren et al., 1991). Hence, trypsin treatment
provides a simple means for testing whether the phosphorylation
observed in Figure 1 was due to an amino acid residue located in the
C-terminal region of the H+-ATPase.
Plasma membranes labeled with 32P as in Figure 1
(lanes 2) were treated with trypsin in the presence of Brij 58, a detergent producing 100% sealed, cytoplasmic-side-out vesicles
(Johansson et al., 1995), thus making the C-terminal region of all
H+-ATPase molecules accessible for trypsin
cleavage. Mild trypsin treatment resulted in a partial removal of the
native 100-kD H+-ATPase band and the appearance
of a 90-kD band (Fig. 2A). This 90-kD
band, representing the H+-ATPase lacking the
C-terminal region, was not phosphorylated (Fig. 2B), indicating that
the phosphorylated amino acid residue was located in the C-terminal
region; the remaining 100-kD H+-ATPase species was not
labeled. The latter result is in agreement with the recent finding
(Jahn et al., 1997; Oecking et al., 1997) that trypsin treatment
causing removal of the C-terminal region from about 50% of the
H+-ATPase molecules was sufficient for the
removal of all bound 14-3-3, indicating that the C termini of
H+-ATPase molecules binding 14-3-3 are more
susceptible to proteolysis.
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| Figure 2.
Trypsin treatment of isolated plasma membrane
vesicles removes all 32P label from the
H+-ATPase. A, Immunostaining of the H+-ATPase;
B, 32P-polypeptides in A, as visualized with a phosphor
imager. Plasma membranes containing
[32P]H+-ATPase were treated with trypsin in
the presence of Brij 58, thus making the C-terminal region of
all H+-ATPase molecules accessible for proteolysis.
Polypeptides were separated by SDS-PAGE and transferred to a membrane
for immunostaining. The positions of the native 100-kD
H+-ATPase and the 90-kD species resulting from proteolytic
removal of the C-terminal region of the H+-ATPase are
indicated.
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To determine the position of the phosphorylation site in the C
terminus, the labeled H+-ATPase was excised from
the gel and digested with the proteinase LysC, cleaving polypeptides
C-terminal to Lys residues. The resulting peptides were separated by
reversed-phase HPLC and the radioactivity in each fraction determined.
Only two radiolabeled peaks, with the sequences GLDIDTIQQNYT and
GLDIDTXQQAYX, were found (data not shown). These amino acid sequences
are homologous to the relatively conserved C-terminal end of the plant
plasma membrane H+-ATPase (Fig.
3), except that the C-terminal Val
residue was missing in both sequences (this residue probably fell under
the detection limit). The sequences obtained differ in at least one
position and probably represent two different
H+-ATPase isoforms in spinach leaves. To identify
the phosphorylated amino acid residue, the radioactivity released
during each sequencing cycle was measured. Most of the radioactivity
applied was released with the 12th amino acid (Fig.
4), i.e. the second Thr in the sequence
corresponding to Thr-948 in the Arabidopsis isoform AHA 1 (compare with
Fig. 3). Thr-948 is a highly conserved residue, present in all
H+-ATPases found in the database (Fig. 3).
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| Figure 3.
Alignment of the amino acid sequences obtained for
the two 32P]H+-ATPase peptides with C-terminal
H+-ATPase sequences. The amino acid sequences obtained for
the two 32P-peptides (32PC-terminal1 and
32PC-terminal2) are compared with C-terminal sequences (the
last 15-21 amino acids, depending on isoform) of plant plasma membrane
H+-ATPases found in the database. GeneWorks
(IntelliGenetics, Oxford Molecular Group, Oxford, UK) was used for the
multiple alignment. Sequence identity is indicated with boxes and dots.
Dashes indicate gaps introduced to maximize sequence identity.
Numbering in the consensus sequence is according to the Arabidopsis
isoform AHA1.
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| Figure 4.
Identification of the in vivo-phosphorylated amino
acid residue. The graphs show the radioactivity released for each cycle
during sequencing of the two 32P-peptides
(32PC-terminal1 and 32PC-terminal2 in Fig. 3)
and identifies Thr-948 (numbering is according to the Arabidopsis
isoform AHA1, Fig. 3) as the phosphorylated amino acid residue. PSL,
Photostimulated luminescence.
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Role of Phosphothreonine-948
Assuming that phosphothreonine-948 is part of a binding motif for
14-3-3 protein, synthetic peptides corresponding to this domain were
tested for their ability to disrupt the
H+-ATPase/14-3-3 complex and deactivate the
H+-ATPase. Thus, plasma membranes isolated from
fusicoccin-treated leaves were incubated with synthetic peptides
corresponding to the last 16 amino acids in the C terminus of the
H+-ATPase
(KLKGLDIDT942IQQAYT948V;
numbering is according to the Arabidopsis isoform AHA1, Fig. 3); one
peptide was not phosphorylated, a second was phosphorylated on Thr-948,
and a third was phosphorylated on Thr-942. However, none of the
peptides (in concentrations up to 100 µM, and incubation
times up to 30 min) had any effect on H+-ATPase activity
nor on the amount of 14-3-3 bound to the plasma membrane (data not
shown). This is in agreement with earlier results, where incubation
with phosphorylated Raf-1 peptide did not remove the bound 14-3-3
after incubation with fusicoccin (Jahn et al., 1997), which
demonstrates the irreversible nature of fusicoccin-induced H+-ATPase/14-3-3 complex formation.
| |
CONCLUDING REMARKS |
Phosphorylation of an amino acid residue in the C-terminal region
of the H+-ATPase has been suggested as a possible
mechanism to regulate H+-ATPase activity
(Palmgren et al., 1991). In vivo phosphorylation of the oat root plasma
membrane H+-ATPase, as well as a pH- and
Ca2+-dependent in vitro phosphorylation of Ser
and Thr residues of the H+-ATPase in isolated
plasma membrane vesicles, was shown by Schaller and Sussman (1988), but
no effect on activity was found. More recently, Vera-Estrella et al.
(1994) and Xing et al. (1996) have described an elicitor-induced, in
vivo dephosphorylation of the plasma membrane H+-ATPase of
cultured tomato cells, a dephosphorylation that caused activation of
the enzyme. Furthermore, Sekler et al. (1994) have demonstrated in vivo
phosphorylation of the H+-ATPase of the
acidophilic alga Dunaliella acidophila. In none of these
cases was the phosphorylated amino acid identified. However, for
D. acidophila, the phosphorylation site was clearly
localized to the C-terminal part of the enzyme.
In this work we present the first identification to our knowledge of an
in vivo phosphorylation site in the plant plasma membrane H+-ATPase. We show that after incubation of
spinach leaf tissue with fusicoccin, the plasma membrane
H+-ATPase may be isolated in a phosphorylated
form (Fig. 1, B and D, lanes 2). Furthermore, we show that the
phosphorylated amino acid is located in the C-terminal region of the
H+-ATPase (Figs. 2 and 3) and is identical to
Thr-948 (Fig. 4), the second amino acid from the C-terminal end in most
isoforms of the H+-ATPase (Fig. 3). Because
phosphothreonine-948 is protected from turnover by 14-3-3 binding, it
may be part of a binding motif for 14-3-3 protein. Alternatively,
phosphothreonine-948 may be shielded from protein phosphatase/kinase
activity due to a conformational change induced by 14-3-3
binding. The putative 14-3-3-binding motif in which
phosphothreonine-948 resides is far from a perfect match to any of
the recently identified optimal binding motifs for 14-3-3 proteins
(Muslin et al., 1996; Yaffe et al., 1997). However, as proposed by
Yaffe et al. (1997), "The nature of 14-3-3 interaction is
determined by the extent to which the binding motif matches the optimal
consensus sequence", and "determines if the role of 14-3-3 is to
act as a sequestering molecule, a chaperone, or an adaptor." For the
H+-ATPase/14-3-3 interaction, where the 14-3-3
protein acts as a regulatory protein, a relatively weak and readily
reversible binding should be expected. A weak interaction is supported
by experimental data showing that the H+-ATPase
when isolated is devoid of 14-3-3 unless the tissue has been
incubated with fusicoccin to induce a stronger interaction (Jahn et
al., 1997; Oecking et al., 1997). Thus, the irreversible binding of
14-3-3 induced by fusicoccin is lethal; the hosts of the fungus
Fusicoccum amygdali, peach and almond trees, wilt and die
because the irreversible activation of the
H+-ATPase in stomatal cells results in a
permanent opening of the stomata (for review, see de Boer, 1997).
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FOOTNOTES |
1
This work was supported by the Swedish
Foundation for Strategic Research, the Swedish Natural Science Research
Council, the Swedish Council for Forestry and Agricultural Research,
and the European Union Biotechnology Program.
*
Corresponding author; anne.olsson{at}plantbio.lu.se; fax
46-462-224-116.
Received April 24, 1998;
accepted July 1, 1998.
| |
ACKNOWLEDGMENTS |
We wish to thank Adine Karlsson for excellent technical
assistance and Dr. Louise Race (Department of Plant Cell Biology, Lund
University, Sweden) for critically reading the manuscript. We thank Dr.
Wolfgang Michalke (Institut für Biologie III,
Albert-Ludwigs-Universität, Freiburg, Germany) for kindly
providing the monoclonal antibody to the
H+-ATPase and Dr. David B. Collinge (Department
of Plant Biology, Royal Veterinary and Agricultural University,
Frederiksberg C, Denmark) for a generous gift of the antiserum to the
14-3-3 protein.
| |
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Top
Abstract
Introduction