Tài liệu Báo cáo khoa học: Characterization of the interaction between the plasma membrane H+-ATPase of Arabidopsis thaliana and a novel interactor (PPI1) doc

Characterization of the interaction between the plasma
membrane H
+
-ATPase of Arabidopsis thaliana and a novel
interactor (PPI1)
Corrado Viotti, Laura Luoni, Piero Morandini and Maria Ida De Michelis
Dipartimento di Biologia ‘L. Gorini’, Universita
`
di Milano, CNR Istituto di Biofisica – Sezione di Milano, Italy
The H
+
-ATPase is the major electrogenic pump in the
plasma membrane (PM) of plant cells. By pumping
protons from the cytoplasm to the apoplast it gener-
ates an electrochemical proton gradient, which drives
the transport of mineral ions and organic solutes, and
plays a crucial role in cytoplasmic and apoplastic pH
homeostasis [1,2]. The PM H
+
-ATPase participates in
a variety of physiological processes such as phloem
loading, stomata opening, mineral nutrition, growth of
root hairs and pollen tubes, salt and osmotolerance,
leaf movements, and acid growth [1,2]. In vivo , its
activity is modulated by several signals such as hor-
mones (auxin, abscisic acid), light, water potential,
acid load, toxins like fusicoccin (FC) and pathogens,
but a molecular description of the mediators involved
is missing for most of these signals [1,2].
Plant genomes contain a large family of PM H
+

-
ATPase genes (12 in Arabidopsis thaliana, 10 in rice
and nine in Nicotiana plumbaginifolia), which can be
grouped in five clusters based on sequence alignments
and intron positions [3,4]. Individual isoforms exhibit
tissue- and developmental-specific expression patterns
and a number of quantitative differences in catalytic
and regulatory properties [1–4]. Thus, the first regula-
tion of proton pumping activity in different cells types
and physiological conditions takes place at both the
transcriptional and translational levels [1–4].
As to post-translational regulation, the best-known
mechanism described to date involves the auto-inhibi-
tory action of the C-terminal domain. The plant PM
H
+
-ATPase is a P-type ATPase with an extended
(approximately 100 amino acids) cytosolic C-terminus
containing two inhibitory regions. Proteolytic cleavage
or genetic deletion of the C-terminus has little effect
on enzyme activity at the acidic pH optimum (pH 6.4–
6.6), but markedly increases enzyme activity in the
physiological range of cytoplasmic pH values (pH 7.0–
7.5), resulting in an alkaline shift of the pH optimum
Keywords
Arabidopsis thaliana;H
+
-ATPase; plasma
membrane; PPI1; 14-3-3 proteins
Correspondence

M. I. De Michelis, Dipartimento di Biologia
‘L. Gorini’, Universita
`
di Milano, CNR Istituto
di Biofisica – Sezione di Milano, via G.
Celoria 26, 20133 Milano, Italy
Fax: +39 02 50314815
Tel: +39 02 50314822
E-mail:
(Received 28 July 2005, revised 13 September
2005, accepted 20 September 2005)
doi:10.1111/j.1742-4658.2005.04985.x
Proton pump interactor, isoform 1 (PPI1) is a novel interactor of the C-ter-
minus of Arabidopsis thaliana plasma membrane H
+
-ATPase (EC 3.6.3.6)
(Morandini P, Valera M, Albumi C, Bonza MC, Giacometti S, Ravera G,
Murgia I, Soave C & De Michelis MI (2002) Plant J 31, 487–497). We pro-
duced two fusion proteins consisting of, respectively, the first 88 amino
acids or the entire protein deleted of the last 24 hydrophobic amino acids,
and we show that the latter protein has a threefold higher affinity for the
H
+
-ATPase. PPI1-induced stimulation of H
+
-ATPase activity dramatically
decreased with the increase of pH above pH 6.8, but became largely
pH-independent when the enzyme C-terminus was displaced by fusicoccin-
induced binding of 14-3-3 proteins. The latter treatment did not affect
PPI1 affinity for the H

+
-ATPase. These results indicate that PPI1 can bind
the H
+
-ATPase independently of the C-terminus conformation, but is not
able to suppress the C-terminus auto-inhibitory action.
Abbreviations
Brij 58, polyoxyethilene 20 cethyl ether; BTP, bis tris propane {1,3-bis[tris(hydroxymethyl)methylamino]propane}; FC, fusicoccin; GST,
glutathione S-transferase; IPTG, isopropyl thio-b-
D-galactoside; NTA, nitrilotriacetic acid; PM, plasma membrane.
5864 FEBS Journal 272 (2005) 5864–5871 ª 2005 FEBS
([1] and references therein). The auto-inhibitory action
of the C-terminus is suppressed, besides by pH, by
lysophospholipids and by 14-3-3 proteins ([1] and ref-
erences therein). The latter are regulatory proteins
present in all eukaryotic systems which modulate the
activity of a number of target proteins, generally bind-
ing to sequence motifs including a phosphorylated Ser
or Thr residue [5–8]. Phosphorylation of the highly
conserved penultimate Thr residue of the PM H
+
-ATP-
ase results in binding of 14-3-3 protein. 14-3-3 binding
is stabilized by the fungal toxin FC which decreases
the dissociation rate, thus inducing the formation of
an almost irreversible complex in which the enzyme is
locked in the same active conformation determined by
cleavage of the C-terminus [1,9–16]. Also, blue-light
activation of PM H
+

-ATPase in guard cells of broad
beans involves protein kinase-mediated phosphoryla-
tion of Ser and Thr residues in the C-terminus of the
pump and 14-3-3 binding [17]. Much less is known
about in vitro and in vivo activation by other effectors:
an increase of PM-associated 14-3-3s has been
observed also in response to cold or osmotic stress,
and their binding to the H
+
-ATPase is suggested by
the parallel increase of the number of FC-binding
sites [18–20]. As to auxin, a soluble auxin receptor has
been reported to bind and activate the PM H
+
-ATPase,
but the site of binding has not been identified so
far [21].
A novel interactor of the PM H
+
-ATPase C-termi-
nus was identified in a two-hybrid screening. The novel
protein, named PPI1 (proton pump interactor, isoform
1), is a 612 amino acids protein rich in charged and
polar residues, except for the extreme C-terminus
where it presents a hydrophobic stretch of 24 amino
acids forming a putative transmembrane domain. PPI1
does not resemble any protein of known function, but
it is probably the first identified member of a new fam-
ily of plant regulatory proteins, as at least five A. thali-
ana genes and many expressed sequence tags (ESTs)

from different plant species encode proteins with signi-
ficant similarity to PPI1 [22].
The N-terminal domain of PPI1, originally identified
by the two-hybrid technique, binds A. thaliana PM
H
+
-ATPase in overlay experiments and stimulates
enzyme activity. The interaction is not suppressed by
controlled tryptic cleavage of the enzyme, indicating
that the PPI1 binding site in the H
+
-ATPase C-termi-
nus is localized upstream of the main tryptic cleavage
site and thus is different from the 14-3-3 binding
site. Moreover, PPI1 further enhances FC-stimulated
H
+
-ATPase activity [22].
Here we report a characterization of the interaction
of PPI1 with the H
+
-ATPase in PM isolated from
control and FC-treated A. thaliana cultured cells,
which indicates that PPI1 is unable to suppress the
auto-inhibitory action of the enzyme C-terminus, but
further enhances the activity of the enzyme whose
C-terminus has been displaced by low pH or by
FC-induced binding of 14-3-3s.
Results
The C-terminus of isoform 1 of the PM H

+
-ATPase
of A. thaliana (AHA1) interacts with the first 88 amino
acids of PPI1 [22], indicating that the PM H
+
-ATPase
binding site of PPI1 is localized therein. Indeed, fusion
proteins containing the first 88 amino acids of PPI1,
linked either to a His-tag (His
6
PPI1
88
) or to GST,
interact with A. thaliana H
+
-ATPase in the PM and
stimulate its activity [22]. However, other parts of the
protein may be important for regulation of the interac-
tion. As the entire PPI1 protein was difficult to handle
due to low solubility (unpublished results from the
authors’ laboratory), we expressed in Escherichia coli
a truncated protein devoid of the hydrophobic C-tail,
with a His
6
-tag at the C-terminal end, far away from
the interaction site (PPI1
588
His
6
). The fusion protein

was purified by Ni-NTA affinity chromatography and
its ability to interact with the H
+
-ATPase C-terminus
was tested against another fusion protein harboring the
last 104 amino acids of AHA1 fused to GST, GST–
AHA1(846–949). Figure 1 shows that PPI1
588
His
6
and
GST–AHA1(846–949) bound each other in overlay
experiments both when a membrane spotted with
GST–AHA1(846–949) was incubated with PPI1
588
His
6
(Fig. 1A) and when PPI1
588
His
6
was spotted and the
AB
Fig. 1. Interaction between PPI1
588
His
6
and the C-terminus of
A. thaliana PM H
+

-ATPase (AHA1). The indicated proteins were
spotted and incubated with 1 l
M PPI1
588
His
6
(A) or 1 lM GST–
AHA(1846–948) (B) as described in Experimental procedures. Inter-
action was detected by immunodecoration with antisera against
the N-terminus of PPI1 (A) or the C-terminus of the H
+
-ATPase (B).
His
6
–ACA8(1–116) reproduces the N-terminus of an A. thaliana PM
Ca
2+
-ATPase [31]. Results are from one experiment, representative
of three giving similar results.
C. Viotti et al. Interaction between plasma membrane H
+
-ATPase and PPI1
FEBS Journal 272 (2005) 5864–5871 ª 2005 FEBS 5865
membrane incubated with GST–AHA1(846–949)
(Fig. 1B); the signals were specific as no signal was
detected when free GST was spotted and the membrane
incubated with PPI1
588
His
6

(Fig. 1A) or when an un-
related His-tagged protein was spotted and the mem-
brane incubated with GST–AHA1(846–949) (Fig. 1B).
The ability of PPI1
588
His
6
to activate the H
+
-ATP-
ase in PM isolated from cultured A. thaliana cells was
compared to that of His
6
PPI1
88
. Figure 2 shows that
both proteins stimulated the H
+
-ATPase activity
assayed at pH 6.4 in a concentration-dependent man-
ner, but PPI1
588
His
6
was about threefold more effect-
ive than His
6
PPI1
88
. The k

0.5
values evaluated from
five independent experiments were 0.4 ± 0.1 lm for
PPI1
588
His
6
and 1.7 ± 0.2 lm for His
6
PPI1
88
. Thus,
all the following experiments were performed with
PPI1
588
His
6
.
The analysis of the effect of PPI1
588
His
6
on the
dependence of PM H
+
-ATPase activity on the concen-
tration of MgATP (Fig. 3) showed that stimulation
decreased with the increase of PPI1
588
His

6
concentra-
tion. Consequently, PPI1
588
His
6
only slightly increased
V
max
but substantially lowered the apparent K
m
for
MgATP.
Activation of the PM H
+
-ATPase by cleavage or by
displacement of the auto-inhibitory C-terminal domain
is strongly pH-dependent, increasing with the increase
of pH beyond the relatively acidic pH optimum of
enzyme activity [1,23–26]. The dependence of H
+
-ATP-
ase activation by PPI1
588
His
6
on the pH of the assay
medium is completely different: Fig. 4 shows that the
effect of PPI1
588

His
6
on H
+
-ATPase activity was very
high at pH 6.0, but decreased with the increase of pH,
virtually disappearing above pH 7.0. As a conse-
quence, the pH optimum for enzyme activity is slightly
more acidic in the presence of PPI1
588
His
6
than in its
absence.
A completely different picture emerged when the
effect of PPI1
588
His
6
on H
+
-ATPase activity was
assayed in PM isolated from FC-treated cells. FC
determines a stable association of 14-3-3 proteins to
the C-terminus of the H
+
-ATPase, locking the enzyme
in an active conformation [9,10,12–16]. Consequently
(Fig. 5), enzyme activity stayed high throughout the
pH range examined (up to pH 7.1). Addition of

PPI1
588
His
6
further enhanced the H
+
-ATPase activity
in a pH-independent manner.
The different conformation of the enzyme C-terminus
in PM isolated from control or FC-treated cells may
alter the accessibility of PPI1
588
His
6
. To test this possi-
bility, we analyzed PPI1
588
His
6
-induced activation of
Fig. 2. Stimulation of A. thaliana PM H
+
-ATPase activity as a func-
tion of the concentration of His
6
PPI1
88
and PPI1
588
His

6
. PM treat-
ment with the specified concentrations of His
6
PPI1
88
(closed
triangles) or PPI1
588
His
6
(open triangles) and H
+
-ATPase activity
assays were performed at pH 6.4. Results are given as percentage
stimulation of H
+
-ATPase activity which in the absence of PPI1
was 665 nmolÆmin
)1
Æmg protein
)1
. Results are from one experi-
ment, representative of five giving similar results.
Fig. 3. Effect of PPI1
588
His
6
on the dependence of PM H
+

-ATPase
activity on the concentration of MgATP. PM treatment with (open
symbols) or without (closed symbols) 2 l
M PPI1
588
His
6
and H
+
-ATP-
ase activity assays (pH 6.4) were performed as described in Experi-
mental procedures, except that ATP concentration was varied
between 0.1 and 2 m
M, as indicated, in the presence of a constant
excess of 2 m
M MgSO
4
. Results are from one experiment, repre-
sentative of three giving similar results. The mean V
max
and ap-
parent K
m
values were, respectively, 1.20 ± 0.04 lmolÆmin
)1
Æmg
protein
)1
and 0.35 ± 0.05 mM in the absence and 1.37 ± 0.06
lmolÆmin

)1
Æmg protein
)1
and 0.13 ± 0.02 mM in the presence of
PPI1
588
His
6
.
Interaction between plasma membrane H
+
-ATPase and PPI1 C. Viotti et al.
5866 FEBS Journal 272 (2005) 5864–5871 ª 2005 FEBS
the H
+
-ATPase in the two PM fractions as a function
of PPI1
588
His
6
concentration. Assays were performed at
pH 7.0 to ensure at the same time effective auto-inhibi-
tion and reliable measurements of PPI1 effect in control
PM. The results reported in Fig. 6 show that stimulation
of the H
+
-ATPase activity in PM isolated from control
or FC-treated cells similarly increased with the increase
of PPI1
588

His
6
concentration; the k
0.5
values evaluated
from three independent experiments were 0.24 ± 0.06
and 0.19 ± 0.02 lm, respectively, for control PM and
PM from FC-treated cells.
Discussion
PPI1 is a modulator of the plasma membrane H
+
-
ATPase, which binds the enzyme C-terminus and stimu-
lates its activity [22]. The available preliminary evidence
indicates that its mechanism of action is different from
that of 14-3-3 proteins, the best known modulators of
the autoinhibitory action of the enzyme C-terminus
([1,22] and references therein), proposing PPI1 as a
novel mechanism of regulation which could play an
important role in the subtle modulation of proton extru-
sion in response to endogenous or exogenous signals.
The two-hybrid screen for interactors of the C-termi-
nus of AHA1 led to the isolation of a cDNA fragment
encoding the first 88 amino acids of PPI1 [22]. This
result, together with the finding that fusion proteins
containing the first 88 amino acids of PPI1 linked to
an His-tag (His
6
PPI1
88

) or to GST interact with
A. thaliana H
+
-ATPase in the PM and stimulate its
activity [22], suggested that the site of interaction with
the PM H
+
-ATPase was localized in the N-terminus
of PPI1. To further characterize the biological activity
Fig. 4. pH dependence of the activation of A. thaliana PM H
+
-ATP-
ase by PPI1
588
His
6
. PM treatment with (open symbols) or without
(closed symbols) 2 l
M PPI1
588
His
6
and H
+
-ATPase activity assays
were performed at the specified pHs. Results are from one experi-
ment, representative of three giving similar results.
Fig. 5. pH dependence of the activation of A. thaliana H
+
-ATPase in

PM purified from FC-treated cultured cells by PPI1
588
His
6
.PM
treatment with (open symbols) or without (closed symbols) 2 l
M
PPI1
588
His
6
and H
+
-ATPase activity assays were performed at the
specified pHs. Results are from one experiment, representative of
three giving similar results.
Fig. 6. Dependence on the concentration of PPI1
588
His
6
of the sti-
mulation of H
+
-ATPase activity in PM purified from control and
FC-treated cultured cells. Assays were performed at pH 7.0.
Results are given as percentage stimulation of H
+
-ATPase activity
which in the absence of PPI1 was 261 (control, open triangles) and
507 (FC-treated, closed triangles) nmolÆmin

)1
Æmg protein
)1
. Results
are from one experiment, representative of three giving similar
results.
C. Viotti et al. Interaction between plasma membrane H
+
-ATPase and PPI1
FEBS Journal 272 (2005) 5864–5871 ª 2005 FEBS 5867
of PPI1 we produced a new fusion protein, containing
the PPI1 protein devoid only of the last 24 amino
acids, a putative transmembrane domain (PPI1
588
His
6
);
the His-tag was fused to the protein C-terminus, to
minimize its effects on the conformation of the protein
N-terminus. The results reported in this paper show
that this fusion protein has an affinity for the H
+
-
ATPase threefold higher than that of His
6
PPI1
88
. This
result suggests that residues downstream of the first 88
amino acids of PPI1 may participate in the interaction

with the H
+
-ATPase and makes PPI1
588
His
6
a suitable
tool to study the mechanism of action of PPI1.
The analysis of the pH dependence of PPI1-
induced activation of the H
+
-ATPase showed that
stimulation decreases dramatically with the increase
of pH above pH 6.8; PPI1-induced activation of the
H
+
-ATPase becomes pH-independent in PM isolated
from FC-treated cells. At pH values above the opti-
mum for H
+
-ATPase activity, the C-terminus exerts
its auto-inhibitory action, presumably by binding to
an intramolecular site [1,23–27]; thus, it might ham-
per the access of PPI1
588
His
6
. FC-induced binding of
14-3-3 displaces the C-terminus [1,9–16] and thus
might facilitate the binding of PPI1. However, the

k
0.5
value for the PPI1–H
+
-ATPase interaction at
pH 7.0 was at least as low as at pH 6.4 and not
affected by FC-induced 14-3-3 binding, indicating
that the affinity of the H
+
-ATPase for PPI1
588
His
6
is not altered by the conformation of the C-termi-
nus. These results indicate that (Fig. 7) PPI1, in
response to an as yet unidentified signal, can interact
with the PM H
+
-ATPase independently from its
activation state, but is not able to suppress the auto-
inhibitory action of the C-terminal domain. PPI1 can
only hyper-activate H
+
-ATPase molecules whose
C-terminus has been displaced by other factors such
as low pH or 14-3-3 proteins.
Experimental procedures
Strains, media and general techniques
Escherichia coli XL10 (Stratagene, La Jolla, CA, USA) was
used for recombinant DNA work while BL21(DE3)pLysS

(Novagen, Madison, WI, USA) and BL21(DE3) Codon
plus
TM
pRil strains (Stratagene) were employed as hosts for
protein expression. All strains were grown in Lennox broth
base (Gibco BRL, Rockville, MD, USA).
Bacterial transformation was according to the protocol
of [28]. Soluble proteins were assayed with the Bio-Rad
protein assay (Bio-Rad, Hercules, CA, USA) with c-glob-
ulin as a standard, while membrane proteins were
assayed according to [29] with bovine serum albumin as a
standard.
Plasmid construction
A DNA fragment coding for the first 588 amino acids of
PPI1 protein was amplified from clones isolated previously
[22] using the following primers: gatggatcccatATGGGTG
TTGAAGTTGTA annealing around the start codon of the
Ppi1 ORF and gactcgagATTAGTCGACTTCTTACGC
annealing just before the putative transmembrane domain
(capital letter in the sequence represent nucleotides match-
ing target sequence). The PCR product was cloned into
pET-32b plasmid (Novagen) deleted of the thioredoxin
gene, using NdeI and XhoI restriction sites. The resulting
plasmid was transferred into E. coli strain XL10 and the
frame and the identity of the cloned fragment verified by
sequencing. The construct with the N-terminal portion of
PPI1 has been previously described [22].
The DNA fragment coding for the last 104 amino acids
(Ser846–Val949) of AHA1 was amplified from EST clones
49E5 from Arabidopsis Biological Resource Center (ABRC,

Ohio State University, OH, USA) using the following prim-
ers: ggatcccatatgAGCGGAAAGGCGTGG and ggatcctca
CACAGTGTAGTGA. The PCR product was cloned into
pGEX-2TK vector for fusion to GST, using the BamHI
restriction site. The frame and identity of the PCR product
were checked by sequencing.
Protein expression and purification
The plasmid encoding the PPI1 protein truncated of its ter-
minal 24 hydrophobic amino acids with a His
6
-tag at its
C-terminus (PPI1
588
His
6
) was transformed into BL21(DE3)
Fig. 7. Schematic model of the mechanism of action of PPI1 on
the PM H
+
-ATPase.
Interaction between plasma membrane H
+
-ATPase and PPI1 C. Viotti et al.
5868 FEBS Journal 272 (2005) 5864–5871 ª 2005 FEBS
Codon plus
TM
pRil strain (Stratagene) and its expression
was induced in liquid cultures at 37 °C (0.6–0.7 D
595
) with

1mm isopropyl thio-b-d-galactoside (IPTG). After 1 h of
induction, cells were cooled on ice, centrifuged and stored
at )80 °C. Cells pellets were lysed in the presence of 0.4%
N-lauroyl sarcosine [30] and sonicated until a clear, non
viscous solution was obtained. Particulate matters were
removed by centrifugation (15 min at 12 000 g) and the sol-
uble fraction loaded onto a Ni
2+
-NTA agarose affinity col-
umn. Protein was purified essentially as described by the
Ni-NTA supplier (Qiagen, Milan, Italy). Eluted fractions
were monitored by SDS ⁄ PAGE, pooled and concentrated
by centrifugation with Vivaspin 15R (cut-off 30 kDa; Viva-
science AG, Hannover, Germany). Imidazole was removed
by repeated cycles of concentration-dilution with 1 mm
BTP-Hepes pH 8.0, glycerol 10% (w ⁄ v) Brij 58 (Aldrich,
Milwaukee, WI, USA) was added to the sample
(0.1 mgÆmL
)1
) in the first concentration cycle.
The expression of the N-terminal portion of the pro-
tein (His
6
PPI1
88
) was done in the same conditions of
PPI1
588
His
6

, but with 3 h of induction. Protein was purified
essentially as described by the Ni-NTA supplier (Qiagen).
Eluted fractions were monitored by SDS ⁄ PAGE, pooled
and concentrated by centrifugation with Vivaspin 6 (cut-off
5 kDa; Vivascience AG). Imidazole was removed as des-
cribed above.
The C-terminus of AHA1 fused to GST, GST–
AHA(1846–949) was expressed in E. coli strain BL21(DE3)-
Codon plus
TM
pRIL (Stratagene). Cells were grown at
37 °C until D
595
of 0.6 was reached, then 1 mm IPTG was
added and the culture grown for 2 h. GST–AHA(1846–949)
was purified by affinity chromatography on Glutathione
Sepharose 4B gel (Amersham Biosciences, Piscataway, NJ,
USA). The purification procedure was performed under
native conditions as described in the manufacturer instruc-
tions except for the addition of 0.1% (w ⁄ v) lysozyme and
0.5% (v ⁄ v) Triton X-100 during cell lysis.
His
6
-ACA8
1)116
was produced as described by Luoni
et al. [31].
Plant material and isolation of PM vesicles
Cell suspension cultures of A. thaliana ecotype Landsberg
were grown as described in [32]. In vivo treatment with FC

was performed for 120 min by adding the phytotoxin to the
culture medium at the final concentration of 10 lm. Cells
were harvested by a double centrifugation at 1000 g for
5 min; highly purified PM fractions were obtained by a
two-step aqueous two-phase partitioning system as des-
cribed in Olivari et al. [15].
Overlay experiments
The interaction between PPI1
588
His
6
and the C-terminus of
the PM H
+
-ATPase was tested both by incubating with
PPI1
588
His
6
a membrane on which GST–AHA(1846–949)
(5 lm) had been spotted, and, vice versa, by incubating with
GST–AHA(1846–949) a membrane on which PPI1
588
His
6
(3 lm) had been spotted. Fusion proteins were spotted
(2 lL of each) onto 0.2 lm nitrocellulose and incubated for
3 h at room temperature in blocking solution [1% (w ⁄ v)
bovine serum albumin (BSA), 0.2 mm EGTA, 50 mm
KNO

3
,2mm MgSO
4
,5mm (NH
4
)
2
SO
4
, 0.1 mm ammo-
nium molybdate, 40 mm BTP ⁄ Mes pH 6.4]. Membranes on
which GST–AHA(1846–949) was spotted were incubated
for 2 h at room temperature in the same blocking solution
with the addition of 1 lm PPI1
588
His
6
and interaction was
detected by immunodecoration with antiserum against the
N-terminus of PPI1. Membranes on which PPI1
588
His
6
was
spotted were incubated for 2 h at room temperature in the
same blocking solution with the addition of 1 lm GST–
AHA(1846–949) and interaction was detected by immuno-
decoration with antiserum against the C-terminus of the
H
+

-ATPase. The antiserum against the N-terminus of PPI1
was raised in rabbit using His
6
PPI1
88
as antigene. Immuno-
decoration was performed by incubating the membrane for
2 h at room temperature with the antiserum diluted
1 : 1000 in 20 mMTris ⁄ HCl pH 7.4, 150 mm NaCl, 3%
(w ⁄ v) BSA and 0.1% (v ⁄ v) Tween20. The antiserum against
the C-terminus of the H
+
-ATPase was raised in rabbit
using as antigene the highly conserved sequence Arg912–
Tyr943 of A. thaliana proton pump isoform 2 (AHA2) con-
jugated to ovalbumin. Immunodecoration was performed
by incubating the membrane for 2 h at room temperature
with the antiserum diluted 1 : 1000 in 20 mMTris ⁄ HCl
pH 7.4, 150 mm NaCl, 3% (w ⁄ v) BSA and 0.1% (v ⁄ v)
Tween20. After several washes, signal detection was per-
formed with an ECL anti-rabbit IgG linked to horseradish
peroxidase (Amersham Biosciences) diluted 1 : 5000 in the
same solution reported above.
PM H
+
-ATPase activity
Unless otherwise specified, PM H
+
-ATPase activity was
assayed in 0.2 mm EGTA, 50 mm KNO

3
, 2.3 mm MgSO
4
,
5mm (NH
4
)
2
SO
4
, 0.1 mm ammonium molybdate,
1 lgÆmL
)1
oligomycin, 100 lgÆmL
)1
Brij 58, 5 lm carbonyl
cyanide p-trifluromethoxy-phenylhydrazone, buffered with
40 mm BTP-Mes (pH 6.4–6.8) or BTP-Hepes (pH 7–7.3), 2
unitsÆmL
)1
pyruvate kinase, 2 mm phosphoenolpyruvate
and 0.3 mm ATP.
Plasma membranes (0.5–1 lg protein) were incubated at
0 °C for 15 min with or without the specified PPI1 fusion
proteins in 90 lL of assay medium in absence of ATP,
pyruvate kinase and phosphoenolpyruvate; all samples con-
tained the same volume of 1 mm BTP-Hepes pH 8.0, 10%
glycerol. The volume was then adjusted to 100 lL with
assay medium containing ATP, pyruvate kinase and phos-
phoenolpyruvate and the reaction was carried out for

60 min at 30 °C. Released P
i
was determined as described
C. Viotti et al. Interaction between plasma membrane H
+
-ATPase and PPI1
FEBS Journal 272 (2005) 5864–5871 ª 2005 FEBS 5869
in De Michelis and Spanswick [33]. The PM H
+
-ATPase
activity was evaluated as the difference between total activ-
ity and that measured in the presence of 100 lm vanadate
(less than 10% of total activity at pH 7; less than 5% of
total activity at pH 6.4). Reported data are the results from
one experiment with three replicates representative of at
least three experiments; SE of the assays did not exceed 3%
of the measured value.
Acknowledgements
This project was supported by the Italian Ministry for
Instruction, University and Research in the FIRB 2001
frame.
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