Synthetic capacity of Arabidopsis phosphatidylinositol synthase 1 expressed in Escherichia coli

Biochimica et Biophysica Acta 1634 (2003) 52 – 60 www.bba-direct.com

Synthetic capacity of Arabidopsis phosphatidylinositol synthase 1 expressed in Escherichia coli Anne-Marie Justin, Jean-Claude Kader, Sylvie Collin * Laboratoire de Physiologie Cellulaire et Mole´culaire des Plantes, Universite´ Pierre et Marie Curie, UMR 7632 CNRS/Paris 6, Tour 53, 4e e´tage, Case Courrier 154, 4, Place Jussieu, 75252 Paris Cedex 05, France Received 15 August 2003; accepted 27 August 2003

Abstract Phosphatidylinositol (PtdIns) synthase 1 from the plant Arabidopsis thaliana has been expressed in Escherichia coli in order to study the synthetic capacities of the enzyme. Analysis of the total fatty acid content of the bacteria shows that PtdIns synthase activity does not have a profound effect on the proportions of the different fatty acids produced, even if the presence of an extra acidic phospholipid leads to a global reduction of the lipid content. A closer analysis carried out on individual phospholipids reveals a global fatty acid composition almost unchanged in the two major bacterial lipids phosphatidylethanolamine (PtdEtn) and phosphatidylglycerol (PtdGro). Phosphatidylinositol has a very unusual composition that shows the ability of the plant enzyme to use CDP-diacylglycerol molecular species absent from plants. We identified the various PtdIns molecular species. They represent a pool of the major molecular species of PtdEtn and PtdGro. These results, together with the determination of the apparent affinity constants of AtPIS1 for myo-inositol and CDP-diacylglycerol, allow us to discuss some of the constraints of PtdIns synthesis in plants in terms of specificity, which will depend on the subcellular localization of the protein. D 2003 Elsevier B.V. All rights reserved. Keywords: Phosphatidylinositol synthase; Arabidopsis; Escherichia coli; Phosphatidylinositol molecular species; Phospholipid synthesis

1. Introduction Biological membranes are characterized by their composition in phospholipids, which differ either by the nature of the polar head group, or at the level of fatty acids, by their position on the glycerol skeleton, the length of the carbon chain and degree of unsaturation. In plants, phosphatidylinositol (PtdIns) is the most highly saturated phospholipid [1]. It is present in membranes as a group of molecular species, whose roles in structure or as the precursors of biologically active phosphoinositides may

Abbreviations: BSA, bovine serum albumin; CDP-DAG, cytidine diphosphodiacylglycerol; CL, cardiolipin; CMP, cytidine monophosphate; (d)CDP-DAG, deoxycytidine diphosphodiacylglycerol; DPtdGro, diphosphatidylglycerol or cardiolipin; GLC, gas liquid chromatography; GPI, glycosylphosphatidylinositol; PtdOH, phosphatidic acid; PtdEtn, phosphatidylethanolamine; PtdGro, phosphatidylglycerol; PtdGroP, phosphatidylglycerophosphate; PtdIns, phosphatidylinositol; PtdIns 4-P, phosphatidylinositol 4-phosphate; PtdSer, phosphatidylserine; TLC, thin layer chromatography * Corresponding author. Tel.: +33-1-44-27-59-13; fax: +33-1-44-2761-51. E-mail address: [email protected] (S. Collin). 1388-1981/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2003.08.006

not be equivalent. In the case of a phosphorylated derivative of PtdIns, PtdIns 4-P, the establishment of different membrane pools is suggested by the wide subcellular distribution of PtdIns 4-kinase [2]. Regulation of the membrane location of the enzyme would be a way of regulating the availability of PtdIns 4-P to different proteins [3]. A pool can be defined by its functional specificity—phosphoinositides have been implicated in numerous cell functions such as vesicle trafficking [4 –6], regulation of enzymatic activities [2], regulation of the cytoskeletal organization, responses to environmental stresses such as cold temperatures or drought [7,8], gravitropism [9,10], hormone response [11], plant defense [12]—or location in membrane domains, like in the case of GPI anchors, or chemical nature. The question therefore arises as to what controls the proportions of the different PI molecular species in membranes. Phosphatidylinositol synthase (CDP-diacylglycerol:myoinositol 3-phosphatidyltransferase, EC 2.7.8.11) is the enzyme responsible for PtdIns synthesis in plants, animals, lower eucaryotes as well as some bacteria. The enzyme uses CDP-diacyglycerol (CDP-DAG) and myo-inositol as substrates, releasing PtdIns and CMP. In Arabidopsis thaliana,

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two genes encoding PtdIns synthase have been identified [13,14]. The existence of two isoforms raises the question of possible specificities of the two proteins, either in location, synthetic capacity or regulation. In the work presented here, we have used a heterologous host, Escherichia coli, to study the enzymatic characteristics of the enzyme coded by gene AtPIS1. Previous laboratories, including ours, have used this prokaryotic PtdIns-deficient host to express PtdIns synthase, to successfully show that the protein is able to catalyze de novo synthesis of PtdIns as well as exchange of the polar inositol head, to study the regulation of the enzyme by its substrates or the effects of PtdGro substitution by the anionic PtdIns in PtdGro-deficient strains [13,15 –18]. The analysis presented here has focused on a different aspect of PtdIns synthesis: in order to examine the synthetic capacity of PtdIns synthase and to identify possible specificities on the product made, we first compared the effect of PtdIns production on the total fatty acid synthesis of the bacterial host. We then analysed the different molecular species made by AtPIS1 in the bacterial context and compared them with the molecular species present in Arabidopsis plantlets. By this approach we expected to see, for example, whether the enzyme had strict constraints on the fatty acids borne by the CDP-DAG precursor since some fatty acids in E. coli are completely absent from plants and vice-versa. The apparent affinity of the enzyme for its substrates has also been calculated. The data show a unexpected plasticity in the PtdIns molecules made that we discuss with respect to phospholipid synthesis in bacteria and plants.

2. Materials and methods 2.1. Genetic nomenclature The cDNA used in this work corresponds to EMBL accession number H36646 [13] and gene AtPIS1. 2.2. Plant material A. thaliana ecotype Columbia:2 was grown as described previously [13] in a growth chamber at a temperature ranging between 22 and 25 jC. The light intensity was between 150 and 200 Amol m 2 s 1. The photoperiod was 16 h light:8 h dark. 2.3. Growth conditions of the bacterial transformants E. coli cells expressing the AtPIS1 cDNA encoding PtdIns synthase 1 (AtPIS1) from A. thaliana were obtained as described in Ref. [13]. The constructions used are the same as that described in the above-mentioned article. Two bacterial strains were used, one expressing the plant cDNA (strain + PIS [18]), and the non-expressing control strain (strain PIS [18]). The cells were grown at 37 jC in LB

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medium (Miller, Difco) with addition of 1 mM myoinositol and 100 Ag ml 1 ampicillin. Expression of the cDNA was induced in fresh transformants as previously described [13] before the cells were washed in 50 mM Tris – HCl pH 8.0 and stored as pellets at 80 jC. These pellets were used for lipid analyses or as a source of membranes. 2.4. Lipid extractions E. coli cells grown on inositol-containing medium were resuspended in 50 mM Tris – HCl pH 8.0 at a cell density of 100 mg fresh cells ml 1. Lysis was achieved by sonication in a Branson Sonifier 250. Intact cells were eliminated by centrifugation at 4500  g for 10 min and total lipids were extracted from the supernatant according to Bligh and Dyer [19]. The upper aqueous phase was reextracted by addition of chloroform before the two chloroformic phases were pooled. Arabidopsis plantlets were fixed in boiling water for 1 min then cooled on ice before lipids were extracted according to Bligh and Dyer [19]. 2.5. Phospholipid analysis Total lipids from Arabidopsis were separated by TLC on silica plates (Merck) according to Lepage [20]. PtdIns was then analyzed by GLC using a Varian 3300 Gas Chromatograph after transmethylation [21] of an aliquot sample of the PtdIns fraction prepared for analysis of the PtdIns molecular species. Separation of total lipids from E. coli was carried out by TLC according to Ohta [22]. Revelation and identification of each lipid species was carried out as described [13]. Spots were collected and analyzed by GLC after transmethylation with BF3/methanol [21]. Quantitation was performed using exogenous heptadecanoic acid (Sigma) as an internal standard. Other standards were cis-9,10methylene octadecanoic acid methyl ester (19:0j), methylated nonadecanoic acid (19:0), oleic acid methyl ester (18:1) and cis-vaccenic acid methyl ester (18:1); all from Sigma. 2.6. Analysis of PtdIns molecular species Total lipids from 1 g of E. coli cells grown in inositolcontaining LB medium, or 4 g of Arabidopsis plantlets were separated by TLC according to Lepage [20]. The PtdIns spot was scrapped and PtdIns eluted at 8 jC overnight from the silica in 10-ml methanol containing one drop of glacial acetic acid. After extraction of the sample according to Ref. [19], PtdIns molecular species were analyzed by a combination of HPLC (ultrasphere C18 4.6  250 mm, 5 Am, Beckman) and GLC as previously described [23]. The solvant was a mixture of methanol/ water/acetonitrile (90.5:4:2.5) containing 20 mM choline chloride.

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2.7. Phosphatidylinositol synthase activity Membrane purifications were carried out at 5 jC. E. coli cells were resuspended at a density of 100 Ag fresh cells ml 1 in sonication buffer containing 50 mM Tris – HCl pH 8.0, 8% (v/v) glycerol and 8 mM 2-mercaptoethanol. 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 mM Tris –HCl pH 8.0, 20% (v/v) glycerol, 8 mM 2mercaptoethanol, aliquoted and stored at 80 jC. The protein concentration was determined according to Ref. [24] using BSA as a standard. Unless otherwise stated, the incubation conditions for de novo PtdIns synthase activity were 50 mM Tris –HCl pH 8.0, 0.3 mM CDP-DAG dipalmitoyl (Sigma), 2.4 mM Triton X-100, 0.5 mM myo-inositol (Amersham, [3H]-labelled, diluted with unlabelled myoinositol to a final activity of 500 Bq nmol 1), 2.5 mM MnCl2 and 10 to 50 Ag of membrane proteins. Samples were incubated in a final volume of 200 Al at 30 jC for 10 min. The reaction was stopped on ice by addition of 3-ml ice-cold methanol/chloroform (2:1). Lipids were then extracted according to Ref. [19]. The chloroform phase was washed with methanol and 1% (w/v) sodium chloride in 1:1:1 proportions. The total radioactivity of each sample was measured using a scintillation counter, after addition of 6 ml of Emulsifier Safek (Packard).

Fig. 1. Phospholipid biosynthesis pathway in E. coli. Phosphatidylinositol synthesis due to expression of the A. thaliana enzyme AtPIS1 is indicated as a dotted line. Adapted from Refs. [25,26]. The names of the genes encoding the endogenous bacterial enzymes are indicated in italics: cdsA, CDP-DAG synthase; cls, cardiolipin synthase; pgpA,B, phosphatidylglycerophosphatase A,B; pgsA, phosphatidylglycerophosphate synthase; plsB, glycerol 3-P acyltransferase; plsC, 1-acylglycerol 3-phosphate acyltransferase; psd, phosphatidylserine decarloxylase; pss, phosphatidylserine synthase.

3. Results 3.1. Characteristics of the experimental system Expression of PtdIns synthase 1 from Arabidopsis (AtPIS1) was carried out in E. coli, a Gram negative bacteria naturally devoid of PtdIns. The biosynthetic pathway of glycerophospholipids in E. coli is presented in Fig. 1, where a drawing adapted from [25,26] shows how the three major lipids, PtdEtn, PtdGro and cardiolipin, are synthesized from a common precursor, CDP-DAG. PtdIns synthase also uses CDP-DAG as one of its substrates, the only one in common with PtdSer synthase and PtdGroP synthase. It is expected to function with enzymatic characteristics that will depend on its own apparent affinities and requirements in the nature of CDP-DAG molecular species, as well as on the regulations of the endogenous bacterial lipid synthesizing enzymes. 3.2. Fatty acid composition of PtdIns synthesized in E. coli or Arabidopsis The results are presented in Table 1. The fatty acid composition of PtdIns isolated from Arabidopsis plantlets is typically composed of a large proportion of 16:0 (45.6 F 0.3%), followed by 18:2 (29 F 1.5%) and 18:3

(21.7 F 1.4%). As previously published in other reports (Refs. [27,28] for example), PtdIns is the richest in 16:0 among phospholipids. Globally the degree of saturation expressed as the percentage of saturated fatty acids was 48%. The presence of 16:1 was not detected. In E. coli, two sets of data were compared: the global levels of each fatty acid in the presence or absence of PtdIns synthase, and the levels of fatty acids in each phospholipid, again in the presence or absence of PtdIns synthase. Globally, PtdIns synthesis did not have a profound effect on fatty acid levels in terms of proportions since very similar values were found for 14:0 (3.3% vs. 3.9%), 16:0 (38.8% vs. 41.3%) and 18:0 (1.2% vs. 1.1%). Variations were nevertheless observed for 16:1 and 18:1, where expression of the enzyme led to a slight increase in the proportion of both fatty acids (the values are 7.0% vs. 2.0% and 22.0% vs. 15.8%, respectively) and for the cyclic fatty acids whose proportions slightly decreased as a consequence of PtdIns synthesis (19.9% vs. 24.6% for 17:0j and 7.8% vs. 11.3% for 19:0j). When one looks at the results obtained for individual phospholipids, no category exactly reflects these global changes. PtdEtn composition was very stable, PtdIns synthesis only leading to a very small increase in 18:1 (16.3% vs. 13.9%). In PtdGro, which uses a different enzymatic pathway also dependent on CDP-

A.-M. Justin et al. / Biochimica et Biophysica Acta 1634 (2003) 52–60 Table 1 Fatty acid composition of lipids isolated from A. thaliana or E. coli expressing ( + PIS) or not expressing (

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PIS) PtdIns synthase 1 from Arabidopsis

Fatty acids (%) 14:0 Arabidopsis PtdIns E. coli Total lipids PtdIns PtdEtn PtdGro CL NL

+ PIS PIS + PIS + PIS PIS + PIS PIS + PIS PIS + PIS PIS

X

16:0

16:1

–

–

45.6 F 0.3

–

3.3 3.9 3.5 F 1.5 4.4 F 0.1 4.3 F 0.0 5.7 F 2.6 2.0 F 0.1 4.8 F 0.1 4.1 F 0.1 11.1 F 4.6 4.6 F 0.2

– – – – – – – – – 46.3 F 7.3 46.6 F 1.8

38.8 41.3 42.2 F 1.6 43.2 F 0.4 44.6 F 0.1 40.4 F 1.8 42.4 F 0.3 42.0 F 0.7 45.8 F 0.8 24.0 F 5.4 25.4 F 0.2

7.0 2.0 13.2 F 2.7 2.1 F 0.6 1.5 F 0.1 4.1 F 1.3 2.7 F 0.4 5.4 F 1.0 1.6 F 1.6 4.2 F 0.5 traces

17:0j

18:0

18:1a

18:2

18:3

–

2.3 F 0.2

1.3 F 0.2

29 F 1.5

21.7 F 1.4

19.9 24.6 14.3 F 3.2 24.9 F 0.5 25.1 F 0.1 16.4 F 0.2 17.8 F 0.4 15.4 F 0.6 15.5 F 0.2 5.0 F 1.5 8.7 F 0.2

1.2 1.1 1.6 F 0.4 1.2 F 0.1 1.3 F 0.1 2.1 F 0.4 1.6 F 0.1 3.7 F 0.7 5.4 F 0.1 5.9 F 0.7 6.2 F 0.7

22.0 15.8 19.9 F 5.0 16.3 F 0.4 13.9 F 0.3 20.1 F 1.5 19.0 F 0.8 20.7 F 0.8 17.0 F 1.2 3.5 F 3.4 6.0 F 0.0

– – – – – – – – – – –

– – – – – – – – – – –

19:0j

7.8 11.3 5.3 F 3.5 7.9 F 1.0 9.3 F 0.3 11.2 F 1.9 14.5 F 0.5 8.0 F 1.2 10.6 F 0.3 traces 2.5 F 2.4

In Arabidopsis, two fatty acids, 16:3 and 20:0, are not included in the table because they were detected in PtdIns neither in the plant nor in E. coli. In the plant they represented 11.5% and 4.6% of total fatty acids, respectively. The identity of X is not known due to the lack of appropriate fatty acid references commercially available. For each lipid category, all values were obtained from two different experiments except the fatty acid composition of PtdIns in E. coli strain + PIS where the results from five independent cultures were used to calculate the average composition. a Oleic acid in Arabidopsis, cis-vaccenic acid in E. coli.

DAG, the changes concerned 19:0j, whose proportion decreased slightly (11.2% vs. 14.5%). Small variations were also observed for cardiolipids. The most obvious effects of PtdIns synthesis concerned neutral lipids, where clearer variations were observed for 16:1, 17:0j and 19:0j, following the increase or decrease seen at the global level. In terms of global fatty acid composition, PtdIns synthesis therefore principally affected neutral lipids, the phospholipids being relatively stable except PtdGro where a decrease in 19:0j was seen. The major fatty acids in PtdIns were 16:0 (42.2 F 1.6%) and 18:1 (19.9 F 5.0%), followed by 17:0j (14.3 F 3.2) and 16:1 (13.2 F 2.7). About 5% of PtdIns fatty acids were

19:0j. The plant enzyme was therefore able to use fatty acids that are not present in plants, namely 14:0 and the two cyclic fatty acids 17:0j and 19:0j. Globally PtdIns resembled PtdGro and cardiolipin in its fatty acid composition, with very similar values except the enrichment in 16:1 and depletion in 19:0j in PtdIns. The other major glycerolipid from E. coli, PtdEtn, also had a very similar composition, except that it was enriched in 17:0j and poorer in 18:1 when compared to PtdIns, PtdGro and cardiolipin. The composition in 19:0j was intermediate between the two. The degree of saturation was 47% in PtdIns, a value identical to that found in plants, about 50% for PtdEtn in both strains, slightly higher for PtdGro in strain + PIS (50%

Table 2 HPLC analysis of PtdIns molecular species of Arabidopsis plantlets or E. coli transformant cells grown in the presence of myo-inositol

The molecular species are listed in order of elution, without taking into account the sn-1 or sn-2 position of fatty acids. Those that are grouped in a shaded box were eluted at the same time. The values are given for two different batches of plants or cultures of E. coli transformant cells. nd: not detectable. a Oleic acid in Arabidopsis, cis-vaccenic acid in E. coli.

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instead of 46%) and, on the opposite, slightly lower for cardiolipin (50% instead of 55%). 3.3. Phosphatidylinositol molecular species A detailed analysis of the PtdIns molecular species found in the two types of organisms showed that seven molecular species can be found in Arabidopsis, the most abundant ones being 16:0/18:2 and 16:0/18:3, which represent almost 90% of the total (Table 2). Similar results have been found in other plants [29,30]. In E. coli, the situation is completely different. Ten molecular species have been identified, none of them present in Arabidopsis since in plants 18:1 is linoleic acid whereas in E. coli it is cis-vaccenic acid. Most species include fatty acids that are not found in the plant molecular species: 14:0, 16:1, 17:0j, 19:0j. The most abundant one is 16:0/18:1 (c. 31%), followed by 16:0/16:1 (c. 29%) and 16:0/17:0j (c. 20%). Globally, the percentage of molecular species carrying one 16:0 is 91% in Arabidopsis, whereas in E. coli it is 92%.

Fig. 3. Phosphatidylinositol de novo synthesis as a function of the CDPDAG concentration in the reaction mixture. The same experiments carried out with protein samples from different cultures gave very similar results. The myo-inositol concentration was 0.5 mM.

maximum incorporation of myo-inositol was observed at 0.2 mM CDP-DAG (Fig. 3). Beyond this value, the synthesis activity decreased to reach a level close to 70% of the maximum in our conditions.

3.4. Kinetics parameters for de novo synthesis

4. Discussion

To better characterize the AtPIS1 protein, the apparent Michaelis constants for myo-inositol and CDP-DAG were determined. The experiments were carried out in the presence of 5 mM EDTA and 7.5 mM MnCl2. We used the Lineweaver and Burke representation to determine the affinity of AtPIS1 for its substrates. At 0.3 mM CDPDAG, the apparent Km for myo-inositol is 0.40 mM (Fig. 2A). The calculated apparent Km for CDP-DAG diC16 is 10 AM when the myo-inositol concentration is 1.5 mM (Fig. 2B). The amplitude of concentrations tested was comprised between 0.01 and 0.15 mM. A very similar value was found for CDP-DAG diC14 (9 AM, data not shown). In the standard incubation conditions described in Section 2, the

4.1. Effects of PtdIns synthesis on phospholipid synthesis

Fig. 2. Apparent Michaelis affinity constant of AtPIS1 for myo-inositol (A) and CDP-dipalmitoylglycerol (B). Each plot was constructed using data from two different cultures. All enzymatic activities were measured in the presence of 5 mM EDTA and 7.5 mM MnCl2. Ten micrograms of membrane proteins was incubated in each reaction, for 10 min at 30 jC, in the presence of 0.3 mM CDP-DAG diC16 (A) or 1.5 mM myo-inositol (B).

Our investigation started with the analysis of the effect of PtdIns synthesis on the fatty acid composition of endogenous lipid of E. coli. Although previous studies have analyzed the functionality of PtdIns synthase in E. coli [15 – 17], to our knowledge, no data has been presented on the consequences on the fatty acid composition of each lipid. Our results show that globally the synthesis of PtdIns in E. coli did not have a profound qualitative effect on the synthesis of the two other major lipids depending on CDPDAG, i.e. PtdEtn and PtdGro, although in quantitative terms it is known that synthesis of an additional lipid using the same precursor as PtdSer and PtdGroP leads to a decrease in PtdEtn as well as PtdGro [13,15,17], particularly in strains BL21(DE3)pLysE [13] and HD30 [17]. In E. coli, PtdSer synthase has been extensively studied [31]. It is known to be distributed between two locations, ribosomes, where it is inactive, and the plasma or inner membrane, where it is bound to acidic phospholipids by electrostatic interactions and active. The key element is the balance between zwitterionic and acidic phospholipids, which allows a negative feedback of the PtdGro/CL pathway on PtdEtn biosynthesis. In our case, in the absence of PtdIns, the value PtdEtn/ (PtdGro + CL) is 3.6 [13]. In the expressing strain, the value calculated without taking PtdIns into account is 4, whereas if PtdIns is used to make the calculation, we find 1.8 [13]. In E. coli BL21(DE3)pLysE, the acidic phospholipid PtdIns does not seem to participate in the regulation of PtdSer synthase activity and PtdEtn synthesis, a result that was expected since PtdIns cannot substitute for PtdGro in E. coli [17].

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4.2. Access of PtdIns synthase 1 to its substrates PtdIns synthase did not seem to sequester particular CDP-DAG molecular species at the expense of PtdSer synthase and PtdGroP synthase. At that point, the question arises of the organization of the bacterial membrane in terms of lipid synthesizing enzymes. What are the affinities of both enzymes for their substrates? Do PtdSer synthase and PtdGroP synthase have access to the same CDP-DAGs or do they function on different pools? The apparent affinity constant of PtdSer synthase for CDP-DAGs has not been calculated as such, but using mixed micelles, Carman and Dowhan [32] assessed the specificity of the enzyme for a number of lipid substrates. They found that the affinity for the micelle surface and the binding constant of the enzyme for the substrate on the surface of the micelles are independent of the fatty acid composition of CDP-DAGs. In another series of experiments, Rilfors et al. [33] measured PtdSer synthase activity from E. coli reconstituted in liposomes, and found that addition of PtdIns did not lead to any change in activity, suggesting that in our experimental system, PtdIns synthase could interfere with PtdSer synthase at the level of CDPDAGs availability, PtdIns itself being unlikely to interfere directly. The apparent Km value of PtdGroP synthase for (d)CDP-DAG has been calculated to be 40 AM [34]. It is 33 AM in yeast [35], 46 AM in rat [36] and 12 and 17 AM for CDP-DAG diC16 for the two Arabidopsis isozymes [37]. These values are close to what we find for PtdIns synthase. We therefore suggest that the use of CDP-DAG by PtdSer synthase, PtdGroP synthase and PtdIns synthase will therefore depend either on specific requirements in the different CDP-DAG molecular species, due for example to the structure of the enzyme in the case of the first two proteins, and/or on the in situ availability of these molecules to the enzymes. A first approach to try to answer this question was to look at the molecular species of PtdIns made by AtPIS1 in the bacterial cells. The three major molecular species identified in E. coli correspond to the molecular species usually found preponderant in the endogenous lipids: 16:0/ 16:1, 16:0/17:0j and 16:0/18:1 in PtdEtn and PtdGro [38 – 42]. The same result was obtained in PtdIns made by AtPIS1 in E. coli. Little has been published on the organization of protein-synthesizing enzymes, lipids or CDP-DAG in domains within bacterial membranes. PtdEtn seems to be distributed randomly in the inner and outer membranes of E. coli strain AB1623 [43], but the existence of lipid domains in bacteria has been visualized [44,45], which could be related to the biological functions of these different lipids [46]. Even if the existence of different PtdOH pools has been suggested, the absence of data on CDP-DAG domains or lipid-synthesizing protein domains does not make it possible at present to directly answer the question of accessibility to CDP-DAG pools to the different enzymes. A direct activation of CDP-DAG synthase has not been

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tested but since the level of lipids in mass is lower in the + PIS strain than in the control strain [13], and since the fatty acid composition of both PtdEtn and PtdGro is almost unchanged, it is unlikely to play a preponderant role in the selectivity of all enzymes. 4.3. Phosphatidylinositol synthesis A comparison of all affinity constants published for PtdIns synthase for de novo synthesis activity in different organisms is summarized in Table 3. Apart from rat brain, liver microsomes and dog pancreas, the apparent Km for myo-inositol is comprised between 60 and 400 AM. The affinity for CDP-DAG is in most cases much higher, except in the case of castor bean endosperm, Chlamydomonas microsomes, yeast microsomes and rat pituitary GH3 cell plasma membrane. The optimal CDP-DAG concentration was 0.2 mM for the Arabidopsis enzyme, a lower value than what was described for PtdIns synthase activity in Chlamydomonas [47]. We observed the same effect of increasing CDP-DAG concentrations in the medium. Inositol incorporation was inhibited at concentrations beyond 20 times the calculated affinity constant. A last point in the discussion of PtdIns synthase specificities in E. coli is the possibility of the enzyme to produce PtdIns via an exchange reaction of the polar head for inositol. This exchange reaction has been described for PtdIns synthase in several organisms [16,18 and references therein, 47] where the protein is able to replace the inositol head of PtdIns for another inositol in an apparently useless reaction. The high levels of PtdEtn in E. coli, as well as the fact that PtdIns isolated from E. coli is close to PtdEtn and PtdGro in its fatty acid composition, raises the question of whether PtdIns could be made by exchange of ethanolamine, glycerol or even serine for inositol on the different phospholipids. Our results show that although the fatty acid Table 3 Apparent Michaelis constants of PtdIns synthase for myo-inositol and CDPDAG in de novo synthesis (in AM) Material Arabidopsis, expressed in E. coli Castor bean endosperm Cauliflower Chlamydomonas microsomes Yeast microsomes Human placenta Dog pancreas Rat pituitary GH3 cells, plasma membrane Rat pituitary GH3 cells, endoplasmic reticulum Rat brain Rat liver microsomes

myo-inositol 400 300a 200b 80 – 100c,d 280f 760g 60h

CDP-DAG 10 9 1350a 45a 370b 70e 36f 18g 210h

260i 4600j 1300k 2500l

9.5k 170l

The small letters refer to the following references: a [56], b [47], c [16], d [57], e [58], f [59], g [60], h [61], i [62], j [63], k [64], l [65].

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composition of each phospholipid is close, they are not identical. PtdIns is different from both PtdEtn and PtdGro in its enrichment in 16:1. PtdEtn is known to be richer in 16:1 than PtdGro, with higher levels of 16:0/16:1 than 16:0/18:1 than this lipid, but in strain BL21(DE3) the levels of 16:1 found in PtdEtn are much lower than in PtdIns. Furthermore, although the fatty acid composition of PtdIns in 18:1 matches that of PtdGro and cardiolipin, the high level of 16:1 still represents a discrepancy in the direct synthesis of PtdIns from PtdGro. The exchange activity of PtdIns synthase on PtdEtn has been studied in rat liver, where the enzyme has been shown not to use PtdEtn as a substrate [48]. Serine and glycerol are very poorly incorporated in place of myo-inositol [49]. In Saccharomyces cerevisiae no exchange between inositol and PtdEtn has been observed when the enzyme was reconstituted in vesicles [50]. Our suggestion is therefore that although a low exchange reaction of PtdIns synthase on lipids other than PtdIns, namely PtdGro and PtdSer, cannot be excluded, the majority of PtdIns arises from true synthesis from CDP-DAG and inositol. 4.4. Implications for the specificity of PtdIns synthase 1 in Arabidopsis In plants, the enrichment of PtdIns in C16:0, C18:2 and C18:3 has been published by many authors. A comparison of the fatty acid composition of PtdIns with that of its precursors shows a selection for particular molecular species in pea leaf, potato tuber and soybean microsomes [51]. This selectivity could be due to enzymatic specificities but the results obtained in E. coli are not in favor of this hypothesis. Some situations have been described in the literature that show variations in PtdIns fatty acid content. In the fad2 mutant of Arabidopsis, characterized by a depletion in 16:0, 18:0, 18:2 and 18:3, but a strong enrichment in C18:1 [52], PtdIns from mitochondrial membranes is also characterized by a strong enrichment in 18:1, a depletion in 18:0 and 18:3, but the proportion of 16:0 is almost unchanged [53], as if a particular constraint existed on the level of this fatty acid in PtdIns. In the fad3 + mutant, which overexpresses the N3 linoleate desaturase [54], the fatty acid composition of phospholipids isolated from cultured cells shows unchanged levels of 16:0 but a decrease in 18:0, 18:2 and an increase in 18:1 and 18:3. In PtdIns from mitochondria, the result is the same, showing in this case again a constant level of 16:0 but also a flexibility in the fatty acid composition of the phospholipid [55]. Our results suggest that the specific composition of PtdIns in plants is the result of more factors than just enzymatic specificity. Of prime importance will be the subcellular localization of the enzyme, which will determine what the competing proteins for CDP-DAG are. In the endoplasmic reticulum (ER), these will be PtdSer synthase and PtdGroP synthase. In his context, the organization of the ER in functional domains suggested by several studies [56]

will provide key elements in understanding PtdIns synthesis in plants.

Acknowledgements The authors wish to thank A. Zachowski for careful reading of the manuscript, D. Petit and A. Jolliot-Croquin for useful advice during the course of this work. We gratefully acknowledge funding from the French Ministe`re de la Jeunesse, de l’Education Nationale et de la Recherche via C.N.R.S. and the University Pierre et Marie Curie (UMR 7632).

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