Phosphatidylinositol synthesis and exchange of the inositol head
are catalysed by the single phosphatidylinositol synthase 1
from
Arabidopsis
Anne-Marie Justin, Jean-Claude Kader and Sylvie Collin
Universite
´
Pierre et Marie Curie and CNRS, Laboratoire de Physiologie Cellulaire et Mole
´
culaire, Paris, France
In order to study some of its enzymatic properties,
phosphatidylinositol synthase 1 (AtPIS1) from the plant
Arabidopsis thaliana was expressed in Escherichia coli, a host
naturally devoid of phosphatidylinositol (PtdIns). In the
context of the bacterial membrane and in addition to de novo
synthesis, the plant enzyme is capable of catalysing the
exchange of the inositol polar head for another inositol. Our
data clearly show that the CDP-diacylglycerol-independent
exchange reaction can occur using endogenous PtdIns
molecular species or PtdIns molecular species from soybean
added exogenously. Exchange has been observed in the
absence of cytidine monophosphate (CMP), but is greatly
enhanced in the presence of 4 l
M
CMP. Our data also show
that AtPIS1 catalyses the removal of the polar head in the
presence of much higher concentrations of CMP, in a
manner that suggests a reverse of synthesis. All of the PtdIns
metabolizing activities require free manganese ions. EDTA,
in the presence of low Mn
2+

synthesis of PtdIns as well as exchange of the inositol polar
head. In addition, we also suggest that placed in the
appropriate conditions, the enzyme is able to catalyse the
reaction reverse of synthesis. The substrates for exchange
can be PtdIns molecular species made by the enzyme or
exogenous molecular species differing in their fatty acid
content. The presence of a chelating agent of manganese,
which is indispensable for de novo synthesis and exchange
activities [1,6,11], had the same effect on both, suggesting
that the enzymatic active site used could be the same in both
cases.
MATERIALS AND METHODS
Genetic nomenclature
The cDNA used in this work corresponds to EMBL
accession number H36646 [11] and gene AtPIS1.
Growth conditions of the bacterial transformants
Escherichia coli cells expressing the AtPIS1 cDNA enco-
ding phosphatidylinositol synthase 1 from Arabidopsis
thaliana (AtPIS1) were obtained in the same way and are
the same as those described [11]. Two bacterial strains were
used, one expressing the plant cDNA (previously called
strain 2, now called strain +PIS), and the nonexpressing
control strain (previously called strain 3a and now called
strain –PIS). The cells were grown at 37 °CinLuria–
Bertani medium (Miller, Difco) supplemented with 1 m
M
myo-inositol and 100 lgÆmL
)1
ampicillin. In the case of
Correspondence to S. Collin, Universite

washed in 50 m
M
Tris/HCl pH 8.0 and stored as pellets at
)80 °C. These pellets were used for lipid analyses or as a
source of membrane proteins.
Enzymatic activities
Membrane purifications were carried out at 5 °C. E. coli
cells were resuspended at a density of 100 lg fresh
cellsÆmL
)1
in sonication buffer containing 50 m
M
Tris/
HCl pH 8.0, 8% (v/v) glycerol and 8 m
M
2-mercaptoeth-
anol. After sonication, intact cells were eliminated by
centrifugation at 4500 g for 10 min and washed once in
sonication buffer. The two supernatants were pooled and
centrifuged at 100 000 g for 1 h. The membrane pellet was
resuspended in 20 m
M
Tris/HCl pH 8.0, 20% (v/v) glycerol,
8m
M
2-mercaptoethanol, aliquoted and stored at )80 °C.
The protein concentration was determined according
to Lowry et al. [13] using BSA as standard.
De novo synthesis. Unless stated otherwise, the incubation
conditions for de novo PtdIns synthase activity were 50 m

incubating 50–200 lg membrane proteins in 50 m
M
Tris/
HCl pH 8.0, 0.36 m
M
PtdIns from soybean (Sigma),
0.5 m
M
myo-inositol containing tritium-labelled myo-inos-
itol for a final activity of 500 BqÆnmol
)1
,2.4m
M
Triton
X100, 2.5 m
M
MnCl
2
in the presence or in the absence of
4 l
M
cytidine monophosphate (CMP), but without CDP-
DAG. The incubation time at 30 °C was 20–30 min.
CMP-dependent PtdIns hydrolysis. This reaction was
followed by incubating 50–200 lg membrane proteins in
50 m
M
Tris/HCl pH 8.0, 3 m
M
CMP, 2.4 m

medium containing the above-mentioned concentrations of
Tris/HCl pH 8.0, Triton X100, MnCl
2
and EDTA were
added the appropriate missing components according to the
reaction studied. Each reaction was started by the addition
of membrane proteins.
Labelling of endogenous PtdIns molecular species
Microsomes from germinating soybean (Glycine max L.
cv. Weber) plantlets were prepared from 5 g seeds as
described [14]. They were resuspended in the same buffer
as E. coli membranes and stored at )80 °C. Membranes
(1.5 mg membrane proteins for E. coli, 3 mg membrane
proteins for soybean microsomes) were incubated in
50 m
M
Tris/HCl pH 8.0, 2.4 m
M
Triton X100, 5 m
M
EDTA, 0.1 m
M
cytidine triphosphate (CTP), 7.5 m
M
MnCl
2
and 2-
3
H(N)-myo-inositol (NEN, 0.46 MBq,
85 · 10

the reverse of synthesis, dependent on higher concentra-
tions of CMP [10]. As the exchange reaction has also been
suggested to occur in plants [7,8], we tested it using
isolated bacterial membranes as a source of enzyme. The
transformation of E. coli, a host naturally devoid of
PtdIns synthase with a single cDNA ensured that the
recombinant protein was the only candidate for the
activities tested.
E. coli cells expressing the AtPIS1 cDNA were cultivated
on M9 minimal medium supplemented with vitamin-free
amino acids to allow isolation of bacterial membranes
lacking endogenous PtdIns, which would interfere with
2348 A M. Justin et al. (Eur. J. Biochem. 269) Ó FEBS 2002
exogenous PtdIns. De novo synthesis was measured in the
incubation conditions described above, in the presence of
CDP-DAG and myo-inositol (Table 1).
The exchange reaction was assayed by incubating
bacterial membranes with
3
H-labelled free myo-inositol
and cold soybean PtdIns, no exogenous CDP-DAG, in
the presence or absence of 4 l
M
CMP. The PtdIns
concentration used was 0.36 m
M
as in Klezovitch et al.
[10]. At the end of the incubation period total lipids were
extracted and the amount of labelled PtdIns was
determined (Table 1). In the absence of CMP, the

)1
Æmg
)1
). On the other hand, mem-
branes lacking AtPIS1 do not induce any variation in the
radioactivity partitioned between each phase during the
incubation time, indicating that the appearance of
3
H-label
in the upper phase is specific to AtPIS1. Several 5¢-CMP
concentrations ranging between 0 and 6 m
M
were tested to
assess the CMP dependence of PtdIns hydrolysis. The
results (data not shown) do indicate that this is the case.
Nevertheless, the appearance of CDP-DAGs could not be
detected (data not shown). This protein-specific release and
CMP-dependent release of label from PtdIns could be
explained by a reaction which is the reverse of synthesis.
The exchange reaction studied by analysis
of radioactive PtdIns molecular species
The exchange reaction was also studied by analysis of the
radiolabelled PtdIns molecular species produced using
various PtdIns substrates (Fig. 1). This experiment is based
on the fact that the Ptdns molecular species synthesized in
E. coli from endogenous CDP-DAG are very different from
those found in plants, in particular soybean, whose PtdIns
composition has been described previously [14]. PtdIns
species made from endogenous CDP-DAG were first
Table 1. Enzymatic reactions catalysed by AtPIS1 expressed in E. coli.

Reverse reaction
Lower phase )108 ± 5 )1
Upper phase 103 ± 13 0
Exchange reaction
– CMP 125 ± 5 0
+4l
M
CMP 1109 ± 138 0
Fig. 1. Radio-HPLC separation of PtdIns molecular species synthes-
ized: (A) by AtPIS1 present in E. coli membranes incubated with CTP
and myo-inositol, but no exogenous CDP-DAGs; (B) by germinating
soybean microsomes, under the same substrate conditions as in (A);
(C) in exchange conditions in the presence of 4 l
M
CMP and soybean
PtdIns by membranes from E. coli +PIS grown on a medium
supplemented with myo-inositol or (D) lacking myo-inositol. The
incubation conditions are as described in Materials and methods.
Ó FEBS 2002 Arabidopsis phosphatidylinositol synthase 1 (Eur. J. Biochem. 269) 2349
studied by incubating membranes from E. coli +PIS
cultivated on medium lacking inositol or microsomes from
germinating soybean in the presence of CTP and
3
H-labelled inositol as in Justin et al. [14]. No exogenous
CDP-DAG was added so that PtdIns could only arise from
an endogenous production following the reactions: phos-
phatidic acid (PtdOH) + CTP fi CDP-DAG + PPi
(pyrophosphate) followed by CDP-DAG +
3
H-inosi-

in sufficient amount to give rise to detectable de novo
synthesized PtdIns molecular species. In Fig. 1C, the
labelled PtdIns of bacterial origin therefore arises from
an exchange reaction.
Effect of EDTA on the enzymatic activities
of PtdIns synthase
The net activity of PtdIns synthase as an exchange enzyme
stimulated by low concentrations of CMP is far from being
negligible when compared to de novo synthesis (Table 1).
For evaluation of net synthesis, one might wish to inhibit
the exchange and one laboratory reported, to this end, that
the addition of 5 m
M
EDTA was sufficient to abolish the
exchange ability of PtdIns synthase when incubated in
conditions of de novo synthesis [8]. In our own experiments,
we have observed that adding 5 m
M
EDTA leads to an
enhancement of inositol incorporation provided that the
concentration of manganese is correspondingly increased to
7.5 m
M
to compensate the chelating effect of EDTA
(unpublished results). We therefore investigated the effect
of EDTA on the reactions catalysed by PtdIns synthase in
the presence of 7.5 m
M
MnCl
2

)1
Æmin
)1
at 0 m
M
EDTA, increasing to 2 nmol PtdInsÆmg
)1
Æmin
)1
between
2.5 and 5 m
M
EDTA and a drop to 0 at 10 m
M
.
Abolition of all exchange activity at 10 m
M
EDTA shows
that the process requires manganese ions. On the other
hand, the CMP-independent activity was much lower,
with a value close to 0.15 nmol PtdInsÆmg
)1
Æmin
)1
at 0 m
M
EDTA which peaked
1
at 0.35 nmol PtdInsÆmg
)1

with no CMP added; m, CMP-dependent PtdIns hydrolysis. (B)
De novo synthesis (light grey), exchange activities (–CMP, striped bar;
+CMP, medium grey) and CMP-dependent PtdIns hydrolysis (dark
grey) normalized to their respective values obtained at 5 m
M
EDTA,
plotted as a function of EDTA concentration in the medium. (C)
Comparison of de novo synthesis activities obtained with different
concentrations of EDTA at constant manganese concentrations:
7.5 m
M
(d)or2.5m
M
(s). (D) Comparison of de novo synthesis
activities as a function of the calculated free Mn
2+
concentration
available in the medium at 7.5 m
M
MnCl
2
and different concentrations
of EDTA (black circles), 2.5 m
M
MnCl
2
and various amounts of
EDTA (s)ordifferentconcentrationsofMnCl
2
[11] (m). At 0 m

)1
could still be measured (Fig. 2C). When this activity was
tested at a lower MnCl
2
concentration with various amounts
of EDTA, the highest activity was not observed at 0 m
M
EDTA and 2.5 m
M
MnCl
2
as seen before [11], but at 2.5 m
M
EDTAwhentheconcentrationinfreemanganeseionsis
close to 0.1 l
M
. An alignment on the same plot of the values
found for de novo synthesis in the presence of 7.5 or 2.5 m
M
MnCl
2
with various amounts of EDTA, or in the presence of
MnCl
2
only [11], as a function of the concentration in free
manganese (Fig. 2D), shows that in the presence of EDTA
concentrations < 2.5 m
M
free Mn
2+

labelled inositol but no CDP-DAG and no PtdIns, no
labelled PtdIns appears, showing that the endogenous level
of CDP-DAG is too low to allow detectable synthesis. The
level of PtdIns in the membranes does not allow synthesis of
detectable PtdIns by exchange of the polar head. As in
Fig. 1D the only source of PtdIns molecular species
characteristic of E. coli that we detect can only come from
the bacterial membrane, we explain this result by a global
stimulation of the exchange reaction by the exogenous
soybean PtdIns.
A question that arises is the relevance of the exchange
of polar head when de novo synthesis is studied. We
calculated that with a linear rate of synthesis of 2 nmol
PtdInsÆmin
)1
Æmg
)1
(Fig. 2), the concentration of CMP in a
reaction mixture of 200 lLis4l
M
after 1 min. with 50 lg
protein. As our incubation conditions use 50 lg protein
incubated for 20 min, although the corresponding PtdIns
concentration does not reach the value used to study
exchange, it is possible that this latter reaction takes place,
possibly interfering with the net de novo synthesis capacity
of PtdIns synthase, especially if the membranes used as a
source of enzyme are rich in PtdIns.
Another question is the reason why CMP stimulates the
exchange reaction and what the exact mechanism for

synthesis is by a different mechanism from base exchange
[20]. Further analysis of the products released from PtdIns
by the Arabidopsis enzyme will allow us to carry out a
deeper study of this particular activity of the protein.
Effect of EDTA on PtdIns synthase reactions
Our data show that at free manganase ion concentrations
far lower than those found to be the optimum when
manganese is used on its own, there are still de novo
synthesis and exchange reactions the activities of which are
close to those seen when no chelating agent is used. This
effect has been studied particularly in the case of de novo
synthesis, where a comparison between data presented here
and data published previously by us clearly show that
EDTA enhances the activity. The pH remained unchanged
between each condition of the EDTA plot, which rules out a
simple pH effect to explain the variations in enzymatic
activity. One possibility therefore is that EDTA could
chelate inhibitory ions, whose identity remains unclear,
maybe with a higher affinity than for Mn
2+
.
If it is clear, for the first time, that a PtdIns synthase from
Arabidopsis is able to catalyse both de novo synthesis of
PtdIns and the exchange of the inositol moiety, but it is still
uncertain by what mechanism this exchange is carried out.
In terms of kinetics, the enzymatic conditions used do not
Ó FEBS 2002 Arabidopsis phosphatidylinositol synthase 1 (Eur. J. Biochem. 269) 2351
favour a reverse reaction followed by re-synthesis. Never-
theless, EDTA seems to have the same effect on both
synthesis and exchange, so further characterization of each

Mixte de Recherche 7632).
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