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First published online August 21, 2009; 10.1104/pp.109.144261 Plant Physiology 151:869-881 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
At4g24160, a Soluble Acyl-Coenzyme A-Dependent Lysophosphatidic Acid Acyltransferase1,[W],[OA]Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India (A.K.G., N.C., R.R.); Institute of Biochemistry, Graz University of Technology, A–8010 Graz, Austria (S.R., G.D.); and School of Science, Monash University, 46150 Petaling Jaya, Malaysia (R.R.)
Human CGI-58 (for comparative gene identification-58) and YLR099c, encoding Ict1p in Saccharomyces cerevisiae, have recently been identified as acyl-CoA-dependent lysophosphatidic acid acyltransferases. Sequence database searches for CGI-58 like proteins in Arabidopsis (Arabidopsis thaliana) revealed 24 proteins with At4g24160, a member of the  /β-hydrolase family of proteins being the closest homolog. At4g24160 contains three motifs that are conserved across the plant species: a GXSXG lipase motif, a HX4D acyltransferase motif, and V(X)3HGF, a probable lipid binding motif. Dendrogram analysis of yeast ICT1, CGI-58, and At4g24160 placed these three polypeptides in the same group. Here, we describe and characterize At4g24160 as, to our knowledge, the first soluble lysophosphatidic acid acyltransferase in plants. A lipidomics approach revealed that At4g24160 has additional triacylglycerol lipase and phosphatidylcholine hydrolyzing enzymatic activities. These data establish At4g24160, a protein with a previously unknown function, as an enzyme that might play a pivotal role in maintaining the lipid homeostasis in plants by regulating both phospholipid and neutral lipid levels.
Acylation of glycerol-3-phosphate (G3P) is the first step in the biosynthesis of glycerolipids in plants. Most of the enzymes involved in this pathway were shown to be membrane bound (Somerville and Browse, 1991  ). However, a soluble G3P acyltransferase has been reported in plants, which acylates G3P to lysophosphatidic acid (LPA) in an acyl-(acyl carrier protein)-dependent manner (Murata and Tasaka, 1997). The role of other soluble enzymes in the glycerolipid biosynthesis pathway is well documented. Cytosolic monoacylglycerol acyltransferase (Tumaney et al., 2001), diacylglycerol acyltransferase (Saha et al., 2006), and LPA phosphatase (Shekar et al., 2002) were shown to be present in the immature seeds of Arachis hypogaea. Recently, cytosolic LPA phosphatase (Reddy et al., 2008) and phosphatidic acid (PA) phosphatase have also been reported in Saccharomyces cerevisiae (Han et al., 2006). In addition, we demonstrated earlier the presence of a soluble LPA acyltransferase (LPAAT) as a part of the cytosolic multienzyme complex for the synthesis of triacylglycerol (TG) in Rhodotorula glutinis (Gangar et al., 2001). In S. cerevisiae, Ict1p catalyzes the acylation of LPA to PA, thereby enhancing phospholipid biosynthesis under cellular stress. A  ict1 deletion strain was shown to be calcofluor white sensitive and exhibited a defective phospholipid biosynthesis, suggesting a role of Ict1p in the maintenance of the cell membranes (Ghosh et al., 2008a).
BLAST analysis of the human genome with the Ict1p sequence resulted in the identification of a gene named CGI-58. Mutations in human CGI-58 are responsible for a rare autosomal recessive genetic disorder known as Chanarin Dorfman syndrome (Zechner et al., 2009
So far, a soluble LPAAT from plants has not been identified, although the importance of such enzymes in other experimental systems has been envisaged (Tumaney et al., 2001
Evolutionary Relationship of CGI-58-Like Proteins from Arabidopsis
To search for the homologs of CGI-58 in plants the Arabidopsis genome was screened as described in "Materials and Methods." After identifying the individual protein sequences, we eliminated the repeated sequences by comparisons with theoretical cDNA and genomic DNA sequences in The Arabidopsis Information Resource database. This strategy led to the identification of 24 proteins that were found to be homologous to CGI-58 (Table I
). All 24 of these proteins belong to the
Multiple sequence alignment of all the 24 proteins revealed three conserved motifs (Fig. 1A ) and several conserved Ser, Asp, and His residues that may serve as a part of the catalytic triad, the active site in most of the known hydrolases/lipases. Motif I is V(X)3HGF, where X represents hydrophobic residues such as Val, Ile, Phe, Met, and Leu. This is supposedly the lipid binding motif of these polypeptides. The motif II is GXSXG, the signature motif of known lipases, phospholipases, and lysophospholipases. The motif III is H(X)4D/E, previously identified as a signature motif of most acyltransferases. Interestingly, among all the 24 proteins identified, the H(X)4D motif was found only in At4g24160, whereas the other polypeptides contained an H(X)4E motif. In order to gain an insight into CGI-58-like proteins in other plants, a global BLAST search analysis was performed using the plant database at the National Center for Biotechnology Information (NCBI). Multiple sequence alignment revealed the presence of all the three motifs that were seen in Arabidopsis to be conserved in other plant species (data not shown).
To examine the relationship among CGI-58-like proteins, a topographic dendrogram was constructed (Fig. 1B). ICT1, CGI-58, and At4g24160 were clustered in the same group. However, CGI-58 and its homologs in nonchordates, amphibians, nematode, and higher mammals diverged to form a different subgroup, indicating a final attainment of function in the evolutionary timescale. Interestingly, none of the other 23 CGI-58 homologs from Arabidopsis was present in the same group, suggesting that At4g24160 diverged from other members of this polypeptide family in the evolutionary history to perform specific catalytic functions that may be important in the physiology of the plant. The dendrogram also suggests that the remaining 23 hydrolases have markedly diverged during evolution, attaining specific functions, a feature common among members of the /β-hydrolase family of proteins. As an example, BODYGUARD 1 domain-containing proteins, At4g24140, At5g41900, and At1g64670, form a distinct group of polypeptides. Multiple sequence alignment of the proteins present in the groups I and II showed that most of the residues were conserved throughout the family (Fig. 1B). Motifs I, II, and III were distinctly present in all the members of the group, suggesting their probable catalytic role per se. An interesting observation made in the sequence alignment was that the Ser of GXSXG motif is replaced by Asn in all the vertebrates starting from amphibians. The effect of such a conversion needs to be examined. The conversion also suggests the divergence of these polypeptides from the other members of Group I.
Using only the information of the amino acid sequence, it has been difficult to assign a function to proteins of the
A detailed domain analysis suggested possible biological roles for these hypothetical proteins. At1g52750 possesses a putative redox active protein domain with a very high expression in the mitochondria and a moderate expression in the nucleus. At3g10840 was found to have a SARS lipid binding domain, suggesting a possible association of the protein with long-chain fatty acids (Meier et al., 2006
The Genevestigator online search tool Meta-Analyzer was used to analyze Arabidopsis Affymetrix microarray data. Relative gene expression was studied in different plant organs, at various growth stages, and under different stress conditions. Many of these genes were found to be up-regulated during biotic stress. The expression profiles of these 24 proteins under different stress conditions are summarized in Table I. At4g24160 was present from the early seedling to the later stages of development but was found to be specifically expressed in the roots. It was up-regulated during Pseudomonas syringae infection and salicylic acid treatment, while abscisic acid (ABA) and zeatin treatment resulted in its down-regulation. The expression pattern of At4g24160 under abiotic stresses like cold, heat, salt, hypoxia, osmotic, genotoxicity, wound, and drought was found to be interesting. At4g24160 is up-regulated under high salt stress with the maximum expression in the roots (eFP database), which is a particularly important observation because salt stress has been shown to be associated with high phospholipid biosynthesis. The other stress conditions that seem to have an effect on the expression of At4g24160 are cold, osmotic stress, and hypoxia.
BLAST analysis of Ict1p in the nonredundant database of NCBI revealed plant At4g24160 and human CGI-58 as its closest homologs (Fig. 2
). At4g24160, a 418-amino acid protein, is a member of the
Biochemical Characterization of At4g24160 As a prerequisite for biochemical investigations, At4g24160 was cloned in pRSET A and overexpressed in BL21 (DE3) cells. Immunoblot analysis using anti-(His)6 monoclonal antibody was performed to confirm the expression. The recombinant protein was purified by Ni2+-nitrilotriacetic acid (Ni2+-NTA) column chromatography (Fig. 3A ).
Lysophospholipid Acyltransferase Activity As one of the most important enzymatic assays, we analyzed lysophospholipid acyltransferase activity of the purified At4g24160 recombinant protein. As can be seen from Figure 3B, At4g24160 was found to be highly specific for LPA as a substrate. Two-dimensional thin-layer chromatography (TLC) analysis confirmed that PA was the product of this enzymatic conversion (Fig. 3C). At4g24160 was found to be capable of using different acyl-CoA donors for the formation of PA; however, the specificity toward oleoyl-CoA was maximum followed by stearoyl-CoA (Fig. 3D). The enzyme showed a protein- and time-dependent incorporation of oleoyl-CoA into PA (data not shown). The LPAAT activity was also analyzed by electron spray ionization-mass spectrometry (ESI-MS; Supplemental Fig. S1, A–C). At this point, it is important to mention that At4g24160 homologs of yeast and human were also shown to exhibit a similar LPAAT activity (Ghosh et al., 2008a , 2008b). ESI-MS analysis also confirmed the LPAAT activity of this recombinant purified enzyme.
Lipase and Phospholipase Activity
A cocktail assay comprising of all the three substrates, i.e. LPA, TG, and PC, was also performed to determine the substrate preference of both At4g24160 and Ict1p. It was observed that the LPAAT activity for proteins was maximum, and they were capable of hydrolyzing TG and PC to a lesser extent. The PLA2 activity of At4g24160 was higher than its TG lipase activity; however, the case was vice versa for Ict1p. Zero minute and no substrate reactions were kept as controls (Fig. 5, A–F ).
Lysophospholipase Activity Since At4g24160 was found to possess a putative lysophospholipase domain, we analyzed the LPA lysophospholipase activity of the purified enzyme using [3H]LPA; however, no lysophospholipase activity was detected. The same experiment was also done by ESI-MS using unlabeled LPA (1-oleoyl), and the entire substrate provided was recovered at the end of the reaction without any product formation, clearly indicating the absence of lysophospholipase activity (Supplemental Fig. S6, A and B).
To study the effect of At4g24160 and Ict1p overexpression on the levels of cellular phospholipids and neutral lipids, S. cerevisiae was transformed with pYES2-At4g24160 and pPS189-ICT1. Immunoblotting with anti-Ict1p antibodies confirmed the expression of the At4g24160 protein (Fig. 6A
). Overexpression of At4g24160 in yeast led to an approximately 2-fold increase in PA as analyzed by [32P]labeling of phospholipids in vivo (Fig. 6, B and C). In addition, total phospholipids were quantified as described by Broekhuyse (1968)
Moreover, yeast cells overexpressing At4g24160 and Ict1p were labeled with [14C]acetate in an induction medium to address the question of their effect on neutral lipid metabolism, which suggested that At4g24160 and Ict1p may also be involved in TG turnover in yeast cells (data not shown).
LPAAT is a crucial enzyme controlling the metabolic flow of LPA into the pool of PA, which plays a key role in many physiological aspects, such as cell signaling, cell polarity, and apoptotic signaling cascades in higher eukaryotes (Park et al., 2004 ). It was also shown that PA specifically regulates the genes involved in modulating cell shape and organization. PA has also been found to target proteins to the membranes, thereby modulating their catalytic activity (Fang et al., 2001; Anthony et al., 2004; Zhang et al., 2004; Huang et al., 2006). A recent study also demonstrated that PA is involved in the regulation of stomatal movements by influencing the ABA level. It binds to the ABA-interacting protein phosphatase to signal ABA-promoted stomatal closure, whereas phospholipase D and PA interact with G protein to mediate ABA inhibition of stomatal opening. This signaling is essential for maintaining the hydration status of plants (Mishra et al., 2006).
In Arabidopsis, five AtLPAAT1 to AtLPAAT5 genes encoding LPAATs have been reported based on their amino acid sequence similarity (Kim and Huang, 2004
Based on the BLAST analysis, 24 proteins were identified in Arabidopsis that are homologs of CGI-58 and Ict1p (Fig. 1A). Among these polypeptides At4g24160 is the closest homolog to the two templates, and in the dendrogram analysis it is grouped (Fig. 1B) in close vicinity to CGI-58 and ICT1. All the three proteins possess no transmembrane domains. Most notably, none of the known AtLPAATs fulfill the latter criterion. Earlier studies have shown that Ict1p in S. cerevisiae is highly expressed during organic solvent exposure (Miura et al., 2000
Interestingly, At4g24160 was also found to possess lipase and phospholipase activities similar to Ict1p (Figs. 4 and 5). Another single polypeptide exhibiting esterase, lipase, and phospholipase activity was reported earlier in the plant system. Thermally stable alkaline lipase from rice bran was shown to hydrolyze para-nitrophenyl palmitate, TG, and phospholipids (Bhardwaj et al., 2001
In plants, PA plays a pivotal role as a multifunctional stress signal. PA is generated either by the action of phospholipase D or by the sequential action of phospholipase C and diacylglycerol kinase (van Leeuwen et al., 2004
Materials
At4g24160 clone was obtained from the Arabidopsis Biological Resource Center. [1-14C]Oleoyl-CoA (54 mCi/mmol), [14C]acetate (51 mCi/mmol), [9,10-3H]triolein (53 Ci/mmol), [2-palmitoyl-9,10-3H]phosphatidylcholine (92.3 Ci/mmol), and [9,10-3H]LPA (47 Ci/mmol) were purchased from Perkin-Elmer Biosciences. [32P]Orthophosphate (5000 Ci/mmol) was obtained from the Board of Radiation and Isotope Technology, Bhabha Atomic Research Centre (Mumbai, India). Silica gel 60F254 TLC plates were from Merck. Oligonucleotide primers, TG, diacylglycerol, phospholipids, lysophospholipids, and solvents were purchased from Sigma-Aldrich. Acyl-CoA donors were obtained from Avanti Polar Lipids Polyclonal antibodies were raised against Ni2+-NTA affinity-purified recombinant Ict1p as described (Ghosh et al., 2008a
Sequence Retrieval, Alignment, and Comparison
Phylogenetic Tree Construction
Examination of Conserved Protein Domains
Gene Expression Analysis
pUNI vector containing At4g24160 open reading frame was used as a template for amplification of the gene. Forward primer (5'-ATGGATCCATGAACTTGAGCCGTTTTGCTT-3') and reverse primer (5'-ATGAATTCCTAAACCAATCGTAGACCATCTAGG-3') were used. PCR (1 min denaturation at 94°C, 1 min annealing at 55°C, and 1 min elongation at 72°C) was performed using Pfu polymerase for 30 cycles with 10 pmol concentration of each primer. The purified PCR product and pRSET A vector (N-terminal His tag) were digested with BamHI and XhoI and ligated directionally. The construct was transformed into Escherichia coli BL21 (DE3) cells and induced with 1 mM isopropylthio-β-galactoside for 4 h at 37°C. The cell pellet was resuspended in lysis buffer containing 50 mM Tris-HCl (pH 8.0) and 300 mM NaCl. Cells were disrupted by sonication. The inclusion bodies were separated and solubilized in lysis buffer containing 6 M urea and 25 mM imidazole. The 10,000g supernatant of solubilized inclusion bodies was allowed to bind to the Ni2+-NTA matrix. The column was washed with lysis buffer containing 25 mM imidazole. The bound protein was eluted with 250 mM imidazole in lysis buffer. Fractions (1 mL each) were collected and analyzed on 12% SDS-PAGE followed by Coomassie Brilliant Blue staining.
For overexpression of At4g24160 in Saccharomyces cerevisiae, full-length At4g24160 cDNA was subcloned from pRSET A into pYES2 vector at the BamHI-EcoRI site and transformed into yeast cells by lithium chloride method (Schiestl and Gietz, 1989
The reaction mixture contained 10 µM [1-14C]oleoyl-CoA (110,000 dpm/assay), 1 to 5 µg enzyme, and 50 µM LPA (1-oleoyl) in assay buffer with a total volume of 100 µL. The reaction was carried out at 30°C for 10 min and terminated by extracting the lipids (Ghosh et al., 2008a
The purified recombinant At4g24160 and Ict1p (10 µg protein) were incubated in a reaction buffer containing 50 mM Tris-HCl, pH 8.0, for 40 min at 30°C in the presence of a sonicated suspension of 1 mM triolein Reactions were stopped by addition of 100 µL of butanol. The butanol layer containing the lipids was washed with 50 mM sodium acetate. The samples were dried and subjected to ESI-MS analysis. The mass by charge peaks obtained were analyzed using the lipid metabolites and pathways strategy. Lipase assay was also performed in a reaction buffer containing 50 mM Tris-HCl, pH 7.5, and 100 µM sodium taurocholate for 40 min at 30°C in the presence of sonicated suspension of 100 µM [9,10-3H]triolein (0.25 µCi/tube). Reactions were stopped by addition of 100 µL of butanol, and the samples were resolved on a silica-TLC plate along with oleic acid standard using chloroform:methanol:ammonia (65:35:5, v/v). The region corresponding to the oleic acid standard was scraped and quantified by liquid scintillation counting. The control value was subtracted from the actual assay value, and the specific activity was calculated after the correction. For esterase assay, para-nitrophenyl stearate (2.5 mM) was used as the substrate, and the hydrolytic product para-nitrophenol was monitored at 410 nm. The absorbance was measured against the reference cell to which buffer had been added instead of dialyzed enzyme solution.
The reaction mixture contained 1 mM sonicated vesicles of dipalmitate and 10 µg enzyme in a total volume of 100 µL assay buffer (0.05 M Tris-HCl, pH 7.5, and 2 mM dithiothreitol). The reaction was carried out at 30°C for 40 min and terminated by extracting the lipids with butanol. The lipids were dried and analyzed by ESI-MS. The mass-to-charge ratio (m/z) peaks of the substrate and the product were identified using lipid maps.
Radiometric assay consisted of 100 µM sonicated vesicles of [2-palmitoyl-9,10-3H]phosphatidylcholine (1 µCi/assay) and 10 µg enzyme in total volume of 100 µL assay buffer (0.05 M Tris-HCl, pH 7.5, and 2 mM dithiothreitol). The reaction was carried out at 30°C for 40 min and terminated by extracting the lipids by the method of Bligh and Dyer (1959)
The purified recombinant At4g24160 and Ict1p (10 µg protein) were incubated in a reaction buffer containing 50 mM Tris-HCl, pH 8.0, for 40 min at 30°C in the presence of a sonicated suspension of 10 µM oleoyl-CoA, 1 mM LPA (1-oleoyl) , 1 mM triolein, and 1 mM PC (dipalmitate). The reaction was terminated by extracting the lipids with butanol. A sodium acetate wash was given to the butanol layer. The lipids were dried and analyzed by ESI-MS. The m/z peaks of the substrate and the product were identified using lipid maps.
The butanol soluble fraction was dried and suspended in HPLC-grade methanol and subjected to ESI-MS analysis (Bruker Esquire 3000 plus electrospray ion trap instrument).The sample was applied directly into the ESI source through a polytetrafluoroethylene line at the rate of 4 µL/min. The ESI-MS settings used were as follows: turbo electrospray inonization source was maintained at 260°C, and the data were collected in the positive ion mode. The experiments were repeated three times, and a similar spectrum was obtained in all the cases. The m/z peaks obtained were analyzed by lipid maps (http://www.lipidmaps.org).
pYES2-At4g24160 and pYES2 transformants were grown to the late log phase in 5 mL SM-U containing 2% (w/v) Glc and then transferred to 50 mL of the same media, such that the absorbance was 0.1. The cells were grown till the absorbance reached 3. A600 = 0.4 cells were inoculated in a fresh medium containing 2% (w/v) Gal and 200 µCi [32P]orthophosphate and grown for 24 h. Cells (A600 = 25) were harvested by centrifugation, and lipids were extracted and analyzed by two-dimensional TLC. The solvents for the first dimension were chloroform:methanol:ammonia (65:35:5, v/v), and solvents for the second dimension were chloroform:methanol:acetic acid:water (40:20:5:0.5, v/v; Ghosh et al., 2008a
The following materials are available in the online version of this article.
Received July 3, 2009; accepted August 17, 2009; published August 21, 2009.
1 This research was supported by a grant from the Department of Biotechnology, Government of India, New Delhi (a program supporting nonconventional yeast to R.R.), and the Fonds zur Förderung der wissenschaftlichen Forschung in Österreich (Projects 18857 and W901–B05 to G.D.).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Ram Rajasekharan (lipid{at}biochem.iisc.ernet.in).
[W] The online version of this article contains Web-only data.
[OA] Open access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.109.144261 * Corresponding author; e-mail lipid{at}biochem.iisc.ernet.in.
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