Tài liệu Báo cáo Y học: Phosphatidylinositol synthesis and exchange of the inositol head are

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 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 lM 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

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 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.

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 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 37
C 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, 4e tage, 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: [email protected]

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)

Eur. J. Biochem. 269, 2347–2352 (2002) FEBS 2002 doi:10.1046/j.1432-1033.2002.02893.x