Regulation of the DPP1-encoded Diacylglycerol Pyrophosphate (DGPP) Phosphatase by Inositol and Growth Phase

The regulation of the Saccharomyces cerevisiae DPP1-encoded diacylglycerol pyrophosphate (DGPP) phosphatase by inositol supplementation and growth phase was examined. Addition of inositol to the growth medium resulted in a dose-dependent increase in the level of DGPP phosphatase activity in both exponential and stationary phase cells. Activity was greater in stationary phase cells when compared with exponential phase cells, and the inositol- and growth phase-dependent regulations of DGPP phosphatase were additive. Analyses of DGPP phosphatase mRNA and protein levels, and expression of β-galactosidase activity driven by a P DPP1 -lacZreporter gene, indicated that a transcriptional mechanism was responsible for this regulation. Regulation of DGPP phosphatase by inositol and growth phase occurred in a manner that was opposite that of many phospholipid biosynthetic enzymes. Regulation of DGPP phosphatase expression by inositol supplementation, but not growth phase, was altered in opi1Δ, ino2Δ, andino4Δ phospholipid synthesis regulatory mutants. CDP-diacylglycerol, a phospholipid pathway intermediate used for the synthesis of phosphatidylserine and phosphatidylinositol, inhibited DGPP phosphatase activity by a mixed mechanism that caused an increase in K m and a decrease inV max. DGPP stimulated the activity of pure phosphatidylserine synthase by a mechanism that increased the affinity of the enzyme for its substrate CDP-diacylglycerol. Phospholipid composition analysis of a dpp1Δ mutant showed that DGPP phosphatase played a role in the regulation of phospholipid metabolism by inositol, as well as regulating the cellular levels of phosphatidylinositol.

The regulation of the Saccharomyces cerevisiae DPP1encoded diacylglycerol pyrophosphate (DGPP) phosphatase by inositol supplementation and growth phase was examined. Addition of inositol to the growth medium resulted in a dose-dependent increase in the level of DGPP phosphatase activity in both exponential and stationary phase cells. Activity was greater in stationary phase cells when compared with exponential phase cells, and the inositol-and growth phase-dependent regulations of DGPP phosphatase were additive. Analyses of DGPP phosphatase mRNA and protein levels, and expression of ␤-galactosidase activity driven by a P DPP1 -lacZ reporter gene, indicated that a transcriptional mechanism was responsible for this regulation. Regulation of DGPP phosphatase by inositol and growth phase occurred in a manner that was opposite that of many phospholipid biosynthetic enzymes. Regulation of DGPP phosphatase expression by inositol supplementation, but not growth phase, was altered in opi1⌬, ino2⌬, and ino4⌬ phospholipid synthesis regulatory mutants. CDP-diacylglycerol, a phospholipid pathway intermediate used for the synthesis of phosphatidylserine and phosphatidylinositol, inhibited DGPP phosphatase activity by a mixed mechanism that caused an increase in K m and a decrease in V max . DGPP stimulated the activity of pure phosphatidylserine synthase by a mechanism that increased the affinity of the enzyme for its substrate CDP-diacylglycerol. Phospholipid composition analysis of a dpp1⌬ mutant showed that DGPP phosphatase played a role in the regulation of phospholipid metabolism by inositol, as well as regulating the cellular levels of phosphatidylinositol.
Mitochondrial membranes also contain phosphatidylglycerol and cardiolipin (1)(2)(3)(4). The synthesis of these phospholipids is a complex process that contains a number of branch points (Fig.  1). PS, PE, and PC are synthesized from PA by the CDP-DG pathway (Fig. 1). CDP-DG is also used for the synthesis of PI and cardiolipin. PE and PC are also synthesized by the CDPethanolamine and CDP-choline pathways, respectively (Fig. 1). The CDP-DG pathway is primarily used by wild-type cells for the synthesis of PE and PC when they are grown in the absence of ethanolamine or choline (1, 2, 4 -6). The CDP-ethanolamine and CDP-choline pathways assume a critical role in phospholipid synthesis when enzymes in the CDP-DG pathway are defective (1,2,4,7). Mutants in the CDP-DG pathway require choline for growth and synthesize PC by way of CDP-choline (8 -15). Mutants defective in the synthesis of PS (8,9) or PE (10,11) can also synthesize PC if they are supplemented with ethanolamine. The ethanolamine is used for PE synthesis by way of CDP-ethanolamine. The PE is subsequently methylated to form PC (Fig. 1).
The CDP-ethanolamine and CDP-choline pathways were once viewed as auxiliary or salvage pathways used by cells when the CDP-DG pathway was compromised (1,2). However, it is now known that these pathways contribute to the synthesis of PE and PC even when wild-type cells are grown in the absence of ethanolamine and choline, respectively (16 -18). For example, the PC synthesized via the CDP-DG pathway is constantly hydrolyzed to free choline and PA by phospholipase D (18,19). Free choline is incorporated back into PC via the CDP-choline pathway, and PA is incorporated into phospholipids via reactions utilizing CDP-DG and DG (2)(3)(4) (Fig. 1).
Genetic, molecular, and biochemical studies have shown that the regulation of phospholipid synthesis is a complex and highly coordinated process (2)(3)(4). The mechanisms that govern this regulation control the mRNA and protein levels of the biosynthetic enzymes, as well as their activity (1)(2)(3)(4). The factors that regulate phospholipid synthesis in S. cerevisiae include water-soluble phospholipid precursors, nucleotides, lipids, and growth phase (1,3,4,7,17).
DGPP is a minor phospholipid recently identified in S. cerevisiae (20). It contains a pyrophosphate group attached to DG (21). DGPP is derived from PA via the reaction catalyzed by PA kinase (20). DGPP phosphatase catalyzes the removal of the ␤-phosphate from DGPP to yield PA and then removes the phosphate from PA to generate DG (20). The function of DGPP has not been established in S. cerevisiae. However, phospholipid composition analysis of a dpp1⌬ mutant devoid of DGPP phosphatase activity (22) has revealed that the DPP1 gene product plays a role in the regulation of phospholipid metabolism (23). The dpp1⌬ mutant exhibits a reduction in the cellular level of PI and an elevation in the levels of PA and DGPP (23). PA plays a central role in phospholipid synthesis as the precursor of all phospholipids synthesized via the CDP-DG, CDP-ethanolamine, and CDP-choline pathways (Fig. 1). Moreover, of all the major phospholipids in S. cerevisiae, PI is the only one that is essential (1,2,24,25).
Because inositol (precursor of PI ( Fig. 1)) has regulatory effects on the expression of many phospholipid biosynthetic enzymes (1,2,4,7,17), we examined the effect of inositol on expression of the DPP1-encoded DGPP phosphatase. We also examined the influence of growth phase on DGPP phosphatase, since it has a major influence on the expression of many phospholipid biosynthetic enzymes (1,2,4,7,17). We discovered that DGPP phosphatase expression was regulated by inositol and growth phase. However, this regulation occurred in an opposite manner to that of most phospholipid biosynthetic enzymes. We also discovered that the activity of DGPP phosphatase was inhibited by CDP-DG and that the activity of PS synthase was stimulated by DGPP. The work reported here increases the understanding of the long and short term regulation of DGPP phosphatase and the influence of the enzyme on phospholipid metabolism.

EXPERIMENTAL PROCEDURES
Materials-All chemicals were reagent grade. Growth medium supplies were purchased from Difco. Radiochemicals were from PerkinElmer Life Sciences. Scintillation counting supplies were from National Diagnostics. Triton X-100, bovine serum albumin, aprotinin, benzamidine, leupeptin, pepstatin, phenylmethylsulfonyl fluoride, diethyl pyrocarbonate, inositol, choline, CDP, and O-nitrophenyl ␤-Dgalactopyranoside were purchased from Sigma. Lipids were purchased from Avanti Polar Lipids. DE52 (DEAE-cellulose) was from Whatman. Protein assay reagent, Zeta Probe TM membranes, electrophoresis reagents, and immunochemical reagents were purchased from Bio-Rad. The NEBlot kit, restriction endonucleases, modifying enzymes, and recombinant Vent DNA polymerase with 5Ј-and 3Ј-exonuclease activity were purchased from New England Biolabs. Primers for polymerase chain reaction were prepared commercially by Genosys Biotechnologies, Inc. Nitrocellulose membranes were purchased from Schleicher & Schuell. ProbeQuant G-50 columns, polyvinylidene difluoride membranes, protein A-Sepharose, and the ECF Western blotting chemifluorescent detection kit were purchased from Amersham Pharmacia Biotech. Silica Gel 60 thin layer chromatography plates were from EM Science.
Strains, Plasmids, and Growth Conditions-The strains and plasmids used in this work are listed in Table I. Methods for yeast growth were performed as described previously (26,27). Yeast cultures were grown in YEPD medium (1% yeast extract, 2% peptone, 2% glucose) containing adenine (40 mg/liter) or in complete synthetic medium minus inositol (28) containing 2% glucose at 30°C. The appropriate amino acid of complete synthetic medium was omitted for selection purposes. Escherichia coli strain DH5␣ was grown in LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl, pH 7.4) at 37°C. Ampicillin (100 g/ml) was added to cultures of DH5␣-carrying plasmids. Media were supplemented with 2% agar for growth on plates. Yeast cell numbers in liquid media were determined spectrophotometrically at an absorbance of 600 nm. Exponential phase corresponded to a cell density of 1 ϫ 10 7 cells/ ml, whereas stationary phase was 1 ϫ 10 8 cells/ml. Stationary phase cultures were harvested 24 h after an initial inoculation density of 1 ϫ 10 6 cells/ml. For the overexpression of CHO1-encoded PS synthase, cells were grown to the exponential phase in complete synthetic medium containing 2% raffinose. Galactose (2%) was then added to the growth medium to induce the expression of PS synthase. Maximum induction (60-fold) was achieved after 2 h.
DNA Manipulations and Amplification of DNA by PCR-Plasmid and genomic DNA preparation, restriction enzyme digestion, and DNA ligations were performed by standard methods (27). Transformation of yeast (29) and E. coli (27) were performed as described previously. Conditions for the amplification of DNA by PCR were optimized as described previously (30). Plasmid maintenance and amplifications were performed in E. coli strain DH5␣.
Construction of Plasmids-Plasmid pJO2 contains the putative promoter of the DPP1 gene fused to the lacZ gene of E. coli. The plasmid was constructed by replacing the EcoRI fragment of plasmid pSD90 with the DPP1 promoter, which was obtained by PCR (primers, 5Ј-GTGAAGAAGCAGGAATTCATAAAGGGACAACACGG-3Ј and 5Ј-GTTTTAATAAACGAAACTGAATTCATTTTGGTCG-3Ј) using strain W303-1A genomic DNA as a template. The PCR primer used in the forward direction corresponds to Ϫ1156 bp to the start codon, and the primer used in the reverse direction corresponds to ϩ25 bp to the start codon. Plasmid pSD90, derived from pMA109, contains the CRD1 promoter. Plasmid pMA109, derived from plasmid YEp357R, contains the PIS1 promoter (31). The correct orientation of the DPP1 promoter in plasmid pJO2 was checked by digestion with PstI and by measurement of ␤-galactosidase activity. The P DPP1 -lacZ construct does not contain any sequences of the DPP1 open reading frame. The pJO2 plasmid was introduced into the indicated strains to examine the expression of the DPP1 gene by measuring ␤-galactosidase activity. A more comprehensive description that includes the synthesis of PA and additional steps in these pathways can be found elsewhere (2,17). The abbreviations used are: PME, phosphatidylmonomethylethanolamine; PDE, phosphatidyldimethylethanolamine; TG, triacylglycerol; PGP, phosphatidylglycerophosphate; CL, cardiolipin. A 1.8-kb insert, containing the CHO1 gene fused to the GAL7 promoter, was released from plasmid YCpGPSS (32) by digestion with SalI/BamHI. This DNA fragment was ligated into the SalI/BamHI sites of pRS425, a multicopy E. coli/yeast shuttle vector containing the LEU2 gene (33) to form plasmid YEpGPSS. This construct was transformed into W303-1A for the overexpression of the CHO1-encoded PS synthase.
RNA Isolation and Northern Blot Analysis-Total yeast RNA was isolated using the methods of Schmitt et al. (34) and Herrick et al. (35). Equal amounts (25 g) of total RNA from each sample were resolved on a 1.1% formaldehyde gel for 2.5 h at 100 V (36). The RNA samples were then transferred to a Zeta Probe TM membrane by vacuum blotting. Pre-hybridization, hybridization with a specific probe, and washes to remove unbound probe were carried out according to the manufacturer's instructions. The DPP1 probe was a 0.87-kb fragment isolated from pDT1-DPP1 by MfeI/BamHI digestion. A TCM1 probe was used as a constitutive standard and a loading control. This probe was generated from an PCR-amplified (primers, 5Ј-CTCACAGAAAGTACGAAG-CACC-3Ј and 5Ј-CAAGTCCTTCTTCAAAGTACC-3Ј) region of genomic DNA. The DPP1 and TCM1 probes were labeled with [␣-32 P]dATP using the NEBlot random primer labeling kit. Unincorporated nucleotides were removed using ProbeQuant G-50 columns. Images of radiolabeled species were acquired by PhosphorImaging analysis.
Preparation of Anti-DGPP Phosphatase Antibodies and Immunoblotting-The peptide sequence SDVTLEEAVTHQRIPDE (residues 263-279 at the C-terminal end of the deduced protein sequence of DPP1) was synthesized and conjugated to carrier protein at Bio-Synthesis, Inc. (Lewisville, TX). Antibodies were raised against the peptide in New Zealand White rabbits by standard procedures (37) at Bio-Synthesis, Inc. The IgG fraction was isolated from antisera by protein A-Sepharose chromatography (37). SDS-polyacrylamide gel electrophoresis (38) using 12% slab gels and immunoblotting (39) using polyvinylidene difluoride membranes were performed as described previously. The anti-DGPP phosphatase antibodies were used at a dilution of 1:1000, and the DGPP phosphatase protein was detected using the ECF Western blotting chemifluorescent detection kit as described by the manufacturer. The DGPP phosphatase protein on immunoblots was acquired by Flu-oroImaging analysis. The relative density of the protein was analyzed using ImageQuant software. Immunoblot signals were in the linear range of detectability.
Preparations of Enzymes-Cells from all strains were disrupted with glass beads (40) in 50 mM Tris maleate buffer, pH 7.0, containing 1 mM Na 2 EDTA, 0.3 M sucrose, 10 mM 2-mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, and 5 g/ml each of aprotinin, leupeptin, and pepstatin. Glass beads and unbroken cells were removed by centrifugation at 1,500 ϫ g for 10 min. The supernatant (cell extract) was used for enzyme assays and immunoblot analysis. DPP1-encoded DGPP phosphatase (20) and CHO1-encoded PS synthase (41) were purified to near homogeneity as described previously. The specific activities of DGPP phosphatase and PS synthase were 60 and 2 mol/min/mg, respectively.

Preparation of Triton X-100/Phospholipid Mixed Micelles-Phospho-
lipids in chloroform were transferred to a test tube, and solvent was removed in vacuo for 40 min. The surface concentration of phospholipids in Triton X-100/phospholipid mixed micelles was varied by the addition of various amounts of an 80 mM solution of Triton X-100 to the dried phospholipids. The total phospholipid concentration in the mixed micelles did not exceed 15 mol % to ensure that the structure of the micelles was similar to that of pure Triton X-100 (44,45). The mol % of a phospholipid in a mixed micelle was calculated with the following formula: Enzyme Assays, Protein Determination, and Analysis of Kinetic Data-DGPP phosphatase activity was measured by following the release of water-soluble 32 P i from chloroform-soluble [␤-32 P]DGPP (10,000 -15,000 cpm/nmol) as described by Wu et al. (20). The reaction mixture contained 50 mM citrate buffer, pH 5.0, 2 mM Triton X-100, 0.1 mM DGPP, and enzyme protein in a total volume of 0.1 ml. ␤-Galactosidase activity was determined by measuring the conversion of Onitrophenyl ␤-D-galactopyranoside to O-nitrophenol (molar extinction coefficient of 3,500 M Ϫ1 cm Ϫ1 ) by following the increase in absorbance at 410 nm on a recording spectrophotometer (46). The reaction mixture contained 100 mM sodium phosphate buffer, pH 7.0, 3 mM O-nitrophenyl ␤-D-galactopyranoside, 1 mM MgCl 2 , 100 mM 2-mercaptoethanol, and enzyme protein in a total volume of 0.1 ml. PS synthase activity was measured by following the incorporation of water-soluble [3-3 H]serine (10,000 -40,000 cpm/nmol) into chloroform-soluble PS as described by Bae-Lee and Carman (41). The reaction mixture contained 50 mM Tris-HCl buffer, pH 8.0, 10 mM MgCl 2 , 3.2 mM Triton X-100, 0.2 mM CDP-diacylglycerol, 0.5 mM serine, and enzyme protein in a total volume of 0.1 ml. MgCl 2 was used instead of MnCl 2 (the alternative cofactor (41)) because DGPP chelates manganese ions. DGPP phosphatase and ␤-galactosidase assays were conducted at 30 and 25°C, respectively. The average standard deviation of the enzyme assays (performed in triplicate) was Ϯ 5%. The enzyme reactions were linear with time and protein concentration. A unit of enzymatic activity was defined as the amount of enzyme that catalyzed the formation of 1 mol of product/min unless otherwise indicated. Specific activity was defined as units/mg protein. Protein concentration was determined by the method of Bradford (47) using bovine serum albumin as the standard. Kinetic data were analyzed with the EZ-FIT enzyme kinetic modelfitting program according to the Michaelis-Menten and Hill equations. EZ-FIT uses the Nelder-Mead Simplex and Marquardt/Nash nonlinear regression algorithms sequentially and tests for the best fit of the data among different kinetic models (48). IC 50 values were calculated from plots of the log of activity versus the inhibitor concentration.
Labeling and Analysis of Phospholipids-Labeling of phospholipids with 32 P i was performed as described previously (8,9,49). Lipids were extracted from labeled cells by the method of Bligh and Dyer (50) as described previously (51). Phospholipids were separated by DEAEcellulose chromatography followed by one-dimensional thin layer chromatography on silica gel plates (52). 2 DEAE-cellulose (acetate form) 2 This method of phospholipid analysis obviated the frustrations en- . PC and PE emerged in fraction 1; PI, PS, and PA emerged in fraction 2, and DGPP and CDP-DG emerged in fraction 3. Chloroform and water were added to each fraction so that the final ratio of chloroform/methanol/ aqueous solvent was 1:1:1 (v/v). The system was mixed; the phases were separated, and the chloroform phase was dried in vacuo. Samples from each fraction were dissolved in chloroform/methanol (9:1) and subjected to one-dimensional thin layer chromatography on silica gel plates using the solvent system chloroform/pyridine/88% formic acid/methanol/water (60:35:10:5:2, v/v). Prior to the application of the samples, 20 l of a 3% solution of butylated hydroxyanisol in methanol was applied to the origin to protect samples against oxidation. The 32 P-labeled phospholipids were visualized and quantified by PhosphorImaging analysis. The positions of the labeled phospholipids on chromatography plates were compared with standard lipids after exposure to iodine vapor.

Effects of Inositol and Growth
Phase on the Level of DGPP Phosphatase Activity-We examined the effect of inositol on the level of DGPP phosphatase activity. Wild-type cells were grown in complete synthetic medium in the absence and presence of various concentrations of inositol. Cultures were harvested in the exponential phase of growth. Cell extracts were prepared and assayed for DGPP phosphatase activity. The addition of inositol to the growth medium resulted in a dose-dependent increase in DGPP phosphatase activity ( Fig. 2A). Maximum DGPP phosphatase activity was found in cells grown with 40 -60 M inositol. This level of activity was 2-fold greater than that found in cells grown without inositol. Concentrations of inositol above 60 M led to a dose-dependent decrease in DGPP phosphatase activity ( Fig. 2A). The reason for this effect was unclear and was not examined further. Studies with the purified DGPP phosphatase enzyme showed that the effect of inositol on the level of DGPP phosphatase activity was not due to a direct effect of inositol on the enzyme (data not shown).
We examined the effect of growth phase on the level of DGPP phosphatase activity. The specific activity of DGPP phosphatase in stationary phase cells was elevated (2.2-fold) when compared with the activity in exponential phase cells (Fig. 2B). In addition, the inositol-dependent regulation of DGPP phosphatase activity was also observed in stationary phase cells (Fig. 2B). The expression of activity reached a maximum be-tween 60 and 100 M inositol. The growth phase-dependent and inositol-dependent regulation of DGPP phosphatase activity appeared to be additive. The specific activity of DGPP phosphatase in inositol-supplemented stationary phase cells was 5.5-fold greater than the activity of the enzyme in exponential phase cells grown without inositol (Fig. 2).
Effect of Inositol and Growth Phase on DGPP Phosphatase mRNA Abundance and Protein Levels-To gain insight into the mechanism of DGPP phosphatase regulation by inositol and growth phase, the amount of DPP1 mRNA was examined. Wild-type cells were grown in complete synthetic medium in the absence and presence of 50 M inositol. This concentration of inositol is commonly used for studies on the regulation of phospholipid synthesis by inositol (1,2). Total RNA was isolated from exponential and stationary phase cells and used for Northern blot analysis with a DPP1 probe. The expression of TCM1 mRNA was also determined and served as a loading control. The TCM1 gene encodes a ribosomal protein that is not regulated by inositol supplementation (53,54). This analysis showed that the presence of inositol in the growth medium resulted in an increase in the relative amount of DPP1 mRNA in both exponential (Fig. 3A) and stationary (Fig. 3C) phase cells. This analysis also showed that the relative amount of DPP1 mRNA in stationary phase cells supplemented with inositol was greater than that found in exponential phase cells grown without inositol. During the course of these experiments, we noted that the total RNA derived from stationary phase cells was especially subject to degradation during its isolation. Thus, it was difficult to quantify the levels of mRNA.
The levels of the DGPP phosphatase protein in response to inositol supplementation and growth phase were examined. Antibodies were generated against a peptide sequence found at the C-terminal end of the DGPP phosphatase protein. These antibodies recognized purified DGPP phosphatase (data not shown), as well as the enzyme in cell extracts. DGPP phosphatase migrates on SDS-polyacrylamide gels as a doublet with a molecular mass of ϳ34 kDa (20,55). Immunoblot analysis using these antibodies showed that the levels of the DGPP phosphatase protein were elevated (4-and 3-fold, respectively) in response to inositol supplementation in both the exponential (Fig. 3B) and stationary (Fig. 3D) phases of growth. This analysis also showed that in cells grown without inositol, the level of the DGPP phosphatase protein was elevated (3-fold) in stationary phase when compared with the exponential phase of growth. The amount of the DGPP phosphatase protein in stationary phase cells supplemented with inositol was 7-fold greater when compared with that of exponential phase cells grown without inositol. These results were consistent with the countered with the poor resolution of phospholipids using two-dimensional thin layer chromatography due to humid summer days in New Jersey.

FIG. 2. Effect of inositol supplementation on the level of DGPP phosphatase activity in the exponential and stationary phases of growth.
Wildtype cells were grown in complete synthetic media with the indicated concentrations of inositol. Cultures were harvested in the exponential (A) and stationary (B) phases of growth. Cell extracts were prepared and assayed for DGPP phosphatase activity. Each data point represents the average of triplicate enzyme determinations from a minimum of two independent experiments Ϯ S.D. levels of DGPP phosphatase activity found in these cells (Fig. 2).
Effect of Inositol and Growth Phase on the Expression of ␤-Galactosidase Activity in Cells Bearing the P DPP1 -lacZ Reporter Gene-We utilized a P DPP1 -lacZ reporter gene to facilitate further studies on the regulation of DPP1 expression. The P DPP1 -lacZ gene in plasmid pJO2 was constructed by fusing the DPP1 promoter in frame with the coding sequence of the E. coli lacZ gene. Thus, the expression of ␤-galactosidase activity was dependent on transcription driven by the DPP1 promoter. The ␤-galactosidase activity in extracts derived from wild-type cells bearing plasmid pJO2 was linear with time and with protein concentration (data not shown). To verify that this reporter gene could be used to examine the expression of the DPP1 gene, we examined the regulation of ␤-galactosidase activity in exponential phase cells supplemented with inositol. The addition of inositol to the growth medium resulted in a dose-dependent increase in ␤-galactosidase activity (Fig. 4). The level of activity in cell extracts derived from cells supplemented with 40 -50 M inositol was 2.6-fold greater than the activity from cells grown in the absence of inositol (Fig. 4). The addition of inositol to cells at concentrations greater than 50 M inositol resulted in a dose-dependent decrease in ␤-galactosidase activity (Fig. 4). These results were consistent with the data on DGPP phosphatase activity (Fig. 2A).
The ␤-galactosidase activity, driven by the P DPP1 -lacZ reporter gene, was also measured in stationary phase cells grown in the absence and presence of 50 M inositol (Fig. 5C). In the absence of inositol supplementation, the ␤-galactosidase activity in stationary phase cells was 4.6-fold greater then the activity found in exponential phase cells (Fig. 5A). The level of ␤-galactosidase activity in inositol-supplemented stationary phase cells was 1.8-fold greater when compared with stationary phase cells grown without inositol (Fig. 5C). The specific activity of ␤-galactosidase in stationary phase cells supplemented with inositol (Fig. 5C) was 8.5-fold greater than that of exponential phase cells grown with inositol (Fig. 5A). These data further confirmed that a transcriptional mechanism was responsible for the changes in DGPP phosphatase activity (Fig. 5,   B and D) that occurred in response to inositol supplementation and growth phase.
For many phospholipid biosynthetic enzymes, the repressive effect of inositol is enhanced by the inclusion of 1 mM choline to the growth medium (1,2,4,7,17). We examined whether choline alone or in combination with inositol affected the expression of the DPP1 gene and DGPP phosphatase activity. This analysis showed that choline supplementation had no effect on the regulation of DGPP phosphatase (data not shown).

Regulation of DGPP Phosphatase by Inositol and Growth Phase in Mutants Defective in the OPI1, INO2, and INO4
Regulatory Genes-We examined the regulation of DGPP phosphatase in mutants defective in the OPI1, INO2, and INO4 regulatory genes. Opi1p, Ino2p, and Ino4p are transcriptional regulators of phospholipid biosynthetic enzymes that are repressed by inositol (1, 2, 4, 7). For example, Opi1p represses the expression of INO1-encoded inositol-1-P synthase and CHO1encoded PS synthase, whereas Ino2p and Ino4p induce the expression of these enzymes (40, 54, 56 -65). An opi1 mutant exhibits elevated expression of phospholipid biosynthetic enzymes, and the expression of these enzymes does not respond to inositol supplementation (1,7,17). Owing to the constitutive low expression of the INO1 gene (53), ino2 and ino4 mutants are inositol auxotrophs (28). These mutants also exhibit repressed levels of the phospholipid biosynthetic enzymes that are repressed by inositol (1,7,17,66,67). Moreover, the expression of the inositol-regulated enzymes in ino2 and ino4 mutants is not affected by inositol supplementation (1,7,17,66,67). For cells grown to exponential phase without inositol, the expression of P DPP1 -lacZ driven ␤-galactosidase activity was 2-fold lower in opi1⌬ mutant cells when compared with wild-type cells (Fig. 5A). In contrast to wild-type cells, the addition of inositol to the growth medium of opi1⌬ cells did not result in an increase in DPP1 expression. Instead, inositol supplementation caused a small decrease in DPP1 expression. We next examined the expression of DGPP phosphatase activity in exponential phase opi1⌬ cells (Fig. 5B). The DGPP phosphatase activity in cells grown in the absence of inositol was slightly higher than that found in wild-type cells. This level of DGPP phosphatase activity did not correlate with the reduced level of ␤-galactosidase activity in opi1⌬ cells when compared with the wild-type control (Fig. 5A). Whether this difference was due to transcriptional control versus translational control will require additional studies. The addition of inositol to opi1⌬ cells resulted in a small reduction in DGPP phosphatase activ- ity (Fig. 5B), which correlated with the small effect of inositol on the expression of ␤-galactosidase activity (Fig. 5A).
The growth phase-dependent regulation of DGPP phosphatase was examined in opi1⌬ mutant cells (Fig. 5, C and D). As in wild-type cells, the expressions of ␤-galactosidase and DGPP phosphatase activities were elevated in stationary phase opi1⌬ cells when compared with these activities in exponential phase cells. However, in contrast to the lower activity found in the exponential phase, the level of expression of ␤-galactosidase activity in stationary phase of opi1⌬ cells was similar to the ␤-galactosidase expression found in wild-type stationary phase cells. Like exponential phase opi1⌬ cells, the expression of ␤-galactosidase and DGPP phosphatase activities did not increase in response to inositol supplementation in stationary phase (Fig. 5, C and D).
The regulation of DGPP phosphatase was examined in ino2⌬ and ino4⌬ mutant cells grown in the presence of 10 and 60 M inositol. The 10 M concentration of inositol was required to support the growth of these inositol auxotrophs (68). This growth condition was considered analogous to the growth condition of wild-type cells grown in the absence of inositol (53). The expression of ␤-galactosidase activity in ino2⌬ mutant cells was reduced in the exponential phase of growth when compared with expression of ␤-galactosidase activity in wild-type cells (Fig. 5A). However, ino4⌬ mutant cells showed ␤-galactosidase activity that was slightly higher than that found in wild-type cells (Fig. 5A). The DGPP phosphatase activity in exponential phase ino2⌬ and ino4⌬ mutant cells was similar to that of wild-type cells (Fig. 5B). The addition of 60 M inositol to the growth medium had small effects on the expression of both ␤-galactosidase (Fig. 5A) and DGPP phosphatase (Fig. 5B) activities in the ino2⌬ and ino4⌬ mutant cells.
The effects of the ino2⌬ and ino4⌬ mutations on DGPP phosphatase regulation by growth phase were examined. The ␤-galactosidase (Fig. 5C) and DGPP phosphatase (Fig. 5D) activities in stationary phase ino2⌬ and ino4⌬ mutant cells were elevated when compared with these activities from exponential phase ino2⌬ and ino4⌬ mutant cells. However, both ␤-galactosidase and DGPP phosphatase activities were lower (2.7-and 1.6-fold, respectively) in ino2⌬ mutant cells when compared with wild-type cells. The ␤-galactosidase and DGPP phosphatase activities in the ino4⌬ mutant were similar to the levels of these activities found in wild-type cells (Fig. 5, C and  D). The expression of ␤-galactosidase and DGPP phosphatase activities in stationary phase ino2⌬ and ino4⌬ mutant cells supplemented with 60 M inositol were elevated but not to the levels observed in wild-type cell supplemented with inositol (Fig. 5, C and D).
Effect of the dpp1⌬ Mutation on the Regulation of the INO1 Gene by Inositol and Growth Phase-We questioned whether the dpp1⌬ mutation affected the expression of genes encoding phospholipid biosynthetic enzymes. Of these genes, INO1 is the most highly regulated by inositol supplementation (1,2), and it was used as a representative gene for this study. Expression of INO1 in the dpp1⌬ mutant was examined by using a P INO1 -lacZ reporter gene in plasmid pJ699Z. The expression of ␤-galactosidase activity in wild-type and dpp1⌬ mutant cells was the same in both exponential and stationary phase cells (data not shown). As described previously (53,56,57,64,69,70), the INO1 gene was repressed by supplementation with 50 M inositol. The dpp1⌬ mutation did not have a major effect on this regulation (data not shown).
Inhibition of DGPP Phosphatase Activity by CDP-DG-We initiated studies to examine the regulation of DGPP phosphatase activity on a biochemical level. Being a membrane-associated enzyme (20,22), DGPP phosphatase has a distinct relationship with its neighboring phospholipids. Thus, we examined whether phospholipids played a role in the biochemical regulation of the enzyme. For these studies, we used purified DGPP phosphatase and Triton X-100/phospholipid mixed micelles so the effects of phospholipids on activity could be examined under well defined conditions. The nonionic detergent Triton X-100 is required to elicit a maximum turnover for DGPP phosphatase activity in vitro (20). The function of Triton X-100 in the assay system for DGPP phosphatase is to form a uniform mixed micelle with the substrate DGPP (20). The Triton X-100 micelle serves as a catalytically inert matrix in which the DGPP is dispersed, preventing a high local concentration of substrate at the active site (71). In addition, this micelle system permitted the analysis of DGPP phosphatase FIG . 5. Effects of the opi1⌬, ino2⌬, and ino4⌬ mutations on the regulation of DGPP phosphatase by inositol and growth phase. Wild-type, opi1⌬, ino2⌬, and ino4⌬ cells bearing the P DPP1 -lacZ reporter plasmid pJO2 were grown in the absence and presence of 50 M inositol. Cultures were harvested in the exponential and stationary phases of growth. Cell extracts were prepared and assayed for ␤-galactosidase activity (A and C) and for DGPP phosphatase activity (B and D). Each data point represents the average of triplicate enzyme determinations from a minimum of two independent experiments Ϯ S.D. WT, wild-type. activity in an environment that mimics the physiological surface of the membrane (71). In Triton X-100/phospholipid-mixed micelles, DGPP phosphatase activity follows surface dilution kinetics (71), where activity is dependent on both the molar and surface concentrations of DGPP (20). In the experiments reported here, DGPP phosphatase activity was measured such that activity was only dependent on the surface concentration of DGPP (20). The concentrations of DGPP and other phospholipids were expressed as a surface concentration (in mol %), as opposed to a molar concentration, since phospholipids form uniform mixed micelles with Triton X-100 (44,45).
DGPP phosphatase activity was assayed in the presence of various phospholipids. The major yeast phospholipids PC, PE, PI, and PS inhibited DGPP phosphatase activity at a final concentration of 10 mol % (Fig. 7A). However, these phospholipids were not considered to be strong inhibitors (30% or less) and were not pursued further. PA, at concentrations up to 10 mol %, does not affect DGPP phosphatase activity (20). On the other hand, the addition of CDP-DG to the assay system resulted in a dose-dependent inhibition (IC 50 ϭ 5.3 mol %) of DGPP phosphatase activity (Fig. 6A). DGPP phosphatase activity was measured in the presence of CDP and DG to examine which part of the CDP-DG molecule was responsible for the inhibition. CDP caused a dose-dependent inhibition (IC 50 ϭ 2.6 mM) of activity (Fig. 6B). DG caused a small increase (25%) in DGPP phosphatase activity at a final concentration of 4 mol % (Fig. 6A). These data indicated that the inhibition by CDP-DG may be attributed to the CDP moiety of the molecule. A kinetic analysis was performed on DGPP phosphatase to explore the mechanism of inhibition by CDP-DG. The dependence of DGPP phosphatase activity on DGPP was examined in the absence and presence of 4 mol % CDP-DG (Fig. 6C). As described previously (20), DGPP phosphatase exhibited saturation kinetics with respect to the surface concentration of DGPP. CDP-DG inhibited DGPP phosphatase activity in a dose-dependent manner at each DGPP concentration. An analysis of the kinetic data showed that CDP-DG was a mixed type of inhibitor (72) causing an increase in K m and a decrease in V max . The K i value for CDP-DG was calculated to be 5 mol %.
Activation of PS Synthase Activity by DGPP-PS synthase is one of the most highly regulated enzymes of phospholipid metabolism (1,2,7,17), and the biochemical regulation of the purified enzyme (41) has been extensively studied (3,4). In light of the fact that CDP-DG regulated DGPP phosphatase activity, we examined the effect of DGPP on PS synthase activity. For these studies, we utilized pure enzyme and the Triton X-100/phospholipid mixed micelle system as discussed above for DGPP phosphatase. The addition of DGPP to the assay system resulted in a dose-dependent stimulation of PS synthase activity (Fig. 7A). The concentration of DGPP that resulted in half-maximum activation (A 0.5 ) was 0.13 mol %. The stimulation by DGPP could be attributed to the pyrophosphate moiety of the molecule since DG does not stimulate PS synthase activity (73). At high concentrations (e.g. 11 mol %), DG inhibits PS synthase activity (73). The dependence of PS synthase activity on the surface concentration of CDP-DG was examined in the absence and presence of 1 mol % DGPP (Fig.  7B). In the absence of DGPP, PS synthase exhibited positive cooperative kinetics (n ϭ 3.2) toward CDP-DG with a K m value of 1.9 mol %. The enzyme was stimulated by DGPP in a dosedependent manner at each CDP-DG concentration. Moreover, DGPP caused a decrease in the cooperative (n ϭ 1.6) kinetic behavior of the enzyme and a decrease in the K m value (0.8 mol %) for CDP-DG. Thus, at low CDP-DG concentrations DGPP had a major stimulatory effect on PS synthase activity. The V max of the reaction was not significantly affected by DGPP. The effect of DGPP (1 mol %) on the kinetics of the enzyme with respect to serine was examined (Fig. 7C). The enzyme exhibited saturation kinetics toward serine in the absence (K m ϭ 2.7 mM) and presence (K m ϭ 2.3 mM) of DGPP. The V max of the reaction in the presence (2.84 units/mg) of DGPP was 2-fold greater than that in absence (1.4 units/mg) of DGPP. These kinetic data indicated that DGPP stimulated PS synthase activity by a mechanism that increased the affinity of the enzyme for CDP-DG (72).
Effect of a dpp1⌬ Mutation on the Phospholipid Composition of Stationary Phase Cells Grown in the Presence of Inositol-We examined the effects of a dpp1⌬ mutation on the phospholipid composition of stationary phase cells. Stationary phase cells that were grown in the absence and presence of 50 M inositol were labeled with 32 P i . Phospholipids were extracted and analyzed as described under "Experimental Procedures." When grown without inositol supplementation, the amounts of the major phospholipids (i.e. PC, PE, PI, and PS) as well as PA and DGPP were not significantly affected by the dpp1⌬ mutation (Table II). The amount of CDP-DG in the dpp1⌬ mutant was nearly 50% greater than that of wild-type cells grown without inositol (Table II). The presence of inositol in the growth medium of wild-type cells resulted in a 4.5-fold increase in PI content and a small increase in CDP-DG content (Table II). On the other hand, the PI content in dpp1⌬ mutant cells increased by only 1.37-fold, and the CDP-DG content did not change when cells were supplemented with inositol (Table II). The PI:PC ratio in the dpp1⌬ mutant grown in the presence of inositol was 4.2-fold lower than that of wild-type cells (Table  II). The amounts of the other phospholipids in the dpp1⌬ mutant supplemented with inositol were not significantly different from that of wild-type cells supplemented with inositol. DISCUSSION The identification and initial analysis of DPP1-encoded DGPP phosphatase indicates that this enzyme plays a previously unidentified role in phospholipid metabolism in S. cerevisiae (20,22). It has been postulated that DGPP may function in a novel lipid-signaling pathway (4,74). Studies with plants, where DGPP was first discovered (21), have shown that this phospholipid accumulates upon G protein activation (75) or hyperosmotic stress (76,77). DGPP accumulation is transient (77), and it is rapidly converted to PA and then to DG (42). In S. cerevisiae, DGPP phosphatase may function to regulate cellular levels of DGPP, PA, and DG (3,4). DGPP has not been identified in mammalian cells. However, Balboa et al. (78) have shown that exogenous DGPP activates mouse macrophages for enhanced secretion of arachidonic acid metabolites, a key event in the immunoinflammatory response of leukocytes.
To gain insight into the role that DGPP phosphatase plays in S. cerevisiae, we examined the effects of inositol supplementation and growth phase on the expression of the enzyme. These two growth conditions have a major impact on the expression of several phospholipid biosynthetic enzymes (1,2,4,7,17). Inositol supplementation to wild-type cells resulted in the elevation of DGPP phosphatase activity in both the exponential and stationary phases of growth. DGPP phosphatase activity was higher in stationary phase cells when compared with exponential phase cells. Moreover, the inositol-and growth phase-dependent regulation of the enzyme were additive. Analyses of the DGPP phosphatase mRNA abundance and protein levels, as well as the expression of ␤-galactosidase activity driven by a P DPP1 -lacZ reporter gene, showed that a transcriptional mechanism was responsible for this regulation. The effects of inositol and growth phase on DGPP phosphatase expression was opposite that of most phospholipid biosynthetic enzymes. For example, CDP-DG pathway enzymes (i.e. CDP-DG synthase, PS synthase, PS decarboxylase, and phospholipid N-methyltransferases) are repressed when wild-type cells are supplemented with inositol and when they enter the stationary phase of growth (1,2,4,7,17). The repression of these enzymes by inositol supplementation or in stationary phase is mediated by a UAS INO cis-acting element (1,64,79) present in the promoters of their genes (1,2,4,7,17). The promoter of the DPP1 gene does not contain a UAS INO element. Additional studies will be required to identify the promoter element(s) responsible for the inositol-and growth phase-dependent regulation of the DPP1 gene.
DGPP phosphatase is not the first example of an enzyme that is regulated by inositol or by growth phase in a manner that is opposite to the co-regulated phospholipid biosynthetic enzymes whose genes contain a UAS INO element (4). These enzymes include 45-kDa Mg 2ϩ -dependent PA phosphatase (51,80) and inositol-1-P phosphatase (81,82). The magnitude of the regulation by inositol supplementation and growth phase observed for DGPP phosphatase was significantly greater than that of these other enzymes. Expression of cardiolipin synthase TABLE II Effect of the dpp1⌬ mutation on phospholipid composition in stationary phase cells grown in the absence and presence of inositol The indicated S. cerevisiae strains were grown to the stationary (1 ϫ 10 8 cells/ml) phase of growth in complete synthetic medium in the absence and presence of 50 M inositol. The steady-state phospholipid composition was determined by labeling cells for 12 generations with 32 P i (5 Ci/ml). The incorporation of 32 P i into phospholipids during the labeling was approximately 2400 cpm/10 8 cells. The phospholipid composition of the cells was determined as described under "Experimental Procedures." The percentages shown for phospholipids were normalized to the total 32 P i -labeled chloroform-soluble fraction that included sphingolipids and other unidentified phospholipids. The values reported are the average of two independent experiments. is not regulated by inositol supplementation, but it is derepressed in stationary phase (83). Phosphatidylglycerophosphate synthase, whose promoter contains a UAS INO element, is repressed by inositol supplementation (84,85) but is derepressed in the stationary phase (86). The UAS INO element contains the binding site for the Ino2p-Ino4p complex, which is necessary for maximum expression of the co-regulated UAS INO -containing genes (4,7,17,53,67). Repression of the co-regulated genes requires the transcription factor Opi1p (59,87). Although Opi1p functions via the UAS INO element (63), it does not bind the element directly and does not interact with Ino2p or Ino4p (88). The DPP1 gene does not have a UAS INO element, yet Opi1p, Ino2p, and Ino4p played a role in its regulation by inositol. In both exponential and stationary phase cells, expression of DPP1 was not regulated normally by inositol in the opi1⌬, ino2⌬, and ino4⌬ mutants. Moreover, the aberrant regulation observed in the ino2⌬ and ino4⌬ mutants differed from each other (expression of DPP1 was greater in the ino4⌬ mutant). This suggests that an Ino2p-Ino4p complex may be required for proper DPP1 regulation. Differential regulatory effects between the ino2⌬ and ino4⌬ mutants have been observed for the 45-kDa Mg 2ϩ -dependent PA phosphatase (51), PI synthase (89), and for cardiolipin synthase. 3 The regulation of DPP1 by growth phase was not affected in the regulatory mutants suggesting that Opi1p, Ino2p, and Ino4p do not play a role in the growth phase-dependent regulation of DGPP phosphatase.
Phospholipid synthesis in S. cerevisiae has been described as existing in two regulatory states designated as "on" and "off" (4). The system is on when the UAS INO -containing genes are maximally expressed and off when they are repressed. When the UAS INO -containing genes are on, the DPP1 gene is off and vice versa. Analysis of phenotypes associated with mutations in the UAS INO -containing genes have led to the hypothesis that PA, or a closely related metabolite, is responsible for generating a signal that causes the derepression of these genes (4,17). According to the model, the system is on when PA is produced more rapidly than it is consumed, and the system is off when PA is consumed more rapidly than it is produced (4). Since DGPP phosphatase is involved with the metabolism of PA, we questioned whether the enzyme was involved in the regulation of the UAS INO -containing genes. The dpp1⌬ mutant did not have a significant effect on the regulation of the INO1 gene by inositol. If DGPP phosphatase were involved, other enzymes (e.g. PA kinase, CDP-DG synthase, and phospholipase D) that catalyze reactions affecting the metabolism of PA may have compensated for the dpp1⌬ mutation. A mutation in the LPP1 gene, which encodes a lipid phosphatase that utilizes PA and DGPP as substrates (23,90), does not affect the regulation of the INO1 gene. 4 CDP-DG and DGPP, substrates of PS synthase and DGPP phosphatase, regulated DGPP phosphatase and PS synthase activities, respectively. CDP-DG was a mixed type inhibitor of DGPP phosphatase. The inhibitor constant for CDP-DG (K i ϭ 5 mol %) was about 12-fold higher than its cellular concentration in stationary phase cells (Table II). On the other hand, the K i value was within the range of the cellular concentration of CDP-DG in exponential phase cells (2-11 mol %) (91,92). Thus, it is unlikely that CDP-DG regulates DGPP phosphatase activity in stationary phase cells, but regulation of the phosphatase by CDP-DG may be physiologically relevant in exponential phase cells. DGPP stimulated PS synthase through a mechanism that involved an increase in the affinity of the enzyme for CDP-DG. The activation constant for DGPP (A 0.5 ϭ 0.13 mol %) was within the range of its cellular concentration in both exponential (0.2-0.4 mol %) (20,22) and stationary (Table II) phase cells. Thus, regulation of PS synthase activity by DGPP may occur in vivo during both phases of growth.
Previous studies have shown that the regulation of PS synthase is the major factor that controls the synthesis of PS and PI from their common precursor CDP-DG (1,3,4). Stimulation of PS synthase by DGPP would favor the synthesis of PS at the expense of PI. DGPP did not affect the activity of PI synthase (data not shown). The partitioning of CDP-DG between PS and PI may be controlled by the activity and/or expression of DGPP phosphatase. For example, reduced levels of DGPP phosphatase would favor the synthesis of PS over PI. Indeed, the major impact of the dpp1⌬ mutation in stationary phase cells supplemented with inositol was a decrease in PI content when compared with wild-type cells. Moreover, this caused a major change in the PI:PC ratio, an index of phospholipid synthesis regulation (93,94).
In summary, the work reported here supported the conclusion that the DPP1-encoded DGPP phosphatase was regulated by genetic and biochemical mechanisms. Moreover, the enzyme played a role in the regulation of phospholipid synthesis by inositol. The synthesis of PI is coordinately regulated with the synthesis of PC by both genetic and biochemical mechanisms (1-3, 7, 17). Clearly, DGPP phosphatase plays a role in this complex regulation.