Lipins from plants are phosphatidate phosphatases that restore lipid synthesis in a pah1 D mutant strain of Saccharomyces cerevisiae Elzbieta Mietkiewska 1 , Rodrigo M. P. Siloto 1 , Jay Dewald 2 , Saleh Shah 3 , David N. Brindley 2 and Randall J. Weselake 1 1 Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Canada 2 Department of Biochemistry, Signal Transduction Research Group, School of Molecular and Systems Medicine, University of Alberta, Edmonton, Canada 3 Plant Biotechnology, Alberta Innovates-Technology Futures, Vegreville, Canada Keywords lipin; phosphatidate phosphatase; Saccharomyces cerevisiae; subcellular localization; triacylglycerol Correspondence R. J. Weselake, Agricultural Lipid Biotechnology Program, Department of Agricultural, Food and Nutritional Science, 4–10 Agriculture ⁄ Forestry Centre, University of Alberta, Edmonton, Alberta T6G 2P5, Canada Fax: +1 780 492 6739 Tel: +1 780 492 4401 E-mail: randall.weselake@ualberta.ca (Received 21 September 2010, revised 10 December 2010, accepted 20 December 2010) doi:10.1111/j.1742-4658.2010.07995.x The identification of the yeast phosphatidate phosphohydrolase (PAH1) gene encoding an enzyme with phosphatidate phosphatase (PAP; 3-sn-phosphati- date phosphohydrolase, EC 3.1.3.4) activity led to the discovery of mam- malian Lipins and subsequently to homologous genes from plants. In the present study, we describe the functional characterization of Arabidopsis and Brassica napus homologs of PAH1. Recombinant expression studies confirmed that homologous PAHs from plants can rescue different pheno- types exhibited by the yeast pah1D strain, such as temperature growth sen- sitivity and atypical neutral lipid composition. Using this expression system, we examined the role of the putative catalytic motif DXDXT and other conserved residues by mutational analysis. Mutants within the carboxy-terminal lipin domain displayed significantly decreased PAP activity, which was reflected by their limited ability to complement different phenotypes of pah1D. Subcellular localization studies using a green fluores- cent protein fusion protein showed that Arabidopsis PAH1 is mostly pres- ent in the cytoplasm of yeast cells. However, upon oleic acid stimulation, green fluorescent protein fluorescence was predominantly found in the nucleus, suggesting that plant PAH1 might be involved in the transcrip- tional regulation of gene expression. In addition, we demonstrate that mutation of conserved residues that are essential for the PAP activity of the Arabidopsis PAH1 enzyme did not impair its nuclear localization in response to oleic acid. In conclusion, the present study provides evidence that Arabidopsis and B. napus PAHs restore lipid synthesis in yeast and that DXDXT is a functional enzymic motif within plant PAHs. Database The nucleotide sequence data have been deposited in the GenBank database under accession numbers HQ113853 and HQ113854 Abbreviations C-LIP, carboxy-terminal lipin domain; DAG, diacylglycerol; DAPI, 4,6-diamidino-2-phenylindole dilactate; DGAT, diacylglycerol acyltransferase; FAME, fatty acid methyl ester; GFP, green fluorescent protein; HAD, haloacid dehalogenase; LPP, lipid phosphate phosphatase; N-LIP, amino-terminal lipin domain; PA, phospatidate; PAH, phosphatidate phosphohydrolase; PAP, phosphatidate phosphatase; PL, phospholipid; TAG, triacylglycerol; WT, wild-type. 764 FEBS Journal 278 (2011) 764–775 ª 2011 The Authors Journal compilation ª 2011 FEBS Introduction Phosphatidate phosphatase (PAP; 3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) catalyzes the dephos- phorylation of phospatidate (PA), yielding diacyl- glycerol (DAG) and inorganic phosphate [1–3]. In eukaryotic cells, PAP activity plays a central role in both lipid metabolism and intracellular signaling mechanisms [4,5]. Two distinct PAP enzyme activities, referred to as PAP1 and PAP2, have been described [2,6–8]. PAP1, now known as PAP activity is Mg 2+ -dependent, utilizes PA as a unique substrate and localizes in the soluble fraction, from where it translocates to internal mem- branes [3,9,10]. By contrast, PAP2, currently known as a family of lipid phosphate phosphatases (LPPs) utilizes many substrates (e.g. PA, lysophosphatidate, sphingo- sine-1-phosphate and DAG pyrophosphate, amongst others), does not require Mg 2+ for activity and is mem- brane-bound [11]. Genes encoding the LPPs have been identified and extensively studied in both yeast and plants [12–15]. Studies in Saccharomyces cerevisiae led to the identifi- cation of the yeast phosphatidate phosphohydrolase (PAH1) gene encoding an enzyme with PAP activity [16]. The S. cerevisiae pah1D strain displays severe defi- ciency in triacylglycerol (TAG), which is apparently caused by a decreased level of DAG, reflecting the importance of this enzyme for de novo synthesis of neu- tral lipids. In addition, the free fatty acid content of pah1D yeast was significantly increased, presumably as a result of the decreased ability of cells to utilize fatty acids for TAG synthesis [16]. The PAH1 enzyme was found in soluble and membrane fractions of the cell and its association with membranes was shown to be of a peripheral nature [2]. The S. cerevisiae PAH1 shares sequence homology with mammalian Lipin-1 in the con- served N-terminal and C-terminal domains of the pro- tein known as N-LIP and C-LIP, respectively [2,3,17]. The C-LIP domain contains a haloacid dehalogenase (HAD)-like catalytic motif found in the superfamily of Mg 2+ -dependent PAPs. This motif consists of DXDXT in which the first aspartate residue is responsible for binding the phosphate moiety during the catalytic reac- tion [18,19]. In addition, a conserved glycine within N-LIP is required for the PAP activity of yeast PAH1 [19]. PAH1 is phosphorylated on seven residues match- ing the minimal Cdk consensus that is required for the efficient transcriptional derepression of key enzymes involved in phospholipid (PL) biosynthesis [20,21]. In mammals, highly phosphorylated forms of Lipin-1 are enriched in the cytoplasm, whereas dephosphorylated forms are found mostly associated with membranes [9,10,22,23]. Homologous PAH1 genes are also present in Arabidopsis thaliana and other plants. Nakamura et al. [24] showed that Arabidopsis PAH1and PAH2 are involved in the eukaryotic pathway of galactolipid bio- synthesis. Recently, it was demonstrated in Arabidopsis that PAHs regulate PL synthesis in a similar manner to that described for S. cerevisiae [25]. In the present study, we demonstrate that, in addition to AtPAH1 and AtPAH2, homologous genes from Bras- sica napus also encode functional PAP enzymes capable of complementing different phenotypes of yeast pah1D strain, including TAG synthesis and temperature-sensi- tive growth. We also provide evidence that the con- served aspartate residues in the HAD-like motif of the plant enzymes are required for PAP function, and demonstrate the importance of conserved glycine and serine residues for the activity of the enzyme. Finally, we show that, when expressed in yeast, AtPAH1 can be detected in cytosolic and membrane fractions, although it is recruited to the nucleus when the cells are cultivated in the presence of oleic acid. A preliminary account of some of the results reported in the present study has been presented at the 19th International Symposium on Plant Lipids [26]. Results and Discussion Arabidopsis and B. napus PAHs are orthologs of yeast PAH1 and mammalian lipins Based on previous studies demonstrating the involve- ment of yeast PAH1 and mammalian lipins in storage lipid accumulation [27], we were initially interested in identifying Arabidopsis genes encoding PAP enzymes that may play a role in de novo DAG synthesis. Two PAH1 homologs were found in Arabidopsis , which are designated in the present study as AtPAH1 (At3g09560) and AtPAH2 (At5g42870), and encode proteins with predicted molecular masses of 100.9 and 101.2 kDa, respectively. In addition, we identified two isoforms of B. napus PAH1 using the expressed sequence tag information available on the Internet (http://brassica.bbsrc.ac.uk). They are designated in the present study as BnPAH1A (GenBank # HQ113853) and BnPAH1B (GenBank # HQ113854), and encode proteins sharing 99% sequence identity and calculated molecular masses of  90.6 kDa. Both Arabidopsis and B. napus PAHs have the conserved N-LIP and C-LIP domains found in yeast PAH1 and mammalian Lipin-1. AtPAH1 and AtPAH2 share 63% and 58% identity in the conserved N-LIP and C-LIP domain, respectively. B. napus PAHs display higher E. Mietkiewska et al. Arabidopsis and B. napus PAHs homologs of PAH1 FEBS Journal 278 (2011) 764–775 ª 2011 The Authors Journal compilation ª 2011 FEBS 765 sequence identity to AtPAH1 at the level of 95% for each N-LIP and C-LIP domain. The C-LIP domain harbors the HAD-like catalytic motif (DXDXT) found in the superfamily of Mg 2+ -dependent phosphatases [17,27]. The HAD-like motif DVDGT is located at positions 707–711, 734–738 and 616–620 of AtPAH1, AtPAH2 and BnPAH1, respectively (Fig. 1). By con- trast to plant PAHs, members of the family of LPPs that do not require Mg 2+ ions for activity in Arabid- opsis and yeast contain a catalytic motif comprising the consensus sequences KXXXXXXRP, PSGH and SRXXXXXHXXXD [2,5,28,29]. Analyses made with several prediction algorithms were unable to detect the presence of potential transmembrane domains in Arabidopsis or B. napus PAHs. This is in agreement with earlier studies on mammalian and yeast homologs [3,16,17]. Unlike Arabidopsis and B. napus PAHs, six putative transmembrane domains were predicted in Arabidopsis LPP2 and LPP3 and in yeast diacylgly- cerol pyrophosphate phosphatase 1 and LPP1 that belong to the family of Mg 2+ -independent PAP2 enzymes [14,16,30]. Arabidopsis and B. napus PAHs complement different phenotypes of pah1-deficient yeast cells To establish the functional relationship between yeast PAH1 and the corresponding plant homologs, the coding region of Arabidopsis and B. napus PAHs were linked to the galactose-inducible GAL1 promoter in the yeast expression vector pYES2 ⁄ NT, which also provides an N-terminal His-tag. The resulting con- structs were transformed into the S. cerevisiae pah1D strain, which displays different phenotypes, such as a reduced level of PAP activity, severe growth deficiency at 37 °C, elevated levels of PA and decreased levels of DAG and TAG [16,19,31]. Transformed yeast cells were cultivated for 16 h after induction with galactose. Immunoblot analysis of yeast homogenates showed that the His-tagged Arabidopsis PAH1 and B. napus PAHs migrated as  130 and 118 kDa proteins, respectively, upon SDS ⁄ PAGE (Fig. 2A, B), and these values are higher than the molecular weights of the predicted polypeptides. A similar shift in mobility was observed in yeast PAH1 and was attributed to post- translational modification of the protein by phosphor- ylation [16,20]. AtPAH2 migrated at a lower molecular weight compared to AtPAH1 and, in this case, a sec- ond band is clearly noticeable on SDS ⁄ PAGE (Fig. 2A, lane 2). The same pattern has been also observed previously for mammalian Lipin-2 [22]. Frac- tionation of yeast homogenate indicated that AtPAH1 was detected in both membrane and soluble fractions of the yeast cell (Fig. 2C). Therefore, to evaluate Mg 2+ -dependent PAP activity, we used crude cell homogenates. The pah1D strain expressing Arabidopsis or B. napus PAHs displayed a significant increase in ScPAH1 AtPAH1 AtPAH2 BnPAH1A BnPAH1B DIDGT N-LIP HAD-like G DVDGT SG DVDGTG DVDGT A A G A DVDGT SG AAAA Fig. 1. PAH1 homologs from plants have similar domain organiza- tion to yeast PAH1 (ScPAH1) polypeptide. Arabidopsis PAH1 (At- PAH1), Arabidopsis PAH2 (AtPAH2) and B. napus PAHs (BnPAH1A and BnPAH1B) are members of the lipin family, containing a conserved N-terminal domain (N-LIP) and a C-terminal catalytic domain with a HAD-like motif usually found in Mg 2+ -dependent phosphatidate phosphatases. The conserved amino acids that were mutated in the present study are indicated. 1212 1 ABC 2 kDa 160 kDa 120 100 kDa 120 160 120 Fig. 2. Plant PAHs expressed in yeast migrate higher on SDS ⁄ PAGE than the predicted molecular masses of polypeptides and are found in both soluble and membrane fractions. Immunoblots of Arabidopsis and B. napus PAHs were carried out using anti-HisG-HRP serum and pro- tein extracts from yeast pah1D expressing the recombinant polypeptides after 16 h of induction. Proteins (40 lg each lane) were run on an 8% SDS ⁄ PAGE gel. Protein molecular mass was calculated using BenchMarkÔ Protein Ladder (Invitrogen). (A) Crude homogenates from cells expressing AtPAH1 (lane 1) and AtPAH2 (lane 2). (B) Crude homogenates from cells expressing BnPAH1A (lane 1) and BnPAH1B (lane 2). (C) Subcellular fractions from cells expressing AtPAH1: microsomal fraction (lane 1) and soluble fraction (lane 2). Membrane and soluble fractions were obtained using 100 000 g centrifugation of the 15 000 g supernatant of crude homogenate. Arabidopsis and B. napus PAHs homologs of PAH1 E. Mietkiewska et al. 766 FEBS Journal 278 (2011) 764–775 ª 2011 The Authors Journal compilation ª 2011 FEBS PAP activity compared to the negative control trans- formed with LacZ (Fig. 3A). The highest difference in PAP activity was observed for BnPAH1A, with an increase of 21.4-fold compared to the negative control. In the pah1D mutant, the PAP activity was reduced by 39% compared to the wild-type (WT) (Inv Sc1 strain) control used in the present study. This was similar to the difference between pah1D and WT parent strain observed by Han et al. [16]. The remaining Mg 2+ - dependent PAP activity in pah1D has been attributed to other enzymes with unknown molecular identities [16]. To evaluate the influence of Arabidopsis and B. na- pus PAHs in the metabolism of neutral lipids, we ana- lyzed the TAG and PL content of yeast cells expressing recombinant PAHs (Fig. 3B). After 48 h of induction, the TAG ⁄ PL ratio in pah1D cells bearing recombinant PAHs was considerably higher compared to the negative control. The most pronounced effect was observed for BnPAH1A, with an increase of 40- fold compared to the TAG ⁄ PL ratio observed in the negative control (LacZ). In addition to the effect on lipid composition, yeast pah1D cells also display reduced growth when culti- vated at 37 °C. To determine whether Arabidopsis and B. napus PAHs could rescue this phenotype, we culti- vated several dilutions of cells expressing plant PAHs at 37 °C. When cells were inoculated in medium supplemented with galactose (induced), lines expressing PAHs from Arabidopsis and B. napus displayed growth on dilutions as low as D 600 = 1.0 · 10 4 (Fig. 3C), whereas cells expressing LacZ grew only at D 600 = 1.0. In medium without galactose (not induced), only WT cells presented appreciable growth, indicating that complementation of temperature-sensi- tive phenotype resulted from Arabidopsis and B. napus PAHs expression. Taken together, these results show that the previ- ously characterized AtPAH1 and AtPAH2 and the two PAH1 homologs from B. napus encode enzymes with PAP activity. Previous work on mammalian lipins indicated that the pah1D yeast expression system could be used as a predictive model for confirming the func- tions of PAH1-homolog genes [22]. Arabidopsis and B. napus PAHs complemented the temperature sensi- tive phenotype from the yeast pah1D strain, which was also observed by Nakamura et al. [24] for AtPAH1 and AtPAH2. Using an Escherichia coli expression sys- tem, Eastmond et al. [25] reported a comparatively higher enzyme activity of AtPAH1 over AtPAH2, which is corroborated by the results obtained in the present study (Fig. 3A). The higher enzyme activity of AtPAH1 compared to AtPAH2 is also evident in the BnPAH1B AtPAH1 AtPAH2 LacZ LacZ BnPAH1A pah1Δ Wild type OD 1 10 10 2 10 3 10 4 Dilutions Induced Not induced C A 0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 SA (nmol·mg –1 ·min –1 ) 0.0 1.0 2.0 3.0 4.0 5.0 LacZ WT B 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 mol TAG/mol PL Fig. 3. Arabidopsis and B. napus PAHs complement different phe- notypes of S. cerevisiae pah1D mutant. (A) PAP activity of crude homogenates from yeast pah1D cells bearing AtPAH1, AtPAH2, BnPAH1A, BnPAH1B or LacZ as a negative control. Yeast homo- genates were prepared from cells induced for 16 h and assayed for PAP activity in the presence of 1 m M MgCl 2 . Total PAP activities were calculated from measurements at three different protein con- centrations. (B) Ratio of TAG to PL of yeast pah1D expressing recombinant PAHs. Total lipids were extracted from yeast cells after 48 h of induction and separated on TLC plates. Spots corre- sponding to TAG and PL were scraped out and transmethylated. FAMEs were analyzed by GC. Each bar represents the mean ± SD from three determinations. (C) Complementation of temperature sensitive phenotypes of pah1D by plant PAH homologs. Yeast pah1D expressing AtPAH1, AtPAH2, BnPAH1A, BnPAH1B or LacZ were cultivated in liquid medium. The density of resulting cultures was adjusted to D 600 = 1 followed by 10-fold serial dilutions. Five microliters of each dilution were spotted onto plates containing 2% galactose (induced) or 2% raffinose (not induced) and incubated for 3 days at 37 °C. WT yeast and pah1D strain previously transformed with LacZ were used as a positive and negative control, respec- tively. E. Mietkiewska et al. Arabidopsis and B. napus PAHs homologs of PAH1 FEBS Journal 278 (2011) 764–775 ª 2011 The Authors Journal compilation ª 2011 FEBS 767 preferential growth at 37 °C of yeast expressing AtPAH1; this was also reported by Nakamura et al. [24]. Taken together, these results collectively suggest that AtPAH1 can complement phenotypes of the pah1D strain more efficiently than AtPAH2. The newly-characterized BnPAH1A and BnPAH1B display relatively stronger complementation over Arabidopsis PAHs in most aspects. Moreover, the restoration of TAG synthesis in the yeast pah1D strain expressing plant PAH homologs suggests an evolutionary conser- vation of the PAP enzyme reaction between yeast and plants. N-LIP and C-LIP are functional domains in Arabidopsis and B. napus PAHs To determine the role of the conserved residues within C-LIP and N-LIP domain of plant PAH1, we exam- ined the mutational effect of selected residues on PAP activity using AtPAH1 and BnPAH1A as models. Using site-directed mutagenesis (Fig. 1), we con- structed mutant AtPAH1 alleles (G83A, D707A, D709A and S752A) and mutant BnPAH1A alleles (G83A, D616A and D618A) and expressed them in the pah1D strain. Yeast cells were harvested after 16 h of induction in medium containing galactose. Cell homo- genates were prepared, verified via immunoblotting and assayed for PAP activity. Immunoblot analysis using anti-HisG serum showed that native and mutant AtPAH1 and BnPAH1A enzymes were expressed at comparable levels (Fig. 4). Although the expression of AtPAH1 and BnPAH1A resulted in PAP activity that was significantly higher compared to LacZ control, the corresponding mutant alleles did not restore PAP activity to comparable levels (Fig. 4). In particular, mutations in the predicted catalytic motif of AtPAH1 (D707A and D709A) and BnPAH1A (D616A and D618A) abolished PAP activity, with levels compara- ble to the negative control. These results are in agree- ment with mutational analysis of the yeast PAH1 catalytic motif [19] and demonstrate that the conserved aspartate residues in the plant homologs are required for their catalytic function. In addition, substitution of the conserved serine 752 with alanine within C-LIP domain of AtPAH1 had a similar negative effect on enzyme activity. The importance of the equivalent con- served serine residue for the enzyme activity in human Lipin-2 and mouse Lipin-1 and Lipin-2 has been described previously [9]. For example, a rare human mutation S734L in LIPIN-2 gene causes Majeed syndrome, a human inflammatory disorder. Recently, Majeed syndrome has been linked to the loss of Lipin- 2-mediated PAP activity [9]. Mutation of the conserved glycine (G83) to alanine in the N-LIP domain of both AtPAH1 and BnPAH1A produced less severe effects on the enzyme activity and resulted in the loss of up to 75% and 54% of the PAP activity of native enzyme, respectively (Fig. 4). Inter- estingly, other mutations in the corresponding position of PAH1 from other organisms appear to have a more 80 100 120A B 0 20 40 60 0 AtPAH1 G83A D707A D709A S752A LacZ 130 kDa 80 100 120 20 40 60 PAP1 activity (%) PAP1 activity (%) 0 BnPAH1A G83A D616A D618A LacZ 118 kDa Fig. 4. Mutations within N-LIP and C-LIP domains affect PAP1 activity of plant PAH1. (A) PAP activity of yeast pah1D strain homo- genates bearing AtPAH1 and its mutant alleles: G83A, D707A, D709A and S752A. Lower: corresponding western blot of the site- directed mutagenized AtPAH1. (B) PAP activity of yeast pah1D strain homogenates bearing BnPAH1A and its mutant alleles: G83A, D616A, D618A. Lower: corresponding western blot of the site-directed mutagenized BnPAH1A. Yeast homogenates were prepared from cells induced for 16 h and assayed for PAP activity in the presence of 1 m M MgCl 2 . The amount of PAP activity of samples bearing AtPAH1 (34.8 nmolÆmg )1 Æmin )1 ) and BnPAH1A (60.0 nmolÆmg )1 Æmin )1 ) were set at 100%. The data shown are the mean ± SD from three determinations. Homogenates of yeast cells expressing LacZ were used as negative controls. Western blot anal- ysis were carried out using anti-HisG-HRP serum and protein extracts (40 lg each lane) from yeast pah1D strain expressing the recombinant polypeptides separated on an 8% SDS ⁄ PAGE gel. Arabidopsis and B. napus PAHs homologs of PAH1 E. Mietkiewska et al. 768 FEBS Journal 278 (2011) 764–775 ª 2011 The Authors Journal compilation ª 2011 FEBS pronounced effect. For example, the G80R yeast PAH1 allele showed essentially no enzyme activity [19]. The same substitution within N-LIP domain was found to be crucial for the fat-regulating function of Lipin-1 in mice [32]. Mild modifications (such as G to A) that do not result in dramatic changes in polarity or steric properties may not be sufficient to completely hinder enzyme activity. To determine whether the previous results with respect to enzyme activity were related to other effects on lipid metabolism, we examined the yeast lipid com- position in stationary phase of pah1D cells expressing mutant alleles of AtPAH1 and BnPAH1A (Fig. 5). As demonstrated previously, the TAG ⁄ PL ratio in the pah1D strain was increased by the expression of AtPAH1 and BnPAH1A genes. However, the expres- sion of their respective mutant alleles within C-LIP domain did not affect TAG ⁄ PL ratio compared to the negative control. Similarly, the expression of mutant AtPAH1 and BnPAH1A C-LIP mutant alleles did not complement the temperature sensitivity of pah1D cells at 37 °C (Fig. 6). The effect of the G83A mutation in BnPAH1A appears to be comparatively mild and might be attributed to unique attributes of BnPAH1A together with a more conservative change as outlined above (Figs 4B, 5B and 6B). In conclusion, amino acid substitutions in the conserved C-LIP domain, including the HAD-like motif of AtPAH1 and BnPAH1A, resulted in the loss of PAP activity. Mutation of G83 in the N-LIP domain also produced a significant reduction in enzyme activity and was less severe for BnPAH1A. The results obtained from temperature growth sensitivity analysis correlated with changes in lipid composition in the pah1D strain and indicate a close relationship between enzyme activity and the different phenotypes. The findings of the present study are in agreement with earlier mutational analysis stud- ies carried out with yeast PAH1 [19]. Furthermore, in the study performed by Han et al. [19], the lack of complementation by the D398E and D400E mutant PAH1 alleles was linked to the specific loss of PAH1- encoded PAP activity. Oleic acid stimulates translocation of GFP-AtPAH1 from the cytosol to the nucleus in the yeast cell We have previously determined that plant PAH1 homologs are present in both soluble and membrane fractions of yeast cells (Fig. 2C). To obtain a more com- prehensive understanding of the subcellular localization of these enzymes, we prepared a construct encoding an N-terminal green fluorescent protein (GFP)-fusion with AtPAH1, and expressed this construct in pah1D under the control of the GAL1 promoter. As shown in Fig. 7A, the GFP-AtPAH1 fusion was present through- out the cytoplasm as a soluble protein, in agreement with the immunoblot on Fig. 2C, and apparently absent in the nucleus. Previous localization studies using yeast PAH1-GFP fusions also indicated that PAH1 was present throughout the cytoplasm [16,33]. Confocal microscopy of GFP-AtPAH1 fusion expressed in Nicotiana benthamiana agrobacterium-infiltrated leaves indicated that the fusion protein was located predomi- nantly in the cytosol [25]. In the case of mammalian PAH1 homologs, both mouse Lipin-1 isoforms can localize to either the cytosol or nucleus. The majority of Lipin-1B is present in the cytosol and the remaining Lipin-1A is prevalent in the nucleus of mature adipo- cytes [3]. PAP activity is primarily cytosolic but, after fatty acid stimulation, it can be largely detected in the 0.0 0.1 0.2 0.3 0.4 0.5 mol TAG/mol PL A 0 0.04 0.08 0.0 0.1 0.2 0.3 0.4 0.5 mol TAG/mol PL B 0 0.04 0.08 Fig. 5. Mutations within N-LIP and C-LIP domains influence the ability of plant PAH1 to restore TAG synthesis in yeast pah1D strain. (A) TAG ⁄ PL ratio of the S. cerevisiae pah1D transformed with AtPAH1 and its mutant alleles: G83A, D707A, D709A and S752A. (B) TAG ⁄ PL ratio of S. cerevisiae pah1D transformed with BnPAH1A and its mutant alleles: G83A, D616A, D618A. Lipids were extracted from yeast cells after 48 h of induction and separated on TLC plates. Spots corresponding to TAG and PL were scraped out and transmethylated. FAMEs were analyzed by GC. Each bar represents the mean ± SD from three determinations. WT yeast and pah1D strain (LacZ) previously transformed with LacZ were used as a positive and negative control, respectively. E. Mietkiewska et al. Arabidopsis and B. napus PAHs homologs of PAH1 FEBS Journal 278 (2011) 764–775 ª 2011 The Authors Journal compilation ª 2011 FEBS 769 endoplasmic reticulum, as described previously in rat hepatocytes [34,35], as well as in the developing seeds of safflower, following stimulation with oleic acid [36]. The yeast expression system used in the present study offers the versatility of controlling environmental conditions and stimuli. Therefore, we aimed to determine whether oleic acid supplementation could affect the subcellular localization of GFP-AtPAH1. When cells were cultivated in the presence of 125 lm oleic acid, fluores- cence was found almost exclusively in the nucleus (Fig. 7B). This suggests that AtPAH1 might be involved in transcriptional gene regulation, although additional studies are required to address this hypothesis. We have shown that conserved residues G83, D707, D709 and S752 are essential for PAP activity of Arabidopsis PAH1 (Fig. 4A). To determine the significance of these conserved residues for nuclear localization, we intro- duced point mutations into a GFP-AtPAH1 fusion at the respective sites of AtPAH1 and investigated whether they exhibited oleate-induced nuclear localization. As shown in Fig. 7C–F, GFP-AtPAH1 mutant alleles localized to the nucleus of yeast cells cultivated in the presence of 125 lm oleic acid. These results demonstrated that conserved amino acid residues G83, D707, D709 and S752 are required for PAP activity of AtPAH1, although they are not required for nuclear localization. Previously, Santos-Rosa et al. [20] indicated that yeast PAH1 could also play a role in transcriptional regulation of PL synthesis. In addition, mammalian Lipin-1 has been also suggested to act as a transcriptional co-activator in the regulation of lipid DICDAPI GFP Merge A B C E D F Fig. 7. Localization of GFP-AtPAH1 fusion in yeast cells is influ- enced by oleic acid supplementation. Yeast pah1D cells expressing GFP-AtPAH1 and the ensuing mutants were cultivated for 16 h in induction medium containing 2% galactose without or with supple- mentation with 125 l M oleic acid. (A) Cells expressing GFP-AtPAH1 cultivated without oleic acid. (B) Cells expressing GFP-AtPAH1 culti- vated with oleic acid. (C–F) Cells expressing GFP-AtPAH1 G83A, GFP-AtPAH1 D707A, GFP-AtPAH1 D709A and GFP-AtPAH1 S752A cultivated with oleic acid, respectively. Nuclei were detected by DNA staining with DAPI in the blue channel and recombinant AtPAH1 was detected with GFP in the green channel. Fluorescence signals were examined using a Leica TCS-SP5 multiphoton confo- cal laser scanning microscope. Arrows indicate the position of a representative nucleus in each micrograph. Scale bar = 2 lm. DIC, Differential interference contrast. AtPAH1 G83A D707A D709A S752A LacZ LacZ pah1Δ Wild type Induced Not induced OD 1 10 10 2 10 3 10 4 Dilutions BnPAH1A G83A D616A D618A LacZ LacZ Wild type pah1Δ A B Fig. 6. Plant PAH1 homologs containing mutations in the N-LIP and C-LIP domains fail to rescue the temperature sensitivity of pah1D cells. (A) Yeast pah1D expressing AtPAH1 and its mutant alleles: G83A, D707A, D709A and S752A. (B) Yeast pah1D expressing BnPAH1A and its mutant alleles: G83A, D616A, D618A. Yeast pah1D expressing AtPAH1, BnPAH1A and their respective mutants were cultivated in liquid medium. The density of resulting cultures was adjusted to D 600 = 1 followed by 10-fold serial dilutions. Five microli- ters of each dilution were spotted onto plates containing 2% galac- tose (induced) or 2% raffinose (not induced) and incubated for 3 days at 37 °C. WT yeast and pah1D strain previously transformed with LacZ were used as a positive and negative control, respectively. Arabidopsis and B. napus PAHs homologs of PAH1 E. Mietkiewska et al. 770 FEBS Journal 278 (2011) 764–775 ª 2011 The Authors Journal compilation ª 2011 FEBS metabolism gene expression [27,37]. Similar to the find- ings of the present study, Donkor et al. [9] have shown that the equivalent serine residue in mouse Lipin-1 and Lipin-2 was required for PAP activity, although it was not required for transcriptional co-activator function. The results obtained in the present study suggest that similar mechanisms of PAH1 trafficking to the nucleus might also be present in plants. Eastmond et al. [25] determined that the expression of AtPAH1 and AtPAH2 is considerably higher in developing seeds, where a substantial flux of FAs to TAG occurs. Similar analyses carried out in our laboratory corroborate these findings (Fig. 8). However, analysis of lipids from seeds of an Arabidopsis Atpah1 ⁄ Atpah2 double knockout showed that TAG content was not substantially reduced relative to WT [25]. Therefore, although mRNA accu- mulation suggests that PAHs might have a key function in seeds, their role in storage lipid metabolism remains unclear. Materials and methods Plant material A. thaliana plants (Columbia-O) were cultivated in a growth chamber at 22 °C with an 18 h photoperiod (120 lEÆm )2 Æs )1 ). Cloning and expression of Arabidopsis and B. napus PAHs A. thaliana PAH1 ORF was amplified from a cDNA clone obtained from the Arabidopsis Biological Resource Center using primers: F1: 5¢-ATA GGTACCTATGGGGTTGGTT GGAAGAG-3¢ (KpnI site is underlined) and R1: 5¢-CGC GCGGCCGCTCATTCTACCTCTTCTATTGGCA-3¢ (NotI site is underlined) and ligated into the pYES2 ⁄ NT (Invitro- gen, Burlington, ON, Canada) yeast expression vector at the KpnI and NotI restriction sites. A. thaliana PAH2 ORF was amplified using a cDNA preparation from developing seeds with primers: F2: 5¢-ATA GGATCCAGATGAATG CCGTCGGTAGG-3¢ (BamHI site is underlined) and R2: 5¢-CGC GCGGCCGCTCACATAAGCGATGGAGGAG-3¢ (NotI site is underlined) and then ligated into the pYES2 ⁄ NT vector at the BamHI and NotI sites. Under the control of the GAL1 promoter, the PAH1 genes in the pYES2 ⁄ NT yeast vector were expressed as an N-terminal fusion protein to the Xpress epitope and polyhistidine (6 · His) tag. B. napus PAHs were isolated using sequence information identified in ESTs database (http://brassica. bbsrc.ac.uk). These partial B. napus PAH1 homolog sequences were used to design primers to amplify the 5¢ and 3¢ ends of the cDNA using the SMART RACE cDNA Amplification kit (Clontech, Palo Alto, CA, USA) and a cDNA preparation from B. napus developing seeds. After sequence assembly to determine the full-length sequence of the cDNA, the ORF was amplified using the primer F3: 5¢-ATA GGTACCTATGAGTTTGGTCGGAAG-3¢ (KpnI site is underlined) and R3: 5¢-CGC GCGGCCGCTCAGT- CAACCTCTTCTACCG-3¢ (NotI site is underlined), and subsequently cloned into the KpnI and NotI sites of pYES2.1 ⁄ NT expression vector. The Arabidopsis and B. napus PAHs in pYES2.1 ⁄ NT were transformed into S. cerevisiae mutant strain pah1D [19] using the S. c. EasyComp transformation kit (Invitrogen). For yeast cells, the pah1D mutant and WT (Inv Sc1 strain; Invitrogen) transformed with pYES2.1 ⁄ NT ⁄ lacZ plasmid (Invitrogen), designated as LacZ and WT in the present study, were used as controls. The transformants were selected and grown as described previously [38]. Briefly, yeast cultures were culti- vated in minimal medium containing 0.67% (w ⁄ v) yeast nitrogen base, 2% (w ⁄ v) raffinose, 20 mgÆL )1 adenine, argi- nine, tryptophan, methionine, histidine and tyrosine, 30 mgÆL )1 lysine and 100 mgÆL )1 leucine. The cultures were grown at 30 °C in a rotary shaker at 250 r.p.m. Expression of the recombinant genes was induced using minimal medium containing 2% (w ⁄ v) galactose and 1% (w ⁄ v) raffinose. Site-directed mutagenesis studies To introduce point mutations into the Arabidopsis PAH1 coding region, a QuikChangeÔ Site-Directed Mutagenesis kit (Stratagene, Mississauga, ON, Canada) was used. The primers used were: G83A (F4: 5¢-ATGTATCTTGA TAATTCTGCTGAAGCATATTTCATCAGG-3¢ and R4: 5¢-CCTGATGAAATATGCTTCAGCAGAATTATCAAG ATACAT-3¢); D707A (F5: 5¢- ACCAAGATAGTGATTT 0 1 2 3 4 5 6 7 8 Leaves Flowers Buds Roots Stems Siliques Relative expression AtPAH1 AtPAH2 Fig. 8. Expression profile of Arabidopsis PAHs. Total RNA was obtained from tissues of mature Arabidopsis plants as well as from developing green siliques. Equal amounts of total RNA were used for cDNA synthesis and serial dilutions of the resulting reaction were used for quantitative RT-PCR. Each bar represents the mean ± SD from three determinations with individual reference genes (At4g34270, At4g33380 and At1g58050). E. Mietkiewska et al. Arabidopsis and B. napus PAHs homologs of PAH1 FEBS Journal 278 (2011) 764–775 ª 2011 The Authors Journal compilation ª 2011 FEBS 771 CAGCTGTTGATGGAACTATAAC-3¢, R5: 5¢-GTTATA GTTCCATCAACAGC TGAAATCACTA TCTTGGT-3¢); D709A (F6: 5¢-TAGTGATTTCAGATGTTGCTGGAACT ATAACTAAATC-3¢, R6: 5¢-GATTTAGTTATAGTTCC AGCAACATCTGAAATCACTA-3¢); and S752A (F7: 5¢-CAGTTACTGTTTTTGGCCGCTCGTGCCATCGTTC-3¢ and R7: 5¢-GAACGATGGCACGAGCGGCCAAAAA CAGTAACTG-3¢). The primers used to introduce a point mutation into BnPAH1A were: G83A (F8: 5¢-TATCTAGA CAATTCCGCGGAAGCGTATTTCATC-3¢ and R8: 5¢- GATGAAATACGCTTCCGCGGAATTGTCTAGATA-3¢); D6161A (F9: 5¢-GATTGTAATTTCAGCTGTTGATGGA ACTATA-3¢ and R9: 5¢-TATAGTTCCATCAACAGCTG AAATTACAATC-3¢); and D618A (F10: 5¢-GTAATTTCA GATGTTGCTGGAACTATAACTAAA-3¢ and R10: 5¢-TTT AGTTATAGTTCCAGCAACATCTGAAATTAC-3¢). Primers were complementary to opposite strands of pYES2.1 ⁄ NT (Invitrogen) yeast expression vector contain- ing either the Arabidopsis PAH1 or B. napus PAH1A gene. The presence of the desired mutation was confirmed by DNA sequencing. Preparation of the GFP-AtPAH1 fusion construct To prepare the GFP-AtPAH1 fusion, the coding sequences of GFP and Arabidopsis PAH1 were PCR amplified sepa- rately using Pfx Platinum polymerase (Invitrogen), which was used for all PCR reactions in the present study. The GFP fragment was generated by PCR with primers F11: 5¢-ATA GGTACCTATGACGCACAATCCCACTATC-3¢ (Kpn I site is underlined) and R11: 5¢-CCAACTCTTCCAACCAACCC CATTTTGTATAGTTCATCCATGCCATG-3¢ (the sequence found in AtPAH1 is in italics). Arabidopsis PAH1 fragment was amplified with primers: F12: 5¢-CATGGCATGGATG AACTATACAAAATGGGGTTGGTTGGAAGAGTTGG- 3¢ (the sequence found in GFP is in bold) and R12: 5¢-C GC GCGGCCGCTCATT CTACCTCTTC TATTGGCA-3 ¢ (NotI site is underlined). The resulting amplicons were combined, re-amplified with primers F11 and R12 and then cloned into the KpnI and NotI sites of pYES2.1 ⁄ NT (Invitrogen). Point mutations: G83A, D707A, D709A and S752A at the corresponding sites of Arabidopsis PAH1 coding region in GFP-PAH1 fusion construct were introduced with primers as described above using QuikChangeÔ Site-Directed Mutagenesis kit (Stratagene) and their presence was con- firmed by DNA sequencing. Immunodetection Total protein (40 lg) was separated onto an 8% SDS ⁄ PAGE gel using standard protocols [39]. After elec- trophoresis, proteins were electrotransferred (1.5 h at 180 mA and 4 °C) to poly(vinylidene difluoride) membrane (GE Healthcare, Baie d’Urfe, Canada) using a Mini Trans- blot (Bio-Rad, Mississauga, ON, Canada) apparatus and transfer buffer [190 mm glycine, 25 mm Tris, 0.1% SDS, 20% (v ⁄ v) methanol]. Anti-His G-HRP serum (Invitrogen) was used at a dilution of 1 : 10 000. The proteins were detected using the Amersham ECL Plus Western Blotting Detection kit (GE Healthcare). The fluorescent signal was detected with the Tyhoon Imaging System (GE Health- care). Gene expression analysis Total RNA was isolated from Arabidopsis tissues with the RNeasy kit (Qiagen, Mississauga, ON, Canada) and used to synthesize single-stranded cDNA with the Superscript II reverse transcriptase followed by RNAse H treatment (both obtained from Invitrogen). The product of these reactions was used for quantitative RT-PCR using the Platinum SYBR Green qPCR (Invitrogen) in accordance with the manufacturer’s instructions. PCR was performed in a 7900HT Fast Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) and efficiency was calculated through serial dilutions of the initial amount of RNA. The relative expression level was calculated using the comparative C t method after normalizing to controls using three reference genes (At4g34270, At4g33380 and At1g58050) with stable expression levels in Arabidopsis [40]. The pair of primers used for each reference gene was: At4g34270 Ref1Fwd 5¢-CATACTGTGGAAGTGAAGTAGTTGAGAA-3¢ and Ref1Rev 5¢-CTTCCCCCTT TGGATTAGC TTT-3¢; At4g33380 Ref2Fwd 5¢-TTTGAAAAG CTTTGAGGA CAAATCT- 3¢ and Ref2Rev 5¢-TT CTCATTGC GCCACGTTT-3 ¢; At1g58050 Ref3Fwd 5¢-GAATTGCCAGTGAACTTTTCTAACG-3¢ and Ref3Rev 5¢-TCAGCAGACACATTCCAATCTTTC-3¢; AtPAH1 AtPAH1Fwd 5¢-TCACCAGATGGCCTATTTC CA-3¢ and AtPAH1Rev 5¢-GATCTTGAACTCATGAGG TGCTCTT-3¢; and AtPAH2 AtPAH2Fwd 5¢-GCCTCAGT CACAAGACAATTTCTAGT-3¢ and AtPAH2Rev 5¢-AGG CCCATCCGGCAAT-3¢. Lipid analysis Total lipids were extracted from induced yeast cells by the method of Bligh and Dyer [41]. The internal standards of 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine (19 : 0-phosphatidylcholine; 100 lg in methanol) and triheptad- ecanoin (17 : 0-TAG; 50 lg in chloroform) were added to each sample to permit quantitative fatty acid analysis. Lipid extracts were separated by 1D TLC on silica gel plates (SIL G25, 0.25 mm; Macherey-Nagel, Du ¨ ren, Germany) using the solvent system hexane ⁄ diethyl ether ⁄ glacial acetic acid (70 : 30 : 1 v ⁄ v). Lipid classes were visualized under UV after spraying with 0.05% primuline solution. Spots corre- sponding to TAG and PL were scraped out and transme- thylated with 3 m methanolic HCl at 80 °C for 1 h. The fatty acid methyl esters (FAMEs) were extracted with Arabidopsis and B. napus PAHs homologs of PAH1 E. Mietkiewska et al. 772 FEBS Journal 278 (2011) 764–775 ª 2011 The Authors Journal compilation ª 2011 FEBS hexane and dried under N 2 . Finally, FAMEs were resus- pended in 1 mL of iso-octane with an internal standard (21 : 0, methyl heneicosanoin, 0.1 mgÆmL )1 ). FAMEs were analyzed on an Agilent6890N Gas Chromatograph (Agilent Technologies, Wilmington, DE, USA) with a 5975 inert XL Mass Selective Detector equipped with an auto sampler. FAMEs were separated using a DB-23 capillary column (30 m · 0.25 mm · 0.25 lm) with a constant helium flow of 1.2 mLÆmin )1 and the temperature program: 165 °C hold for 4 min, 10 °CÆmin )1 to 180 °C, hold 5 min and 10 °CÆmin )1 to 230 °C hold 5 min. Integration events were detected and identified between 2 and 19.5 min, and com- pared against a Nu-Chek 463 gas-liquid chromatography standard (Nu-Chek Prep, Inc., Elysian, MN, USA). Preparation of yeast homogenates and PAP enzyme assay Yeast homogenates were prepared essentially as described by Han et al. [16]. Briefly, cells were harvested and washed with 5 mL of ice-cold isolation buffer (50 mm Tris ⁄ HCl, pH 7.5, 300 mm sucrose, 2 mm dithiothreithol and 0.5 mm phenylmethylsulfonyl fluoride), pelleted by centrifugation and resuspended in 500 lL of isolation buffer. All buffers were pre-treated with AG 50W-X8 (Bio-Rad) ion exchange resin Na + salt form to minimize the presence of Mg 2+ . Cells were broken using three 60-s pulses with a Mini- BeadbeaterTM (BioSpec Products, Bartlesville, OK, USA) using 0.5 mm glass beads. The homogenate was collected and briefly centrifuged to remove unbroken cells. The protein concentration of each lysate preparation was determined using the Bio-Rad method [42]. For the PAP enzyme assay, initially, we used the procedure described by Han et al. [16]. Essentially, this procedure was designed to study the kinetic of purified PAP using a surface dilution kinetic model in which PA is dispersed in micelles of Triton X100. The Mg 2+ -dependent activity, which distin- guishes PAP from LPP activity, was approximately half of the total activity and therefore our differential assay was subject to a larger error than anticipated. We then compared this assay with one that we had designed to measure PAP in homogenates of mammalian cells [43,44]. This latter assay maximizes the level of PAP activity and decreases that of LPP activity. Our optimized assay system contained in a final volume of 0.1 mL: 100 mm Tris buffer, pH 7.5, 1 mm MgCl 2 , 200 lm tetrahydrolipstatin (to inhibit diacylglycerol lipases), 2 mgÆmL )1 fatty acid-poor bovine serum albumin and 0.6 mm PA labeled with [ 3 H]palmitate ( 1 · 10 5 d.p.m. ⁄ assay), which was dispersed in 0.4 mm phosphatidylcholine, and 1 mm EDTA plus 1 mm EGTA that was used to prepare the lipid substrate. Mg 2+ was removed from all buffers by treating with AG 50W-X8 (Bio-Rad) ion exchange resin Na + salt form [44]. Reactions were stopped after incubation at 30 °C with 2.2 mL of chloroform containing 0.08% olive oil as a carrier for neu- tral lipids. Next, 0.8 g of basic alumina was added to absorb the PA and any [ 3 H]palmitate formed by phospholi- pase A type activities [44]. The tubes were centrifuged and 1 mL of the chloroform, which contained the [ 3 H]DAG product, was dried and quantified by scintillation counting. The times of incubation (normally 30 min) were adjusted so that < 15% of the PA was consumed during the incuba- tion. Total PAP activities were calculated from measure- ments at three different protein concentrations to ensure the proportionality of the assay. Parallel incubations were performed in the absence of Mg 2+ to block PAP activity and to measure the LPP activity, which had to be sub- tracted from the total to give the PAP activity. This method gave  10-fold greater total activity than the Triton X-100 micelle assay. The Mg 2+ -independent LPP activity was only  10% of the total activity. Therefore, this assay provided us with a more accurate method of determining PAP activity in homogenates where the measurement of kinetic constants was not required. Confocal microscopy Yeast pah1D cells expressing the GFP-AtPAH1 fusion were induced for 16 h using minimal medium containing 2% (w ⁄ v) galactose, 1% (w ⁄ v) raffinose, 0.6% ethanol ⁄ tylox- apol (5 : 1, v ⁄ v) without or with supplementation with 125 lm oleic acid. A Leica TCS-SP5 multiphoton confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany) was used to examine the subcellular localization of GFP fusions in yeast cells. For the imaging of GFP, a 488 nm laser excitation was used at 30% and 520–570 nm emission. Nuclei were identified by DNA staining with 4,6-diamidino-2-phenylindole dilactate (DAPI; Sigma- Aldrich, Oakville, ON, Canada). Briefly, 5 lL of fresh cell suspension were mixed with 5 lL of 80% glycerol contain- ing 50 ngÆmL )1 of DAPI. The mix was placed onto speci- men slides, covered with a cover glass and visualized immediately. Imaging of DAPI was conducted using a 405 nm laser excitation at 10% and 420–450 nm emission. Data were acquired using a · 63 ⁄ 1.2 HCX PL APO objec- tive. Acknowledgements We are grateful to Dr S. Siniossoglou for providing the pah1D mutant yeast strains. D.N.B. is a Senior Scientist for the Alberta Heritage Foundation for Medical Research. This work was supported by Alberta Innovates Bio Solutions, the Natural Sciences and Engi- neering Research Council of Canada, the Canada Research Chairs Program, the Canada Foundation for Innovation and the University of Alberta. We also thank Crystal Snyder for her critical assessment of the manuscript. E. Mietkiewska et al. Arabidopsis and B. napus PAHs homologs of PAH1 FEBS Journal 278 (2011) 764–775 ª 2011 The Authors Journal compilation ª 2011 FEBS 773 [...]... 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