Regulation of the PIS1-encoded Phosphatidylinositol Synthase in Saccharomyces cerevisiae by Zinc*

In the yeast Saccharomyces cerevisiae, the mineral zinc is essential for growth and metabolism. Depletion of zinc from the growth medium of wild type cells results in changes in phospholipid metabolism, including an increase in phosphatidylinositol content (Iwanyshyn, W. M., Han, G.-S., and Carman, G. M. (2004) J. Biol. Chem. 279, 21976–21983). We examined the effects of zinc depletion on the regulation of the PIS1-encoded phosphatidylinositol synthase, the enzyme that catalyzes the formation of phosphatidylinositol from CDP-diacylglycerol and inositol. Phosphatidylinositol synthase activity increased when zinc was depleted from the growth medium. Analysis of a zrt1Δ zrt2Δ mutant defective in plasma membrane zinc transport indicated that the cytoplasmic levels of zinc were responsible for the regulation of phosphatidylinositol synthase. PIS1 mRNA, its encoded protein Pis1p, and the β-galactosidase activity driven by the PPIS1-lacZ reporter gene were elevated in zinc-depleted cells. This indicated that the increase in phosphatidylinositol synthase activity was the result of a transcriptional mechanism. The zinc-mediated induction of the PPIS1-lacZ reporter gene, Pis1p, and phosphatidylinositol synthase activity was lost in zap1Δ mutant cells. These data indicated that the regulation of PIS1 gene expression by zinc depletion was mediated by the zinc-regulated transcription factor Zap1p. Direct interaction between glutathione S-transferase (GST)-Zap1p687–880 and a putative upstream activating sequence (UAS) zinc-responsive element in the PIS1 promoter was demonstrated by electrophoretic mobility shift assays. Mutations in the UAS zinc-responsive element in the PIS1 promoter abolished the GST-Zap1p687–880-DNA interaction in vitro and abolished the zinc-mediated regulation of the PIS1 gene in vivo. This work advances understanding of phospholipid synthesis regulation by zinc and the transcription control of the PIS1 gene.

In the yeast Saccharomyces cerevisiae, the mineral zinc is essential for growth and metabolism. Depletion of zinc from the growth medium of wild type cells results in changes in phospholipid metabolism, including an increase in phosphatidylinositol content ( 279, 21976 -21983). We examined the effects of zinc depletion on the regulation of the PIS1-encoded phosphatidylinositol synthase, the enzyme that catalyzes the formation of phosphatidylinositol from CDP-diacylglycerol and inositol. Phosphatidylinositol synthase activity increased when zinc was depleted from the growth medium. Analysis of a zrt1⌬ zrt2⌬ mutant defective in plasma membrane zinc transport indicated that the cytoplasmic levels of zinc were responsible for the regulation of phosphatidylinositol synthase. PIS1 mRNA, its encoded protein Pis1p, and the ␤-galactosidase activity driven by the P PIS1 -lacZ reporter gene were elevated in zinc-depleted cells. This indicated that the increase in phosphatidylinositol synthase activity was the result of a transcriptional mechanism. The zinc-mediated induction of the P PIS1 -lacZ reporter gene, Pis1p, and phosphatidylinositol synthase activity was lost in zap1⌬ mutant cells. These data indicated that the regulation of PIS1 gene expression by zinc depletion was mediated by the zinc-regulated transcription factor Zap1p. Direct interaction between glutathione S-transferase (GST)-Zap1p 687-880 and a putative upstream activating sequence (UAS) zinc-responsive element in the PIS1 promoter was demonstrated by electrophoretic mobility shift assays. Mutations in the UAS zinc-responsive element in the PIS1 promoter abolished the GST-Zap1p 687-880 -DNA interaction in vitro and abolished the zinc-mediated regulation of the PIS1 gene in vivo. This work advances understanding of phospholipid synthesis regulation by zinc and the transcription control of the PIS1 gene.
The enzyme responsible for the synthesis of PI in S. cerevisiae is the essential PIS1-encoded PI synthase (CDP-diacylglycerol:myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11) (6,(21)(22)(23). This endoplasmic reticulum-associated (24) enzyme catalyzes the formation of PI and CMP from CDP-diacylglycerol and inositol (25) (Fig. 1). The regulation of PI synthase activity in vivo is largely governed by the availability of its substrates inositol and CDP-diacylglycerol (26 -28). Cellular inositol levels are controlled by expression of the INO1 gene encoding inositol-3-phosphate synthase and by inositol supplementation (26 -28). The levels of CDP-diacylglycerol are controlled through its utilization by the PI synthase enzyme itself and the competing activity of PS synthase (26 -29) (Fig. 1). PS synthase catalyzes the committed step in the synthesis of PC via the CDP-diacylglycerol pathway (27) (Fig. 1). Indeed, the coordinate regulation of the PI synthase and PS synthase enzymes is part of an overall mechanism by which the synthesis of PI is coordinately regulated with the synthesis of PC (3, 27, 30 -34).
Zinc is an essential nutrient required for the growth and metabolism of S. cerevisiae and of higher eukaryotes (35). It is a cofactor for hundreds of enzymes (e.g. alcohol dehydrogenase, carbonic anhydrase, proteases, RNA polymerases, superoxide dismutase) (35) and a structural constituent of many proteins (e.g. transcription factors, chaperones, lipid-binding proteins) (36,37). Zinc deficiency in rats is associated with oxidative damage to DNA, lipids, and proteins (38), and in humans, it is manifested by defects in appetite, cognitive function, embryonic development, epithelial integrity, and immune function (39). Despite its essential nature, zinc is toxic to cells when accumulated in excess amounts (35).
Recent studies have revealed that the synthesis of phospholipids in S. cerevisiae is influenced by zinc deficiency (40). In particular, PI synthase activity is elevated in zinc-depleted cells, whereas several enzyme activities (e.g. PS synthase, PS decarboxylase, PE methyltransferase, and phospholipid methyltransferase) in the CDP-diacylglycerol pathway for PC synthesis are reduced in response to zinc depletion (40). The regulation of these activities by zinc availability contributes to alterations in the cellular levels of the major membrane phospholipids PI (elevated) and PE (reduced) (40). For the PS synthase enzyme, the reduction in activity in response to zinc depletion is controlled at the level of transcription through the UAS INO element in the CHO1 promoter and by the transcription factors Ino2p, Ino4p, and Opi1p (40). In this work, we explored the mechanism by which PI synthase activity is regulated in response to zinc depletion. Our data indicated that this regulation occurred by a transcriptional mechanism that was mediated by the transcriptional activator Zap1p.

EXPERIMENTAL PROCEDURES
Materials-All chemicals were reagent grade. Growth medium supplies were from Difco, and yeast nitrogen base lacking zinc sulfate was purchased from BIO 101. Restriction endonucleases, modifying enzymes, and the NEBlot kit were purchased from New England Biolabs, Inc. RNA size markers were purchased from Promega. The Yeastmaker yeast transformation kit was obtained from Clontech. Plasmid DNA purification and DNA gel extraction kits were from Qiagen, Inc. The QuikChange site-directed mutagenesis kit was from Stratagene. Oligonucleotides for PCRs and electrophoretic mobility shift assays were prepared by Genosys Biotechnology, Inc. ProbeQuant G-50 columns, polyvinylidene difluoride membranes, an enhanced chemifluorescence Western blotting detection kit, and glutathione-Sepharose 4 fast flow were purchased from GE Healthcare. DNA markers for agarose gel electrophoresis, protein molecular mass standards for SDS-PAGE, Zeta Probe blotting membranes, protein assay reagents, electrophoretic reagents, immunochemical reagents, isopropyl 1-thio-␤-D-galactopyranoside, and acrylamide solutions were purchased from Bio-Rad. Ampicillin, aprotinin, benzamidine, bovine serum albumin, leupeptin, o-nitrophenyl ␤-D-galactopyranoside, pepstatin, phenylmethylsulfonyl fluoride, reduced glutathione, IGEPAL CA-630, and Triton X-100 were purchased from Sigma. Mouse monoclonal anti-HA antibodies (12CA5) and ImmunoPure goat anti-mouse IgG (HϩL) antibodies were purchased from Roche Applied Science and Pierce, respectively. Radiochemicals and scintillation counting supplies were purchased from PerkinElmer Life Sciences and National Diagnostics, respectively. Liqui-Nox detergent was from Alconox, Inc.
Strains, Plasmids, and Growth Conditions-The strains and plasmids used in this work are presented in Table I. Yeast cells were grown according to standard methods (41,42) at 30°C in YEPD medium (1% yeast extract, 2% peptone, 2% glucose) or in synthetic complete medium containing 2% glucose. Appropriate nutrients were omitted from synthetic complete medium for the selection of cells bearing plasmids. Zinc-depleted medium was synthetic complete medium prepared with yeast nitrogen base lacking zinc sulfate. For zinc-depleted cultures, cells were first grown for 24 h in synthetic complete medium supplemented with 1.5 M zinc sulfate. Standard synthetic growth medium contains 1.4 M zinc sulfate. Saturated cultures were harvested, washed in deionized distilled water, diluted to 1 ϫ 10 6 cells/ml in medium containing 0 or 1.5 M zinc sulfate, and grown for 24 h. Cultures were then diluted to 1 ϫ 10 6 cells/ml and grown again in medium containing 0 or 1.5 M zinc sulfate. This growth routine with medium lacking zinc was used to deplete internal stores of zinc (43). Cells in liquid medium were grown to the exponential phase (1 ϫ 10 7 cells/ml), and cell numbers were determined spectrophotometrically at an absorbance of 600 nm. Plasmids were maintained and amplified in Escherichia coli strain DH5␣ 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 bacterial cultures that contained plasmids. Yeast and bacterial media were supplemented with 2% and 1.5% agar, respectively, for growth on plates. Glassware were washed with Liqui-Nox, rinsed with 0.1 mM EDTA, and then rinsed several times with deionized distilled water to prevent zinc contamination.
DNA Manipulations and Amplification of DNA by PCR-Plasmid and genomic DNA preparation, restriction enzyme digestion, and DNA ligation were performed by standard methods (42). Conditions for the amplification of DNA by PCR were optimized as described previously (44). Transformation of yeast (45) and E. coli (42) was performed using standard protocols.
RNA Isolation and Northern Blot Analysis-Total RNA was isolated from cells (46,47), resolved by agarose gel electrophoresis (48), and then transferred to Zeta Probe membranes by vacuum blotting. The PIS1 and CMD1 probes were labeled with [␣-32 P]dTTP using the NEBlot random primer labeling kit, and unincorporated nucleotides were removed using ProbeQuant G-50 columns. Prehybridization, hybridization with the probes, and washes to remove nonspecific binding were carried out according to the manufacturer's instructions. Images of the radiolabeled mRNAs were acquired by phosphorimaging analysis.
Anti-PI Synthase Antibodies and Immunoblotting-The peptide sequence AALILADNDAKNANE (residues 201-215 at the C-terminal end of the deduced amino acid sequence of PIS1) was synthesized and used to raise antibodies in New Zealand White rabbits by standard procedures at Bio-Synthesis, Inc. The IgG fraction was isolated from the antiserum using protein A-Sepharose CL-4B (49). SDS-PAGE (50) using 10% slab gels and the transfer of proteins to polyvinylidene difluoride membranes (51) were performed as described previously. The membrane was probed with 12.5 g/ml purified anti-PI synthase IgG fraction. Mouse monoclonal anti-HA antibodies were used at a dilution of 1:1,000. Goat anti-rabbit and anti-mouse IgG-alkaline phosphatase conjugates were used as secondary antibodies at a dilution of 1:5,000. The PI synthase protein (Pis1p) was detected using the enhanced chemifluorescence Western blotting detection kit, and the signals were acquired by FluorImaging. The relative density of the signal was analyzed using ImageQuant software. Immunoblot signals were in the linear range of detectability.
Preparation of Cell Extracts and Protein Determination-Cell extracts were prepared as described previously (52). Cells were suspended in 50 mM Tris-maleate buffer, pH 7.0, containing 1 mM EDTA, 0.3 M sucrose, 10 mM 2-mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 5 g/ml aprotinin, 5 g/ml leupeptin, and 5 g/ml pepstatin. Cells were disrupted by homogenization with chilled glass beads (0.5-mm diameter) using a Biospec Products Mini-Bead-Beater-8. Samples were homogenized for 10 1-min bursts followed by a 2-min cooling between bursts at 4°C. The cell extract (supernatant) was obtained by centrifugation of the homogenate at 1,500 ϫ g for 10 min. The protein concentration was determined by the method of Bradford (53) using bovine serum albumin as the standard.
Enzyme Assays-All assays were conducted in triplicate at 30°C in a total volume of 0.1 ml. PI synthase activity was measured by following the incorporation of [2-3 H]inositol (10,000 cpm/nmol) into PI as described previously (54). The assay mixture contained 50 mM Tris-HCl, pH 8.0, 2 mM MnCl 2 , 0.5 mM inositol, 0.2 mM CDP-diacylglycerol, 2.4 mM Triton X-100, and enzyme protein. ␤-Galactosidase activity was measured by following the formation of o-nitrophenyl from o-nitrophenyl ␤-D-galactopyranoside spectrophotometrically at a wavelength of 410 nm (55). The assay mixture contained 100 mM sodium phosphate, pH 7.0, 3 mM o-nitrophenyl ␤-D-galactopyranoside, 1 mM MgCl 2 , 100 mM 2-mercaptoethanol, and enzyme protein. All assays were linear with time and protein concentration. The average standard deviation of all assays was Ϯ 5%. A unit of PI synthase activity was defined as the amount of enzyme that catalyzed the formation of 1 nmol of product/ min, whereas a unit of ␤-galactosidase activity was defined in mol/ min. Specific activity was defined as units/mg of protein.
Expression and Purification of GST-Zap1p 687-880 from E. coli-The GST-Zap1p 687-880 fusion protein was expressed in E. coli BL21(DE3)pLysS bearing plasmid pGEX-687. A 500-ml culture was grown to A 600 ϳ 0.8 at 28°C, and the expression of GST-Zap1p 687-880 was induced for 1 h with 0.1 mM isopropyl 1-thio-␤-D-galactopyranoside. The culture was harvested, and the resulting pellet was resuspended in 20 ml of phosphate-buffered saline (10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , 140 mM NaCl, 2.7 mM KCl, pH 7.3). Cells were disrupted with a French press at 20,000 pounds/square inch, and unbroken cells and cell debris were removed by centrifugation at 12,000 ϫ g for 30 min at 4°C. The supernatant (cell lysate) was mixed for 1 h with 1 ml of a 50% slurry of glutathione-Sepharose with gentle shaking. The glutathione-Sepharose resin was then packed in a 10-ml Poly-Prep disposable column and was washed with 25 ml of phosphate-buffered saline. Proteins bound to the column were eluted (0.5-ml fractions) with 50 mM Tris-HCl, pH 8.0, buffer containing 10 mM reduced glutathione. SDS-PAGE analysis indicated that the 48-kDa GST-Zap1p 687-880 fusion protein was purified to ϳ90% of homogeneity. The purified GST-Zap1p 687-880 preparation was dialyzed against phosphate-buffered saline containing 10% glycerol and 2.5 mM dithiothreitol.
Electrophoretic Mobility Shift Assays-Double-stranded oligonucleotides (Table II) were prepared by annealing 25 M complementary single-stranded oligonucleotides in a total volume of 0.1 ml containing 10 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 1 mM EDTA. The reaction mixtures were incubated for 5 min at 100°C in a heat block and then for 2 h in the heat block that was turned off. Annealed oligonucleotides were designed to contain a 5Ј-overhanging end, and they were labeled by incorporating [␣-32 P]dTTP to the ends. Annealed oligonucleotides (100 pmol) were incubated with 5 units of Klenow fragment and [␣-32 P]dTTP (400 -800 Ci/nmol) for 30 min at room temperature. Labeled oligonucleotides were purified from unincorporated nucleotides using ProbeQuant G-50 spin columns.
Analyses of Data-Statistical significance was determined by performing Student's t test using SigmaPlot software. p values Ͻ 0.05 were taken as a significant difference.

Effect of the zrt1⌬ zrt2⌬ Mutations on the Expression of PI Synthase Activity in Response to Zinc Depletion-Iwanyshyn et
al. (40) identified PI synthase as an enzyme whose activity increased in wild type cells when zinc was depleted from the growth medium. To confirm that this regulation was governed by the intracellular levels of zinc, the expression of PI synthase activity was examined in a zrt1⌬ zrt2⌬ double mutant (56). This mutant lacks both the high affinity (Zrt1p) and low affinity (Zrt2p) plasma membrane zinc transporters that are primarily responsible for regulating the cytoplasmic levels of zinc in S. cerevisiae (56,57). For this and subsequent experiments, the growth medium lacked inositol and choline supplementation to preclude the regulatory effects that these phospholipid precursors have on phospholipid synthesis (3,27,30,31). As described previously (40), depletion of zinc from the growth medium of wild type cells caused a 2-fold increase in the expression of PI synthase activity (Fig. 2). The level of PI synthase activity in the zrt1⌬ zrt2⌬ mutant grown in the presence of zinc was similar to that expressed in the wild type control   This study cells that were depleted for zinc (Fig. 2). This result indicated that the intracellular levels of zinc were responsible for regulating the expression of PI synthase activity.

Effect of Zinc Depletion on the Expression of PI Synthase
Protein and PIS1 mRNA-Antibodies were generated against a peptide sequence found at the C-terminal end of the PI synthase protein. These antibodies recognized a protein with a subunit molecular mass of 24 kDa, the predicted size of the PIS1 gene product (6). To confirm the identity of this 24-kDa protein as the PI synthase protein, an immunoblot experiment was performed using a cell extract derived from wild type cells that overexpressed the PIS1 gene on a high copy plasmid. Consistent with the overexpression of the PIS1 gene, the amount of the 24-kDa protein that was recognized by the anti-PI synthase antibodies was elevated ϳ7-fold. As a further confirmation, an immunoblot experiment was performed using a cell extract from wild type cells that expressed the PIS1 HA gene on a single copy plasmid. The anti-PI synthase antibodies recognized both the native and HA-tagged versions of the PI synthase protein. HA-tagged PI synthase migrated with a molecular mass of 25 kDa because of the HA epitope. The identity of the HA-tagged PI synthase protein was confirmed by immunoblot analysis using anti-HA antibodies.
The expression of the PI synthase protein was analyzed by immunoblotting to examine the mechanism by which PI synthase activity was regulated in response to zinc depletion. Zinc depletion resulted in a nearly 2-fold increase in the amount of the PI synthase protein compared with cells grown with zinc (Fig. 3A). This indicated that the increase in PI synthase activity was a result of an increase in enzyme level.
We next examined the level of PIS1 mRNA to determine whether the increase in enzyme content was caused by an increase in gene expression. CMD1 mRNA (encodes calmodulin) was measured in this analysis as a loading control because its expression level is not affected by zinc availability (58,59). Northern blot analysis of total RNA isolated from exponential phase cells showed that the relative amount of PIS1 mRNA in zinc-depleted cells was almost 2-fold greater compared with that found in cells grown with zinc (Fig. 3B). These results indicated that a transcriptional mechanism was responsible for the regulation of PI synthase in zinc-depleted cells.
Effect of Zinc Depletion on the Expression of ␤-Galactosidase Activity in Cells Bearing the P PIS1 -lacZ Reporter Gene-The analysis of PIS1 expression was facilitated by the use of plasmid pMA109, which bears a P PIS1 -lacZ reporter gene where the expression levels of ␤-galactosidase activity are dependent on transcription driven by the PIS1 promoter (60). To examine further the effect of zinc depletion on the expression of the PIS1 gene, we measured ␤-galactosidase activity from wild type cells bearing plasmid pMA109 which were grown with various concentrations of zinc. Reduction for zinc in the growth medium resulted in a dose-dependent increase in ␤-galactosidase activity (Fig. 4). The activity found in cells grown in the absence of zinc was 3.5-fold greater than the activity in cells grown in the presence of 1.5 M zinc (Fig. 4). Concentrations of zinc above 1.5 M did not result in a further reduction in ␤-galactosidase activity.
Effects of the ino2⌬, ino4⌬, and opi1⌬ Mutations on the Regulation of PI Synthase by Zinc Depletion-The PI synthase enzyme is found at a branch point in phospholipid synthesis where it competes with another enzyme, PS synthase, for the common liponucleotide substrate CDP-diacylglycerol (27). Unlike PIS1, the expression of the PS synthase gene (CHO1) is repressed in wild type cells when zinc is depleted from the growth medium (40). The regulation of PS synthase expression by zinc depletion is mediated through a UAS INO element in the CHO1 promoter and by the positive transcription factors Ino2p and Ino4p and the negative transcription factor Opi1p (40). Because the PIS1 promoter contains a UAS INO element (60) and the synthesis of PI and PS is regulated coordinately in S. cerevisiae (3, 27, 27, 30, 31), we questioned whether the regulation of PI synthase expression by zinc depletion was   FIG. 2. Effect of the zrt1⌬ zrt2⌬ mutations on the expression of PI synthase activity in response to zinc depletion. Wild type (WT) and zrt1⌬ zrt2⌬ mutant cells were grown in the presence (1.5 M) and absence of zinc as indicated. Cell extracts were prepared and used for the assay of PI synthase activity. Each data point represents the average of triplicate enzyme determinations from a minimum of two independent experiments Ϯ S.D .   FIG. 3. Effect of zinc depletion on the expression of PI synthase protein and PIS1 mRNA. Wild type cells were grown in the presence (1.5 M) and absence of zinc. A, cell extracts were prepared, and 50-g samples were used for immunoblot analysis using anti-PI synthase antibodies (12.5 g/ml). A portion of the immunoblot is shown, and the position of the PI synthase (Pis1p) protein is indicated. The signals of the PI synthase protein from cells grown with and without zinc were quantified using ImageQuant software. The amount of PI synthase protein found in cells grown with zinc was arbitrarily set at 1. The data shown are representative of two independent experiments. B, total RNA was extracted, and 25-g samples were used for Northern blot analysis to determine the abundance of PIS1 mRNA. Portions of Northern blots are shown, and the positions of PIS1 and CMD1 (loading control) mRNAs are indicated. The relative amounts of PIS1 and CMD1 mRNAs from cells grown with and without zinc were determined by ImageQuant analysis of the data. The relative amount of PIS1 to CMD1 mRNA in cells grown with zinc was arbitrarily set at 1. The data shown are representative of two independent experiments.
FIG. 4. Dose-dependent induction of ␤-galactosidase activity in cells bearing the P PIS1 -lacZ reporter gene in response to zinc depletion. Wild type cells bearing the P PIS1 -lacZ reporter plasmid pMA109 were grown in the absence and presence of the indicated concentrations of zinc sulfate. Cell extracts were prepared and used for the assay of ␤-galactosidase activity. Each data point represents the average of triplicate enzyme determinations from a minimum of two independent experiments Ϯ S.D. mediated by Ino2p, Ino4p, and Opi1p. To address this question, PI synthase activity was measured in ino2⌬, ino4⌬, and opi1⌬ mutant cells that were grown in the presence and absence of zinc. In all three regulatory mutants, the PI synthase enzyme was elevated in response to zinc depletion similar to that observed in wild type cells (data not shown). These results indicated that the induction of PI synthase in zinc-depleted cells was not mediated by Ino2p, Ino4p, and Opi1p.
Effects of the zap1⌬ Mutation on the Regulation of PI Synthase by Zinc Depletion-Zap1p is a positive transcription factor that is expressed maximally in zinc-depleted cells and repressed in zinc-replete cells (61). Zap1p directly regulates UAS ZRE -containing genes (e.g. ZRT1, ZRT2, ZRT3, ZRC1, FET4, DPP1) whose expression is induced by zinc depletion (43,58,(62)(63)(64). Inspection of the PIS1 promoter revealed that it contains sequences that bear resemblance to the consensus UAS ZRE (see below). Accordingly, we questioned whether the regulation of PI synthase expression by zinc was dependent on Zap1p function. In the first set of experiments, the zap1⌬ mutant bearing the P PIS1 -lacZ reporter gene was grown in the presence and absence of zinc followed by the measurement of ␤-galactosidase activity. In contrast to wild type cells, zinc depletion did not result in the induction of ␤-galactosidase activity (Fig. 5A). In a second set of experiments, PI synthase protein and activity levels were measured in cell extracts derived from zap1⌬ mutant cells grown in the presence and absence of zinc. Unlike wild type cells, the depletion of zinc from the growth medium of the zap1⌬ mutant did not result in elevated levels of PI synthase protein (Fig. 5B) and activity (Fig. 5C). These results indicated that the zinc-mediated regulation of PIS1 expression was dependent on the Zap1p transcription factor.

Effects of Mutations in the Putative UAS ZRE Elements in the PIS1 Promoter on the Zinc-mediated Regulation of PIS1 Expression-The effects of mutations in UAS ZRE
1 , UAS ZRE 2 , and UAS ZRE 3 in the PIS1 promoter on the zinc-mediated regulation of PIS1 expression was examined. P PIS1 -lacZ reporter genes were constructed with mutations in each of the three putative UAS ZRE elements. For each element, the core sequences were changed to the nonconsensus sequence of 5Ј-CAATTCCAATT-3Ј. Cells bearing the wild type or mutant P PIS1 -lacZ reporter FIG. 5. Effect of the zap1⌬ mutation on the regulation of PI synthase by zinc depletion. Wild type (WT) and zap1⌬ mutant cells were grown in the presence (1.5 M) and absence of zinc. A, cell extracts were prepared from cells bearing the P PIS1 -lacZ reporter plasmid pMA109 and used for the assay of ␤-galactosidase activity. Each data point represents the average of triplicate enzyme determinations from a minimum of two independent experiments Ϯ S.D. B, 50-g samples of cell extracts were used for immunoblot analysis using anti-PI synthase antibodies (12.5 g/ml). The signals of the PI synthase protein from wild type and zap1⌬ mutant cells grown with and without zinc were quantified using ImageQuant software. The amount of PI synthase protein found in wild type cells grown with zinc was arbitrarily set at 1. The data shown are representative of two independent experiments. C, cell extracts were prepared and assayed for PI synthase activity. Each data point represents the average of triplicate enzyme determinations from a minimum of two independent experiments Ϯ S.D. genes were grown in the presence and absence of zinc; cell extracts were prepared and assayed for ␤-galactosidase activity. The mutations in UAS ZRE 3 in the reporter plasmid pPZM3 abolished the induction of ␤-galactosidase activity which was observed in zinc-depleted cells bearing the wild type P PIS1 -lacZ reporter plasmid pMA109 (Fig. 8). Although the expression of the ␤-galactosidase activities found in cells bearing the reporter plasmids with mutations in UAS ZRE 1 (pPZM1) and UAS ZRE 2 (pPZM2) was somewhat attenuated, the PIS1 gene was still induced when cells were depleted for zinc (Fig. 8). These data indicated that the zinc-mediated regulation of PIS1 expression was mediated primarily by the UAS ZRE 3 sequence in its promoter. DISCUSSION The yeast S. cerevisiae has the ability to cope with a variety of stress conditions (e.g. nutrient deprivation) by regulating the expression of enzyme activities including those involved in phospholipid synthesis (4,27,40,40,43,66,67). In particular, the stress condition of zinc depletion results in an increase in PI content which is attributed to elevated expression of PI synthase activity (40). Analysis of the zrt1⌬ zrt2⌬ mutant defective in the major plasma membrane zinc transporters Zrt1p and Zrt2p indicated that a decrease in the intracellular levels of zinc was responsible for the induction of PI synthase activity. That PIS1 mRNA, its encoded protein Pis1p, and the ␤-galactosidase activity driven by the P PIS1 -lacZ reporter gene were elevated in zincdepleted cells indicated that the increase in PI synthase activity was the result of a transcriptional mechanism.
The zinc-mediated induction of the P PIS1 -lacZ reporter gene and PI synthase protein and activity was lost in zap1⌬ mutant cells. These data indicated that the regulation of PIS1 gene expression by zinc was mediated by the Zap1p transcription factor. Zap1p is a zinc-sensing and zinc-inducible regulatory protein that binds to a UAS ZRE found in the promoter of zincregulated genes to drive their transcription (58, 61, 68 -71). Zap1p plays a major role in regulating the intracellular levels of zinc in S. cerevisiae (61,71). For example in zinc-depleted cells, Zap1p mediates increased expression and activity of the high affinity (Zrt1p) and low affinity (Zrt2p, Fet4p) zinc trans-porters in the plasma membrane and of the efflux zinc transporter Zrt3p in the vacuole membrane to elevate the cytoplasmic levels of zinc (56,57,62,68,71,72).
The promoter of the PIS1 gene does not contain a consensus UAS ZRE . However, three putative UAS ZRE sites were identified in the PIS1 promoter sequence by a motif search using the Vector NTI computer program. Electrophoretic mobility shift assays with DNA probes containing the putative UAS ZRE sites and purified recombinant GST-Zap1p 687-880 showed that UAS ZRE 3 in the PIS1 promoter was required for GST-Zap1p 687-880 binding in vitro. Moreover, mutations in UAS ZRE 3 to a nonconsensus sequence abolished the GST-Zap1p 687-880 -DNA interactions in vitro and abolished the induction of PIS1 gene expression (as reflected in ␤-galactosidase activity) in response to zinc depletion. A genomewide cDNA microarray analysis of gene expression identified 46 direct Zap1p target genes that are induced by zinc depletion (58). The PIS1 gene was not identified in that microarray study (58). This might be attributed to the relatively modest level of PIS1 induction (ϳ2-fold) compared with the Ͼ10-fold inductions of other Zap1p target genes (e.g. ZRT1, DPP1) (43,58). The differences between the magnitudes of induction of the PIS1 gene and other Zap1p target genes correlated with the relative binding efficiencies of GST-Zap1p 687-880 with the PIS1 promoter UAS ZRE 3 sequence compared with this sequence mutated to a consensus UAS ZRE sequence. Notwithstanding, the 2-fold induction of the PIS1 gene in response to zinc depletion correlated with the ϳ2-fold increase in the PI content of yeast cells depleted for zinc (40). The steady-state composition of PI in S. cerevisiae is tightly regulated (ϳ2-3-fold changes) (2,3,27). In this regard, we found that the expression of PI synthase did not respond to zinc depletion when the PIS1 gene was overexpressed from a plasmid.
Inositol, the water-soluble substrate of the PI synthase enzyme reaction, plays a major role in the regulation of phospholipid synthesis and composition in S. cerevisiae (2)(3)(4)(5)27). The addition of inositol to the growth medium of wild type cells causes an increase in the level of PI and a decrease in the levels of PS, PE, and PC (28,52). The decreased levels of PS, PE, and PC are primarily the result of a repression mechanism that involves the positive transcription factors Ino2p and Ino4p, the negative transcription factor Opi1p, and a UAS INO element found in the promoter of genes (i.e. CHO1, PSD1, CHO2, and OPI3) encoding the enzymes in the CDP-diacylglycerol pathway for PC synthesis (3, 27, 30 -32) (Fig. 1). The coordinate repression of the CDP-diacylglycerol pathway enzymes by inositol requires the ongoing synthesis of PC (73,74) and is en-  ZRE 3 . C, the experiment was performed with 0.6 g of recombinant GST-Zap1p 687-880 and sequences for wild type (WT) and mutated forms of UAS ZRE 3 . The wild type UAS ZRE 3 sequence was mutated from 5Ј-ACATGAGAGGT-3Ј to the nonconsensus sequence 5Ј-CAATTCCAATT-3Ј (M1) and to a consensus sequence 5Ј-ACCTTGAAGGT-3Ј (M2). Interaction of GST-Zap1p 687-880 with the labeled oligonucleotides was determined by electrophoretic mobility shift assay using a 6% polyacrylamide gel. The data shown are representative of two independent experiments. in the PIS1 promoter on the zinc-mediated regulation of ␤-galactosidase activity in cells bearing the P PIS1 -lacZ reporter gene. Wild type cells bearing the indicated P PIS1 -lacZ reporter plasmids were grown in the presence (1.5 M) and absence of zinc. The UAS ZRE 1 , UAS ZRE 2 , and UAS ZRE 3 sequences in the PIS1 promoter of plasmid pMA109 were mutated to the nonconsensus sequence 5Ј-CAATTCCAATT-3Ј in plasmids pPZM1, pPZM2, and pPZM3, respectively. Cell extracts were prepared and used for the assay of ␤-galactosidase activity. Each data point represents the average of triplicate enzyme determinations from a minimum of two independent experiments Ϯ S.D.
hanced by the inclusion of choline in the growth medium (3, 27, 30 -32). The increased level of PI in response to inositol/choline supplementation is not caused by increased expression of PIS1 mRNA (75) and the PI synthase enzyme (76). Transcription of the PIS1 gene is insensitive to inositol/choline, and it does not require the UAS INO element in its promoter or the transcription factors Ino2p and Opi1p (60). The regulation of PI synthesis by inositol is the result of a biochemical mechanism (28). Given the low intracellular levels of inositol and the relatively high K m value for inositol, the synthesis of PI by the PI synthase enzyme is regulated by the availability of inositol (28). Moreover, inositol is an inhibitor of the PS synthase enzyme, and this regulation also contributes to the decrease in the synthesis of PS and ultimately PE and PC (28). These observations raised the suggestion that PI synthase is a constitutively expressed enzyme (3,30,31). However, as shown here, the level of the PI synthase enzyme is regulated by zinc availability.
This is not the first study to show that the expression of the PIS1 gene is subject to transcriptional regulation. Anderson and Lopes (60) have shown that expression of PIS1 is regulated in response to growth medium carbon source. Compared with glucose, glycerol represses PIS1 expression, whereas galactose induces expression (60). The transcription factor Mcm1p mediates the glycerol-dependent repression of PIS1 gene expression, whereas the transcription factor Sln1p mediates the galactose-mediated induction of gene expression (60). The expression of the PIS1 gene is also regulated by oxygen availability (77). Gene expression is induced when cells are grown under anaerobic conditions and repressed under aerobic conditions. Repression is dependent on transcription factor Rox1p and its binding site in the PIS1 promoter (77). Similar to that observed in cells deprived for zinc (40), a reduction in oxygen availability results in elevated levels of PI (77). The induction of PIS1 gene expression may represent one of the mechanisms by which cells cope with the stress conditions of zinc and oxygen deficiencies given that PI is a precursor to several lipid molecules (sphingolipids, phosphoinositides, and glycosylphosphatidylinositol anchors) that are essential to the growth and metabolism of this eukaryotic organism (3, 9 -20).