Phosphatidylinositol synthesis and exchange of the inositol head are catalysed by the single phosphatidylinositol synthase 1 Anne-Marie Justin, Jean-Claude Kader and Sylvie Collin Univer
Trang 1Phosphatidylinositol synthesis and exchange of the inositol head are catalysed by the single phosphatidylinositol synthase 1
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 thalianawas 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 catalysingthe
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 lMCMP 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 metabolizingactivities require free manganese ions EDTA,
in the presence of low Mn2+concentrations, also has an enhancingeffect
Keywords: Arabidopsis; exchange reaction; phosphatidy-linositol; phospholipid synthesis; reverse reaction; synthase
Phosphatidylinositol (PtdIns) labellinghas longbeen
known to occur via two possible mechanisms: a de novo
synthesis, catalysed by phosphatidylinositol synthase (EC
2.7.8.11), also known as CDP-diacylglycerol
(DAG)/myo-inositol 3-phosphatidyltransferase, and an exchange
reac-tion whereby the sugar head is exchanged between
pre-existingPtdIns molecules and free inositol, leadingin
a test tube and in the absence of CDP-diacylglycerol
(CDP-DAG) to the synthesis of labelled PtdIns when radioactive
inositol is used [1] In animal [2–6] as well as in plant tissues
[7,8] and in the green algae Chlamydomonas reinhardtii [9],
this exchange reaction has been associated to the
endoplas-mic reticulum (ER) or to endoplas-microsomal fractions rich in ER,
where de novo synthesis of PtdIns is most active Thorough
enzymatic characterization of synthesis and exchange has
attempted to understand if both reactions are mediated by
the same enzyme, but only one study has shown that one
gene product, phosphatidylinositol synthase from the yeast
Saccharomyces cerevisiae, carries both activities [10]
In plants, for 20 years, the lack of cloned sequences had
prevented the investigations necessary to determine whether
the situation is the same as in yeast; however, the cloning and expression of a cDNA encodingPtdIns synthase in Arabidopsis thaliana[11] has enabled us to do so The results presented here clearly show that AtPIS1 is able, when expressed in a bacterial system, to catalyse both de novo 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 chelatingagent 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
M A T E R I A L S A N D M E T H O D S 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 expressingthe AtPIS1 cDNA enco-dingphosphatidylinositol 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 expressingthe 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 37C in Luria– Bertani medium (Miller, Difco) supplemented with 1 mM
myo-inositol and 100 lgÆmL)1 ampicillin In the case of
Correspondence to S Collin, Universite´ Pierre et Marie Curie,
Laboratoire de Physiologie Cellulaire et Mole´culaire, UMR 7632
CNRS, Paris 6, Tour 53, 4etage, Case Courrier 154, 4, Place Jussieu,
75252 Paris Cedex 05, France.
Fax: + 33 1 44 27 61 51, Tel.: + 33 1 44 27 59 13,
E-mail: scollin@snv.jussieu.fr
Abbreviations: PtdIns, phosphatidylinositol; PtdOH, phosphatidic
acid; CDP-DAG, CDP-diacylglycerol; CMP, cytidine
monophosphate; CTP, cytidine triphosphate.
Enzymes: phosphatidylinositol synthase or CDP-diacylglycerol/
myo-inositol 3-phosphatidyltransferase (EC 2.7.8.11).
(Received 4 October 2001, revised 13 March 2002,
accepted 20 March 2002)
Trang 2experiments requiringbacterial membranes devoid of
PtdIns, the cells were grown in M9 minimal medium [12]
supplemented with 2 gÆL)1vitamin-free casein (Sigma) and
100 lgÆmL)1 ampicillin The water used was ultrapure,
filtered on a Millipak Filter Unit 0.22 lm rated (Millipore)
Expression of the cDNA was induced in fresh
transform-ants as described previously [11] before the cells were
washed in 50 mMTris/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 5C E coli
cells were resuspended at a density of 100 lgfresh
cellsÆmL)1 in sonication buffer containing50 mM Tris/
HCl pH 8.0, 8% (v/v) glycerol and 8 mM
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 mMTris/HCl pH 8.0, 20% (v/v) glycerol,
8 mM2-mercaptoethanol, aliquoted and stored at)80 C
The protein concentration was determined according
to Lowry et al [13] usingBSA as standard
De novo synthesis Unless stated otherwise, the incubation
conditions for de novo PtdIns synthase activity were 50 mM
Tris/HCl pH 8.0, 0.3 mMCDP-DAG dipalmitoyl (Sigma),
2.4 mM Triton X-100, 0.5 mM myo-inositol (Amersham,
[3H]-labelled, diluted with unlabelled myo-inositol to a final
activity of 500 BqÆnmol)1), 2.5 mM MnCl2 and 10–50 lg
membrane proteins Samples were incubated in a final
volume of 200 lL at 30C for 20 min The reaction was
stopped on ice by the addition of 3 mL ice-cold methanol/
chloroform (2 : 1) Lipids were then extracted accordingto
Sambrook et al [12] The chloroform phase was washed
with methanol and 1% (w/v) sodium chloride in the
proportion 1 : 1 : 1 Radioactivity was measured on a
100-lL aliquot mixed with 6 mL of Emulsifier SafeTM
(Packard) usinga scintillation counter
Exchange reaction The exchange reaction was assayed by
incubating50–200 lgmembrane proteins in 50 mM Tris/
HCl pH 8.0, 0.36 mM PtdIns from soybean (Sigma),
0.5 mMmyo-inositol containingtritium-labelled
myo-inos-itol for a final activity of 500 BqÆnmol)1, 2.4 mM Triton
X100, 2.5 mMMnCl2in the presence or in the absence of
4 lMcytidine monophosphate (CMP), but without
CDP-DAG The incubation time at 30C was 20–30 min
CMP-dependent PtdIns hydrolysis This reaction was
followed by incubating50–200 lgmembrane proteins in
50 mMTris/HCl pH 8.0, 3 mMCMP, 2.4 mMTriton X100,
2.5 mM MnCl2 and 42 lM PtdIns [soybean PtdIns from
Sigma diluted with [myo-inositol-2-3H(N)] PtdIns, (NEN)
to a final activity of 240 BqÆnmol)1] at 30C for 20–40 min
After extraction of total lipids as described above, the
reverse activity was measured on the lower phase after
concentration under nitrogen and mixing with 6 mL
Emulsifier SafeTM or on the upper phase washed with
chloroform (2 : 1), concentrated and mixed with 12 mL
Emulsifier SafeTM
Effect of EDTA on enzymatic reactions All buffers and aqueous solutions were prepared in distilled water (AutostillTMAutofour, Jencons) To the incubation medium containingthe above-mentioned concentrations of Tris/HCl pH 8.0, Triton X100, MnCl2 and EDTA were added the appropriate missingcomponents accordingto 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 gseeds as described [14] They were resuspended in the same buffer
as E coli membranes and stored at )80 C Membranes (1.5 mgmembrane proteins for E coli, 3 mgmembrane proteins for soybean microsomes) were incubated in
50 mM Tris/HCl pH 8.0, 2.4 mM Triton X100, 5 mM
EDTA, 0.1 mM cytidine triphosphate (CTP), 7.5 mM
MnCl2 and 2-3H(N)-myo-inositol (NEN, 0.46 MBq,
85· 104MBqÆmmol)1) in a final volume of 1.6 mL for
30 min at 30C The reaction was stopped by the addition of 4.8 mL methanol/chloroform (2 : 1) as before and lipids were extracted 3H-Labelled PtdIns molecular species were identified by radio-HPLC
Analysis of radioactive PtdIns molecular species After separation by TLC accordingto Lepage [15], the PtdIns spot was scrapped and PtdIns eluted at 8C overnight from the silica in 5 mL methanol containing one drop of glacial acetic acid After re-extraction of the sample accordingto Bligh & Dyer [16], PtdIns molecular species were analysed by radio-HPLC as described previ-ously [17]
R E S U L T S Enzymatic activities catalysed by AtPIS1 Previously published results indicate that the yeast PtdIns synthase placed in a microbial environment is capable of catalysingseveral enzymatic reactions: PtdIns de novo synthesis, usingCDP-DAG and free inositol as substrates and producingPtdIns and CMP; a CMP-independent exchange reaction whereby the inositol molecule of PtdIns can be replaced by another inositol polar head without net synthesis of PtdIns; a CMP-dependent exchange reaction, far greater in quantitative terms than the CMP-independent one; and, finally, a reaction which is 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 colicells expressingthe AtPIS1 cDNA were cultivated
on M9 minimal medium supplemented with vitamin-free amino acids to allow isolation of bacterial membranes lackingendogenous PtdIns, which would interfere with
Trang 3exogenous 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 3H-labelled free myo-inositol
and cold soybean PtdIns, no exogenous CDP-DAG, in
the presence or absence of 4 lM CMP The PtdIns
concentration used was 0.36 mM 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
presence of AtPIS1 allows the incorporation of labelled
inositol into PtdIns, accordingto the enzymatic reaction
called exchange This reaction is greatly enhanced by the
presence of 4 lM CMP, about ninefold under our
conditions
The assay for a reverse reaction was carried out by
measuringthe CMP-dependent release of label from
3H-PtdIns in the aqueous phase of total lipid extractions
as in Bligh & Dyer [16] Radioactivity was measured in
the lower chloroform and upper aqueous phases In the
absence of CMP and in both +PIS and –PIS strains, a
release of label in the aqueous phase was observed after
incubation To calculate the liberation of radioactivity
due to a putative reverse reaction, the amount of label
released in the absence of CMP was substracted from that
released in the presence of 3 mM CMP (Table 1)
Mem-brane samples containingPtdIns synthase catalyse the
liberation of label in the upper phase (which would
correspond to 130 pmol inositolÆmin)1Æmg)1),
counterbal-anced by an equivalent disappearance of PtdIns in the lower
phase ()108 pmolÆmin)1Æmg)1) On the other hand,
mem-branes lackingAtPIS1 do not induce any variation in the
radioactivity partitioned between each phase duringthe
incubation time, indicatingthat the appearance of3H-label
in the upper phase is specific to AtPIS1 Several 5¢-CMP
concentrations ranging between 0 and 6 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 colifrom 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.
Transformant E coli were cultivated on a medium lackinginositol,
membranes were purified as described in Materials and methods and
incubated under various conditions with different substrates The
values indicated and in pmol PtdInsÆmin)1Æmg)1protein In the upper
phase they are given as pmol inositolÆmin)1Æmg)1 Each value is the
mean ± SD of four (+PIS) or three (–PIS) experimental points using
the same protein sample Enzymatic reactions carried out with
mem-branes isolated from different cultures of transformant E coli cells
gave very similar results Conditions for: reverse reaction,
+3H-PtdIns + 3 m M CMP; exchange reaction, +3H-Ins + 0.36 m M
PtdIns.
+ CDP-DAG
– CDP-DAG
Reverse reaction
Exchange reaction
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.
Trang 4studied by incubatingmembranes from E coli +PIS
cultivated on medium lackinginositol or microsomes from
germinating soybean in the presence of CTP and
3H-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-tol fi 3H-PtdIns + CMP PtdIns molecular species were
then separated by radio-HPLC In E coli (Fig 1A), C14:0/
C16:0 PtdIns elutes almost at the same time as the C16:0/
C18:3 molecular species in soybean (Fig 1B), but other
molecular species are eluted at times which allow a perfect
distinction between the two sets of PtdIns molecules: the
C16:0/C17c + C18:1/C17c and C16:0/C19c peaks are
characteristic of E coli (A.-M Justin, unpublished data)
whereas the C16:0/C18:2 and C18:0/C18:2 PtdIns are
characteristic of soybean [14]
In a second experiment, membranes isolated from
E coli+ PIS grown on Luria–Bertani medium and
inos-itol (Fig 1C) or M9 medium lacking inosinos-itol (Fig 1D)
were incubated in conditions of exchange in the presence of
PtdIns isolated from soybean The radio-HPLC elution
profile of PtdIns shows that when endogenous PtdIns is
present in the bacterial membrane, molecular species
typical of E coli and others of soybean become labelled
(Fig 1C) The bacterial membrane contains endogenous
PtdIns that could be used as a substrate for exchange of the
inositol head but also endogenous CDP-DAG molecules
which can be used for de novo synthesis of PtdIns
Nevertheless, when bacterial membranes
lackingendo-genous PtdIns were incubated in the same exchange
conditions, the only labelled PtdIns molecular species
detected were of soybean origin (Fig 1D), showing: (a)
that exogenous soybean PtdIns is used for exchange; (b)
that the endogenous bacterial CDP-DAGs are not present
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 mMEDTA 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 adding5 mM EDTA leads to an
enhancement of inositol incorporation provided that the
concentration of manganese is correspondingly increased to
7.5 mM to compensate the chelatingeffect of EDTA
(unpublished results) We therefore investigated the effect
of EDTA on the reactions catalysed by PtdIns synthase in
the presence of 7.5 mM MnCl2 The results are shown in
Fig 2A and B
At 0 mMEDTA, i.e., 7.5 mMMnCl2, de novo synthesis
reached 0.83 nmol PtdInsÆmg)1Æmin)1 (Fig 2A) As the
EDTA concentration increased, synthesis increased up to
a maximum activity of 2.0 nmol PtdInsÆmg)1Æmin)1 between 2.5 and 5 mM EDTA At 10 mM, synthesis decreased to 0 In the conditions used here we detected two exchange activities, one stimulated by CMP and one independent of CMP The CMP-dependent activity showed
an incorporation profile of labelled inositol that closely paralleled that of the de novo synthesis activity (Fig 2A), with values close to 0.8 nmol PtdInsÆmg)1Æmin)1at 0 mM
EDTA, increasingto 2 nmol PtdInsÆmg)1Æmin)1 between 2.5 and 5 mM EDTA and a drop to 0 at 10 mM Abolition of all exchange activity at 10 mMEDTA 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)1Æmin)1at 0 mM
EDTA which peaked
between 2.5 and 5 mM Comparatively, the CMP-depend-ent PtdIns hydrolysingactivity was the lowest of all in our conditions
To evaluate better the effect of the chelator on each reaction, each activity was normalized to that obtained at
5 mM EDTA and plotted as a function of the free Mn2+ concentration (Fig 2B) The data show that synthesis and exchange with or without CMP behave in the same way accordingto the concentration in available Mn2+
Fig 2 Effect of EDTA on the enzymatic reactions catalysed by PtdIns synthase The results shown here are typical of those observed for at least two independent cultures (A) All activities were measured in the presence of 7.5 m M MnCl 2 Fifty lgmicrosomal proteins were incu-bated for 20 min as described in Methods d, De novo synthesis; j, exchange reaction in the presence of 4 l M CMP; h, exchange reaction 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) or 2.5 m M (s) (D) Comparison of de novo synthesis activities as a function of the calculated free Mn2+ 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) or different concentrations of MnCl 2 [11] (m) At 0 m M
Mn 2+ , all enzyme activities are null Because of the x axis chosen, activities obtained at submicromolar Mn2+ concentrations in the experiments involvingfixed concentrations of manganese chloride and varyingamounts of EDTA appear on the y axis just above 0.
Trang 5Effect of EDTA as a function of the concentration
in free manganese ions
The concentration of free mang anese ions was calculated at
each EDTA concentration usingan apparent EDTA
stability constant K1 of 5· 1011ÆM )1 for manganese at
pH 8.0[18] At 7.5 mMEDTA, when the calculated
con-centration of free manganese ions is in the submicromolar
range, a synthesis activity of 1.53 nmol PtdInsÆmg)1Æmin)1
could still be measured (Fig 2C) When this activity was
tested at a lower MnCl2concentration with various amounts
of EDTA, the highest activity was not observed at 0 mM
EDTA and 2.5 mMMnCl2as seen before [11], but at 2.5 mM
EDTA when the concentration in free mang anese ions is
close to 0.1 lM An alignment on the same plot of the values
found for de novo synthesis in the presence of 7.5 or 2.5 mM
MnCl2with various amounts of EDTA, or in the presence of
MnCl2only [11], as a function of the concentration in free
manganese (Fig 2D), shows that in the presence of EDTA
concentrations < 2.5 mMfree Mn2+give higher activities
than those observed with Mn2+ alone, suggesting an
activatingeffect of EDTA on enzymatic activity
D I S C U S S I O N
The exchange reaction
Usingthe same incubation conditions for PtdIns and CMP
as those published by Klezovitch et al [10] for examination
of the PtdIns synthase from yeast expressed in a bacterial
system, we found that the Arabidopsis enzyme expressed in
E coli is able to catalyse not only de novo synthesis of
PtdIns but also the exchange reactions in the absence or in
the presence of CMP In plants, the exchange reaction is
known to occur [7,8] but it has never been shown to be
catalysed by the same enzyme as that responsible for de novo
synthesis The experimental conditions used reproduce the
parameters defined as optimum by Sexton & Moore [7] and
Sandelius & Morre´ [8] with a pH of 8.0 and the presence of
manganese ions In the range of concentrations tested, we
found an optimum free manganese ion concentration of
2.5 mM, a value that is in the same range as the
concen-trations found by the authors cited above
In a preliminary work (data not shown), we have
observed that when E coli membranes prepared from cells
grown in inositol-containing medium are incubated with
labelled inositol but no CDP-DAG and no PtdIns, no
labelled PtdIns appears, showingthat 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)1Æmg)1(Fig 2), the concentration of CMP in a
reaction mixture of 200 lL is 4 lMafter 1 min with 50 lg
protein As our incubation conditions use 50 lgprotein
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 interferingwith 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 exchange is Our data does not allow us to distinguish between a reverse reaction followed by re-synthesis, as suggested by Paulus & Kennedy [19], and a real exchange The fact that EDTA has the same effect, in particular
a stimulation between 2.5 and 5 mM, on all reactions catalysed by PtdIns synthase seems to suggest that the chemical reactions involved are not very different, but that the enzymatic parameters in favour of synthesis, reverse reaction or exchange are quite different in terms of concentrations of the reactants in each case; a simple reverse reaction followed by synthesis is puzzlingor in any case very difficult to analyse in terms of kinetics From a functional point of view, if a reaction reverse of synthesis followed by synthesis usingCDP-DAGs different from those liberated was responsible for the exchange activity we detected, it could be a mechanism whereby in defined conditions cells could specifically destroy particular PtdIns molecular species to replace them by others more adapted to new physiological or environmental parameters
Removal of the polar head The data presented here are not an absolute proof that PtdIns synthase catalyses a true reverse reaction The enzymatic activity we detected is nevertheless strictly dependent on CMP, and strictly associated with the AtPIS1 protein The dependence of the PtdIns synthase reverse reaction has been extensively studied in rat pituitary GH3 cells, where the authors have identified the reverse products and shown that the CMP activation of the reverse of 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 chelatingagent 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 Mn2+
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
Trang 6favour 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
activity is needed to see whether PtdIns synthase uses two
different active sites, as made possible by the existence of
two hydrophilic pockets in the protein [11], whether the
same active site can adopt different conformations
accord-ingto substrate concentrations or physico-chemical
condi-tions or, finally, whether exchange is, mechanistically, an
inverse synthesis followed by re-synthesis The work carried
out on the rabbit lungand rat liver microsomal enzyme
seemed to suggest different enzymes as the pH and divalent
cation requirements were different for the two activities
[4,21] but their results could perhaps be explained by two
different catalytic sites Sexton & Moore also suggested for
the castor bean endosperm enzyme that exchange is not a
reversal of the transferase activity [7] No obvious sequence
similarities with other proteins catalysingan exchange of
phospholipid head, such as phosphatidylserine synthase
(CMP-PtdOH/L-serine 3-phosphatidyltransferase), came up
when alignments were made (data not shown) so the
question remains open until further data, currently being
compiled in our laboratory, are obtained, with the aim of
understandinghow PtdIns synthase functions and is
regulated
A C K N O W L E D G E M E N T S
The authors thank A Zachowski, E Ruelland and A Jolliot-Croquin
for helpful discussions and advice This project was funded by the
French Ministries of Research and Education via CNRS and the
University Pierre et Marie Curie (Unite´ Mixte de Recherche 7632).
R E F E R E N C E S
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2 Holub, B.J (1974) The Mn2+-activated incorporation of inositol
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4 Bleadsdale, J.E & Wallis, P (1981) Phosphatidylinositol-inositol
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6 Irvine, R.F (1998) Manganese-stimulated phosphatidylinositol headgroup exchange in rat liver microsomes Biochim Biophys Acta 1393, 292–298.
7 Sexton, J.C & Moore, T.S Jr (1981) Phosphatidylinositol synthesis by a Mn2+-dependent exchange enzyme in castor bean endosperm Plant Physiol 68, 18–22.
8 Sandelius, A.S & Morre´, D.J (1987) Characteristics of a phos-phatidylinositol exchange activity of soybean microsomes Plant Physiol 84, 1022–1027.
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