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
+

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: [email protected]
(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 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)

+
-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

-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

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

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

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
+

)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

) 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

)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

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

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

, 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

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
+

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

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C. Viotti et al. Interaction between plasma membrane H
+
-ATPase and PPI1
FEBS Journal 272 (2005) 5864–5871 ª 2005 FEBS 5871