From the Unité de Biochimie Physiologique, Université Catholique de Louvain, Croix du Sud 2-20, B-1348 Louvain-la-Neuve, Belgium
Received for publication, August 24, 2000, and in revised form, October 26, 2000
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ABSTRACT |
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The major plant plasma membrane
H+-ATPases fall into two gene categories, subfamilies
I and II. However, in many plant tissues, expression of the two
subfamilies overlaps, thus precluding individual characterization.
Yeast expression of PMA2 and PMA4, representatives of the two plasma
membrane H+-ATPase subfamilies in Nicotiana
plumbaginifolia, has previously shown that (i) the isoforms have
distinct enzymatic properties and that (ii) PMA2 is regulated by
phosphorylation of its penultimate residue (Thr) and binds regulatory
14-3-3 proteins, resulting in the displacement of the autoinhibitory
C-terminal domain. To obtain insights into regulatory differences
between the two subfamilies, we have constructed various chimeric
proteins in which the 110-residue C-terminal-encoding region of PMA2
was progressively substituted by the corresponding sequence from PMA4.
The PMA2 autoinhibitory domain was localized to a region between
residues 851 and 915 and could not be substituted by the corresponding
region of PMA4. In contrast to PMA2, PMA4 was poorly phosphorylated at
its penultimate residue (Thr) and bound 14-3-3 proteins weakly. The
only sequence difference around the phosphorylation site is located two
residues upstream of the phosphorylated Thr. It is Ser in PMA2 (as in
most members of subfamily I) and His in PMA4 (as in most members of subfamily II). Substitution of His by Ser in PMA4 resulted in an enzyme
showing increased phosphorylation status, 14-13-3 binding, and ATPase
activity, as well as improved yeast growth. The reverse substitution of
Ser by His in PMA2 resulted in the failure of this enzyme to complement
the absence of yeast H+-ATPases. These results show
that the two plant H+-ATPase subfamilies differ
functionally in their regulatory properties.
The plant plasma membrane H+-ATPase
(PMA)1 is an electrogenic
pump, which couples ATP hydrolysis to proton transport out of the cell. The resulting electrochemical gradient is then used by
secondary transporters, acting as uniporters or as proton symporters and antiporters, to move ions and metabolites across the plasma membrane (for review, see Refs. 1-3).
The 33 plant H+-ATPases cDNA clones obtained as yet
have been classified into two different gene subfamilies (I and II),
which diverged before the separation of monocot and dicot plants (4, 5). However, the fact that members of both subfamilies are simultaneously expressed in the same organ (4, 6) hinders the
determination of the enzymatic and regulatory properties of individual
isoforms in plant material. This limitation was overcome by the
heterologous expression of H+-ATPases from
Arabidopsis thaliana (7) and Nicotiana
plumbaginifolia (8) in the yeast Saccharomyces cerevisiae.
PMA2 and PMA4, the two most widely expressed N. plumbaginifolia genes, each representing one of the two
subfamilies, were able to replace the yeast H+-ATPase
genes, while still allowing yeast growth. However, clear differences
were observed between the two PMAs in terms of proton pumping, pH
dependence of ATPase activity, and growth capacity of the yeast at low
pH (9).
The plant H+-ATPase is regulated by an autoinhibitory
domain located in its C-terminal region (10). In the plant,
phosphorylation of the penultimate residue (a threonine) is required
for the binding of regulatory 14-3-3 proteins (11, 21, 27). These
highly conserved proteins function as a dimer in all eukaryotic cells and serve diverse regulatory functions (for review, see Ref. 12). However, studies performed using plant material have not attempted to
differentiate between the different isoforms, and yeast expression of
plant H+-ATPases was therefore also used to address their
regulatory features. For instance, deletions or point mutations within
the inhibitory C-terminal region of H+-ATPases from
Arabidopsis (13, 14) and N. plumbaginifolia (15,
16) resulted in enzyme activation and improved yeast growth. Using this
heterologous system, it was recently demonstrated that, as with
H+-ATPases expressed in the plant, phosphorylation of the
H+-ATPase penultimate residue (Thr) is necessary for 14-3-3 binding and ATPase activation (17, 20). In the case of PMA2, the
phosphorylation and complex formation with 14-3-3 proteins was readily
observed in yeast in the absence of fusicoccin, a fungal toxin often
used to stabilize the plant H+-ATPase·14-3-3 complex
(17). Given the kinetic differences between PMA2 and PMA4, it was
important to determine whether these representatives of the two
subfamilies were regulated in the same way. Here, we show that, in
contrast to PMA2, PMA4 was poorly phosphorylated and bound 14-3-3 proteins only weakly. This regulatory difference could be linked to a
single residue located two positions upstream of the phosphorylated Thr.
Strains--
YAK2 (8) is a yeast strain in which the
H+-ATPase genes, PMA1 and PMA2, have
been deleted. This strain is kept alive by yeast PMA1 under
the control of the GAL1 promoter in a URA3
centromeric plasmid PRS-316 (18). The strain YAKpma2Ntagged was
previously described (17).
For His tagging of PMA4, plasmid 2µp(PMA1)pma4, containing
wild-type N. plumbaginifolia pma4 cDNA (9), was modified
by PCR to introduce a sequence coding for 6 histidine residues
at the N terminus (between codons 2 and 3). Two primers: T4-PCR2, 5'-GCGCCTAGGCACCTCGTCTCTACCGTGTAGTGGTAGTGGTAGTGTTTCG-3' and T4-PCR3, 5'-GACAAGTGAGGTTGCGGG-3' were used to generate the PCR product. The fragment was digested with BamHI/PstI and
then used to replace the corresponding fragment in plasmid
2µp(PMA1)pma4 (9) to give
2µp(PMA1)pma4Ntagged.
We produced four chimeras in which the 110 residues at the C-terminal
region of PMA2 were progressively substituted by the corresponding
region from PMA4. Recombinant DNA molecules were created by sequential
PCR amplifications (Table I).
2µp(PMA1)pma2Ntagged (17) served as the template for the
PCR1 series and 2µp(PMA1)pma4 for the PCR2 series. For
each chimera, both PCR products (which partly overlapped) were combined
and amplified as a single fragment using primers PH2-1 and PH4-2. The
new fragments were digested with BglII/XbaI, then
used to replace the corresponding fragment in plasmid
2µp(PMA1)pma2Ntagged (17).
Plasmid 2µp(PMA1)pma2Ntagged (17) was modified by PCR to
replace Ser-953 with Ala (PMA2-S953A), Asn (PMA2-S953N), or His (PMA2-S953H). N-tagged PMA2-E14D was modified to replace Ser-953 with
His (PMA2-E14D/S953H). His-949 in PMA4 was replaced in plasmid 2µp(PMA1)pma4Ntagged and chimera (PMA2+
Transformants with the different constructions were obtained as
follows: YAK2 was transformed by the plasmid bearing the plant H+-ATPase gene under the control of the PMA1
promoter and was plated on MGal-His, Leu, Ura, Trp. Transformants were
replicated on MGlu pH 6.5 or 5.5 supplemented with 0.1% 5-fluoroorotic
acid to counterselect the URA3 plasmid PRS-316 and obtain a
yeast strain expressing only a plant plasma membrane
H+-ATPase.
Media and Growth Conditions--
Yeast cells were grown either
in rich medium containing 2% (w/v) glucose and 2% (w/v) yeast extract
(YGlu) or in minimal medium containing 0.7% (w/v) yeast nitrogen base
without amino acids (Difco) and 0.115% (w/v) drop mix (19),
supplemented with all amino acids and nucleotides required for growth.
Solid media contained 2% agar. The 5-fluoroorotic acid medium was
prepared as described by Treco (19). The pH of the medium was adjusted
with KOH/HCl to pH 6.5 for PMA2, pH 5.5 for PMA4 or to another pH when
indicated. For growth comparison, liquid cultures at the early
exponential stage (15-30 × 106 cells/ml) were
diluted in sterile water to 1 × 106 cells/ml and then
spread on different plates.
Solubilization and Purification of His-tagged
H+-ATPase--
Plasma membranes were prepared according to
Morsomme et al. (16). Solubilization and purification of the
His-tagged H+-ATPase were performed according to Maudoux
et al. (17).
Electrophoresis and Western Blotting--
Electrophoresis
analysis and Western blotting were performed as described in Morsomme
et al. (16). Quantification was performed using
125I-protein A (Amersham Pharmacia Biotech) detected by
PhosphorImager® (Bio-Rad Molecular Imager®
System GS-S25).
ATPase Assays--
ATPase assays were performed at 32 °C in a
reaction mixture consisting of 50 mM MES/MOPS/Tris, 6 mM MgATP, 1 mM free Mg2+
(MgCl2), 10 mM sodium azide (mitochondrial
ATPase inhibitor), 20 mM KNO3 (vacuolar ATPase
inhibitor), and 0.2 mM molybdate (phosphatase inhibitor) at
pH 6.4. The reaction was started by adding plasma membrane proteins (14 µg) to 160 µl of reaction mixture. After 3, 6, and 9 min, 50-µl
aliquots were mixed with 60 µl of 5% trichloroacetic acid to stop
the reaction.
PMA4 Is Weakly Phosphorylated and Shows Weak 14-3-3 Binding--
Heterologous expression in yeast has been previously used
to express N. plumbaginifolia PMA2 and PMA4 separately,
making it possible to study individual enzymatic and physiological
properties. His tagging of PMA2 permitted its easy purification (17).
To compare the solubilized isoforms, a His6 tag was added
to the N-terminal region (between residues 2 and 3) of PMA4. In
addition, we tagged an activated PMA4 mutant (A129P) bearing a mutation (Ala-129 replaced by Pro) that resulted in a more active enzyme and
improved yeast growth at low pH (data not shown), similar to two other
activated PMA4 mutants described previously (T861D and Gln882stop, Ref.
9). We chose to use A129P in this study because, contrary to the two
other PMA4-activated mutants, the amino acid substitution is not
localized in the regulatory C-terminal region. However, we must be
aware that this does not exclude a three-dimensional proximity.
No significant differences in growth rate or ATPase activity were
observed between yeast strains expressing tagged or untagged PMA4,2 showing that, as with
PMA2 (17), His tagging did not interfere with enzyme performance. After
purification on a Ni2+-NTA column, PMA4 was found to
interact weakly with the two yeast 14-3-3 proteins (BMH1 and BMH2)
compared with PMA2 (Fig. 1A). This was not because of a loss of 14-3-3 proteins during purification because Western blotting analysis of membranes also revealed the low
abundance of these regulatory proteins compared with the strain expressing PMA2 (Fig. 1B). The difference in binding amount
of 14-3-3 proteins between the two H+-ATPase isoforms was
not because of a difference in the total amount of 14-3-3 proteins in
the yeast cells expressing either PMA2 or PMA4 because the same amount
of 14-3-3 proteins was found in a total yeast extract of both strains
(data not shown). As the binding of 14-3-3 to PMA2 depends on
phosphorylation of Thr-955 (17), we determined the phosphorylation
status of PMA4 using anti-phosphothreonine antibodies. In contrast to
PMA2, phosphothreonine in PMA4 was only weakly detectable (Fig.
1B). A129P, a PMA4-activated mutant, displayed higher 14-3-3 binding and phosphorylation status than the corresponding wild-type
protein (Fig. 1B), suggesting that the mutation resulted in
increasing accessibility of the C-terminal region to
phosphorylation.
Defining the PMA2 C-terminal Inhibitory Domain--
To define
putative differences between the inhibitory C-terminal region of PMA2
and PMA4, we constructed four chimeras in which the 110-residue
C-terminal region of PMA2 was progressively replaced by the
corresponding region of PMA4. These chimeras were defined on the basis
of (i) sequence alignment of the C-terminal domains of the two isoforms
and (ii) information on mutated residues in this region (Fig.
2A). We defined three zones
(Fig. 2B), the first two (residues 851-871 and 879-895)
containing all but one of the identified PMA2-activating mutations in
the C-terminal region (15, 16) and the third (residues 896-915) was
highly divergent between the two isoforms. The chimeras used are shown in Fig. 2C. As shown in Fig.
3, chimera (PMA2+1), in which the entire
PMA2 C-terminal region was replaced with that from PMA4, showed much
better growth than PMA2, suggesting that the PMA2 inhibitory domain
could not be functionally replaced by the corresponding region from
PMA4. A chimera retaining the first zone in which activating PMA2
mutants were found (chimera PMA2+3/4) still showed stimulation
of growth compared with PMA2, but to a lesser extent. Retention of both
activating mutant zones (chimera PMA2+1/2) reduced growth to the
level observed for wild-type PMA2. Finally, when only the third
C-terminal region was replaced (chimera PMA2+ The Nature of the Fourth Residue from the C terminus
(C
We therefore mutated His-953 in chimera (PMA2+ In this study, N. plumbaginifolia PMA2 and PMA4 were
chosen as representatives of the two major plant H+-ATPase
subfamilies. Their direct characterization in plant material has been
hampered by their partially overlapping expression in many plant
tissues (4). Heterologous expression in yeast is a good alternative,
because it has previously allowed us to compare their kinetics (9) and
to show that PMA2 is activated in yeast, as in plants, by
phosphorylation of its penultimate residue (Thr) and binds regulatory
14-3-3 proteins (17). Moreover, 14-13-3 binding was observed in the
absence of fusicoccin, a fungal toxin often used to artificially
stabilize the H+-ATPase/14-3-3 complex. This therefore
validates the yeast model.
The first aim of this study was to delineate the autoinhibitory
C-terminal region. Replacing the entire PMA2 C-terminal region with
that from PMA4 fully activated the enzyme and resulted in a much better
yeast growth. This demonstrates that the autoinhibitory C termini of
PMA2 and PMA4 are not interchangeable. Progressive reduction of
contribution of the PMA4 C-terminal region was correlated to a decrease
of yeast growth or even lethality when only the last 41 residues of
PMA2 were replaced by those from PMA4. From these data, we can conclude
that the inhibitory domain covers more than 50 residues and includes
the first-half of the C-terminal region in which 19 single-point PMA2
mutations, which result in activated ATPase and better yeast growth,
have been localized (15, 16). Our data show that the inhibitory domain
extends to the region encompassing residues 895-915, because adding
back this region of PMA2 (PMA2+ The second finding of this paper concerns the regulatory
phosphorylation site of the H+-ATPase family. Sequence
comparison of all plant H+-ATPase cDNAs available in
the database shows that they can be grouped into two subfamilies and a
similar dichotomy is observed in the first half of the C-terminal
region. In contrast, the last 30 C-terminal residues are highly
homologous except for the fourth residue from the C terminus (PMA2-953
or PMA4-949), which is Ser or Ala in subfamily I and His or Asn in
subfamily II. This amino acid is 2 residues removed from the
penultimate Thr, which is phosphorylated by a kinase and therefore
belongs to the 14-3-3 binding site defined at the H+-ATPase
C terminus (11, 17, 20, 21). The significance of the difference at this
position is supported by two arguments. Firstly, the divergence between
the two subfamilies is ancient, because it preceded the separation
between dicot and monocot species and yet the consensus has been
strictly retained within each subfamily. Secondly, we showed that these
differences have functional consequences on PMA regulation and yeast
growth. For instance, converting Ser-953 of PMA2 into Ala (the
alternative residue for subfamily I) did not cause any change, whereas
converting it into either His or Asn (subfamily II residues) prevented
yeast growth. The observation that PMA2-S953H did not support yeast
growth whereas PMA4 (which also displayed His at C An additional fact has to be considered. PMA2-E14D and PMA4-A129P, both
activated mutants, were more phosphorylated and bound more 14-3-3 proteins than their respective wild-type enzymes. As it was shown
previously that the PMA2-E14D H+-ATPase had a C-terminal
region more accessible to trypsin cleavage (16), we can suggest that in
the wild type, the C-terminal region is not fully accessible to the
kinase. Two parameters have to be taken into account: (i) the
accessibility to the C-terminal region and (ii) the phosphorylation
consensus sequence. Both effects seem to be additive because E14D (more
accessible C-terminal region and Ser at 953) conferred a better growth
than PMA2 (Ser at 953) or E14D/S953H (more accessible C-terminal
domain). Weak accessibility and poor consensus (PMA2-S953H) combined
did not allow yeast growth.
In most cases, 14-3-3 proteins recognize their target through a
specific phosphorylated sequence. The two most widespread motifs are
RSXpSXP (23) and
RX(Y/F)XpSP (24). The plant H+-ATPase
is an exception, because the 14-3-3 dimer interacts with the last part
of the C-terminal region (11, 17, 20, 21), which does not contain the
above sequence. Within the last 30 residues, which are highly conserved
between both subfamilies, position C How can we apply the data obtained in the yeast system to plants? The
existence of several isoforms expressed in various plant organs
complicates the comparison of their individual regulatory properties.
For instance, in spinach, the formation of a
H+-ATPase/14-3-3 complex is increased by fusicoccin, a
fungal toxin that makes 14-3-3 binding irreversible. After tryptic
cleavage, two H+-ATPase C-terminal peptides were
identified, one with Ala and the other with Asn at position
C More generally, how could differential regulation be explained? At
least two models can be proposed. In the first one, different kinases
would be involved, each of which specifically recognizes members of
only a single subfamily, allowing completely independent transducing
systems. In the second model, a single kinase would be involved and, if
behaving like in the yeast enzyme, would favor the phosphorylation of
subfamily I members unless the modification of subfamily II members
(e.g. previous phosphorylation by another kinase at another
H+-ATPase site) renders their C terminus more accessible to
the kinase. This hypothesis is realistic, because we showed that a single point mutation (A129P) of PMA4 resulted in increased
phosphorylation and 14-3-3 binding.
It is clear that any progress toward the understanding of differential
regulation will require tools that allow members of a single family to
be specifically studied. Genetic methods that silence the expression of
a whole subfamily might be one approach. However, the silencing of
pma4 in tobacco induced pleiotropic effects and therefore
might disturb the regulatory systems (26). A better approach might be
to express in the plant single H+-ATPase isoforms tagged in
such a way (e.g. His6) that they can be
specifically followed.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Primers used to obtain chimera constructs
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
PMA4 interacts weakly with the 14-3-3 proteins. A, affinity-purified N-tagged wild-type PMA2
and PMA4 (10 µg) were analyzed by SDS-polyacrylamide gel
electrophoresis and stained with Coomassie Blue. PMA and 14-3-3 proteins are indicated. The two intermediate bands for PMA2 are
H+-ATPase degradation products (17). MW
corresponds to molecular mass markers. The sizes in kDa are indicated
on the right. B, plasma membranes from the
strains expressing PMA2, PMA4, and the mutant PMA4-A129P (1.5 µg)
were analyzed by Western blotting, using antibodies against
H+-ATPase, phosphorylated threonine, or 14-3-3 proteins.
Quantification of phosphorylation status (filled bars) and
14-3-3 binding (open bars) is shown below
(n = 3). 100% corresponds to the signal obtained for
PMA2. The values (mean ± S.D.) are corrected for by
H+-ATPase levels.
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Fig. 2.
Alignment of C-terminal amino acid sequences
of PMA2 and PMA4 and schematic depiction of the four chimeras.
A, the last 110 residues of the C-terminal regions of PMA2
and PMA4 are aligned. Identical residues are shown as a dot.
The residues in squares represent the point mutations
resulting in H+-ATPase activation (9, 15, 16). The
circled residue is the phosphorylated Thr (17).
B, the C-terminal region of PMA2 schematically divided into
four regions. Two regions (residues 851-871 and 879-895) contain most
of the H+-ATPase-activating mutations (dark
gray), the third (residues 895-915) is not well conserved between
the two isoforms (medium gray), whereas the fourth (residues
915-956) (light gray) is highly conserved. C,
schematic diagram of the PMA chimeras used in this study. The junction
between PMA2 and PMA4 sequences is detailed above (two residues are
displayed for each isoform). The PMA4 region is represented by an
open rectangle. See text for detail.
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Fig. 3.
Growth of yeast strains expressing chimeric
PMA2-PMA4 H+-ATPases. Yeast cells expressing N-tagged
wild-type PMA2 or chimeras (PMA2+1), (PMA2+3/4), and
(PMA2+1/2) were grown on YGlu pH 6.5, replicated on solid YGlu
medium at the indicated pH, and grown at 30 °C for 4 days.
4) Determines Phosphorylation--
As shown in the
PMA2/PMA4 alignment (Fig. 2A), chimera (PMA2+
4,
position 953) appeared of interest as it was the only one that was
highly conserved within, but different between, the two plant H+-ATPase subfamilies: Ser (55%) or Ala (45%) in
subfamily I (here represented by PMA2) and His (87.5%) or Asn (12.5%)
in subfamily II (here represented by PMA4).
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Fig. 4.
Growth of yeast strains expressing mutated
(PMA2+
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Fig. 5.
Effect of mutation of residue C-4
in PMA2-E14D. A, the strains expressing PMA2-E14D and
PMA2-E14D/S953H were grown in YGlu medium pH 6.5 and replicated on
solid YGlu medium at pH 5.5. B, plasma membrane fractions
containing PMA2-E14D or PMA2-E14D/S953H (1.5 µg) were
electrophoresed, transferred on a nitrocellulose membrane and
immunodetected using antibodies against H+-ATPase,
phosphorylated Thr or 14-3-3 proteins. Quantification
(n = 3) of phosphorylation status (filled
bars) and 14-3-3 binding (open bars) is shown below.
100% corresponds to the signal obtained for PMA2-E14D. The values
(mean ± S.D.) are corrected for by H+-ATPase
levels.
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Fig. 6.
Effect of mutation of residue C-4
in PMA4. A, the strains expressing PMA4 and PMA4-H949S
were grown in YGlu medium and replicated on a solid YGlu medium at pH
6.0. B, plasma membrane fractions containing PMA4 and
PMA4-H949S (1.5 µg) were electrophoresed, transferred on a
nitrocellulose membrane, and immunodetected using antibodies against
H+-ATPase, phosphorylated Thr, or 14-3-3 proteins.
Quantification of phosphorylation status (filled bars) and
14-3-3 binding (open bars) is shown below (n = 3). 100% corresponds to the signal obtained for PMA4. The values
(mean ± S.D.) are corrected for by H+-ATPase levels.
C, specific ATPase activity was determined for the two
strains on four independent plasma membrane preparations.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
4) did,
was expected because we showed previously that PMA4 has a higher
intrinsic ATPase activity than PMA2 (9). To bypass the lethality of
PMA2-S953H, mutation of Ser-953 was obtained in an activated mutant
(PMA2-E14D). In this case, His at C
4 decreased
phosphorylation of Thr-955 and binding of 14-3-3 proteins, and reduced
yeast growth. Reciprocal results were obtained when His-949 of PMA4 was
mutated to Ser. We can therefore conclude that the differences observed
between the two subfamilies at residue C
4 are
functionally significant. As the position C
4 is only two
residues upstream from the phosphorylated Thr, the most direct
interpretation of these data is to consider that the Ser/His
substitution modifies the recognition site for kinase. A major
difference between Ala/Ser and His/Asn is that the latter are larger
and thus possibly interfere with kinase binding or catalysis.
Interestingly, another case has been reported recently (22) in which
the presence of either a Ser or Asn residue seems to be related to
different 14-3-3 binding properties. The barley lypoxygenase isoform
13-lox interacts with 14-3-3 proteins, whereas the 9-lox isoform does
not. Sequence analysis revealed a putative 14-3-3 binding site
(RKPSDSKP) in 13-lox, which differed by a single residue in 9-lox,
where the Ser two positions N-terminal to the putative phosphorylated
Ser in 13-Lox was replaced by Asn.
4 is the only one
showing a clear difference between subfamilies. We therefore propose
that the formation of the H+-ATPase/14-3-3 complex, which
depends on the phosphorylation of Thr at position C
2, is
differentially regulated between the two subfamilies, the consensus
being QQ(S/A)YpTV for subfamily I and QQ(H/N)YpTV for subfamily II.
4 (11). Although H+-ATPase genes have not
been cloned from spinach, we can suggest that these two peptides belong
to subfamilies I and II, respectively, and that both plant
H+-ATPase types can be phosphorylated and interact with
14-3-3 proteins. However, being a rather extreme approach, fusicoccin
is not a physiological regulator and is therefore not a good tool for
revealing possible differential regulation between
H+-ATPase subfamilies. The case might be different with
blue light activation, which, in guard cells from Vicia
faba, led to the identification of two H+-ATPase
isoforms, VHA1 and VHA2, that were phosphorylated in the C-terminal
region (25). In this case, both isoforms belong to subfamily II,
suggesting that only this subfamily was responding to this
environmental factor in this cell type.
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FOOTNOTES |
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* This work was supported in part by grants from the Belgian National Fund for Scientific Research, the European Commission (BIOTECH program), the Human Frontier Science Program, and the Interuniversity Poles of Attraction program of the Belgian Government Office for Scientific, Technical, and Cultural Affairs.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.
Recipient of a fellowship from the Belgian Fonds pour la Formation
à la Recherche dans l'Industrie et dans l'Agriculture.
§ To whom correspondence should be addressed. Tel.: 32-10-473621; Fax: 32-10-473872; E-mail: boutry@fysa.ucl.ac.be.
Published, JBC Papers in Press, November 15, 2000, DOI 10.1074/jbc.M007740200
2 M. Feuermann and M. Boutry, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: PMA, plasma membrane H+-ATPase; PCR, polymerase chain reaction; MOPS, 4-morpholinepropanesulfonic acid.
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REFERENCES |
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