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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Biol Chem. Author manuscript; available in PMC Sep 13, 2005.
Published in final edited form as:
PMCID: PMC1201514
NIHMSID: NIHMS3453

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

Abstract

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 due to 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 GST-Zap1p687–880 and a putative UASZRE in the PIS1 promoter was demonstrated by electrophoretic mobility shift assays. Mutations in the UASZRE 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.

Phosphatidylinositol (PI)1 is the third most abundant phospholipid in the cellular membranes of the yeast Saccharomyces cerevisiae (13), and it is essential for the growth and metabolism of this model eukaryote (46). In addition to being a major structural component of the membrane, PI serves as the precursor for sphingolipids (7, 8), the D-3, D-4, and D-5 phosphoinositides (3, 912), and glycosyl PI anchors (13, 14)(Fig. 1). Several of these PI-derived lipids and their metabolic products are prominent signaling molecules in S. cerevisiae and in higher eukaryotes that contribute to essential physiological functions (12, 1520).

FIG. 1
Pathways for the synthesis of PI and PC in S. cerevisiae

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, 2123). This ER-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 (2628). Cellular inositol levels are controlled by expression of the INO1 gene encoding inositol-3-phosphate synthase and by inositol supplementation (2628). The levels of CDP-diacylglycerol are controlled through its utilization by the PI synthase enzyme itself and the competing activity of PS synthase (2629)(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, 3034).

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 UASINO element 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 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, 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-β-D-thiogalactoside, and acrylamide solutions were purchased from Bio-Rad. Ampicillin, aprotinin, benzamidine, bovine serum albumin, leupeptin, O-nitrophenyl β-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 (H+L) antibodies were purchased from Roche 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-deplete 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 μM zinc sulfate. Standard synthetic growth medium contains 1.4 μM zinc sulfate. Saturated cultures were harvested, washed in deionized distilled water, diluted to 1 × 106 cells/ml in media containing 0 or 1.5 μM zinc sulfate, and grown for 24 h. Cultures were then diluted to 1 × 106 cells/ml and grown again in media containing 0 or 1.5 μM zinc sulfate. This growth routine with medium lacking zinc was used to deplete internal stores of zinc (43). Cells in liquid media were grown to the exponential phase (1 × 107 cells/ml), and cell numbers were determined spectrophotometrically at an absorbance of 600 nm. Plasmids were maintained and amplified in Escherichia coli strain DH5α grown in LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl, pH 7.4) at 37 °C. Ampicillin (100 μg/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.

TABLE I
Strains and plasmids used in this work

DNA Manipulations and Amplification of DNA by PCR

Plasmid and genomic DNA preparation, restriction enzyme digestion, and DNA ligations 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) were performed using standard protocols.

Construction of Plasmids

Plasmid pWMI1 contains a 2.2-kb DNA fragment for the PIS1 gene with sequences for an HA epitope tag inserted after the start codon. Genomic DNA prepared from strain W303-1A was used as a template to produce a 5′ fragment of PIS1 HA (primers: 5′-CCCCCCGGGCTAATGCATGAGCCAATAGAG-3′ and 5′-AGCGTAGTCTGGGACGTCGTATGGGTACATCTTGTACTATCACACTTTCCCTCTTAT-3′) and a 3′ fragment of PIS1 HA (primers: 5′-TACCCATACGACGTCCCAGACTACGCTAGTTCGAATTCAACACCAGAAAAGGTTACT-3′ and 5′-CGTCTAGAGTGCAAGTTGGAGAGAATCGCTTCCG-3′). The 5′- and 3′-fragments of PIS1HA were digested with XmaI/AatII and AatII/XbaI, respectively, and inserted into the XmaI/XbaI sites of pRS416 to generate the plasmid pWMI1. The Stratagene QuikChange site-directed mutagenesis kit was utilized according to the manufacturer’s instructions to generate plasmids pPZM1-pPZM3. These plasmids were derivatives of pMA109 (PPIS1-lacZ) and contained mutations in UASZRE1, UASZRE2, and UASZRE3 of the PIS1 promoter. Plasmids pPZM1 (mutagenic primers: 5′-TTTTTCTTCCTTTTCCCTAACAATTCCAATTGCTTCTCTTCTCTTCTCCTT-3′ and 5′-AAGGAGAAGAGAAGAGAAGCAATTGGAATTGTTAGGGAAAAGGAAGAAAAA-3′), pPZM2 (mutagenic primers: 5′-TTTTAGCCATGGACACTTCTCAATTCCAATTTGTTGATGTCCATGGCTAAAA-3′ and 5′-TCAATGGCAGTTTTATCAACCAATTGGAATTGGAATTGAGAAGTGTCCATGGCTAAAA-3′), and pPZM3 (mutagenic primers: 5′-ATATAAGTAAAACATAAAAACAATTCCAATTGGTATGGTTTATTTGCCGTC-3′ and 5′-GACGGCAAATAAACCATACCAATTGGAATTGTTTTTATGTTTTACTTATAT-3′) were constructed by amplification of plasmid pMA109 by PCR. Plasmid pMA109 was eliminated from the mutant plasmid reactions by digestion with DpnI. The mutant plasmids were amplified in E. coli, and the purified plasmids were sequenced to confirm the mutations in the PIS1 promoter.

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 [α-32P]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 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 μg/ml of the purified anti-PI synthase IgG fraction. Mouse monoclonal anti-HA antibodies were used at a dilution of 1:1000. Goat anti-rabbit and anti-mouse IgG-alkaline phosphatase conjugates were used as secondary antibodies at a dilution of 1:5000. 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 μg/ml aprotinin, 5 μg/ml leupeptin, and 5 μg/ml pepstatin. Cells were disrupted by homogenization with chilled glass beads (0.5 mm diameter) using a Biospec Products Mini-BeadBeater-8. Samples were homogenized for ten 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 × g for 10 min. 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 [2-3H]inositol (10,000 cpm/nmol) into PI as described previously (54). The assay mixture contained 50 mM Tris-HCl (pH 8.0), 2 mM MnCl2, 0.5 mM inositol, 0.2 mM CDP-diacylglycerol, 2.4 mM Triton X-100, and enzyme protein. β-galactosidase activity was measured by following the formation of O-nitrophenyl from O-nitrophenyl β-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 β-D-galactopyranoside, 1 mM MgCl2, 100 mM 2-mercaptoethanol, and enzyme protein. All assays were linear with time and protein concentration. The average standard deviation of all assays was ± 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 β-galactosidase activity was defined in μmol/min. Specific activity was defined as units/mg of protein.

Expression and Purification of GST-Zap1p687–880 from E. coli

The GST-Zap1p687–880 fusion protein was expressed in E. coli BL21(DE3)pLysS bearing plasmid pGEX-687. A 500-ml culture was grown to A600 ~ 0.8 at 28 °C, and the expression of GST-Zap1p687–880 was induced for 1 h with 0.1 mM isopropyl-1-thio-βD-galactopyranoside. The culture was harvested, and the resulting pellet was resuspended in 20 ml of phosphate-buffered saline (10 mM Na2HPO4, 1.8 mM KH2PO4, 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 × 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- Zap1p687–880 fusion protein was purified to ~90% of homogeneity. The purified GST- Zap1p687–880 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 μM 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 5′ overhanging end, and they were labeled by incorporating [α-32P]dTTP to the ends. Annealed oligonucleotides (100 pmol) were incubated with 5 units of Klenow fragment and [α-32P]dTTP (400–800 Ci/nmol) for 30 min at room temperature. Labeled oligonucleotides were purified from unincorporated nucleotides using ProbeQuant G-50 spin columns.

TABLE II
Oligonucleotides used for electromobility shift assays

Formation of the protein-DNA complexes was allowed for 15 min at room temperature in a total volume of 10 μl containing 1 pmol of radiolabeled DNA probe (2.5 × 105 cpm/pmol), 10 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 50 mM KCl, 1 mM DTT, 0.025 mg/ml poly(dI-dC)·poly(dI-dC), 0.2 mg/ml bovine serum albumin, 0.04% IGEPAL CA-630, 10% glycerol, and the indicated concentrations of purified GST- Zap1p687–880. The reaction mixtures were resolved on 6% polyacrylamide gels (1.5-mm thickness) in 0.5× Tris-Borate-EDTA buffer at 100 V for 45 min. Gels were dried onto blotting paper, and the radioactive signals were visualized by phosphorimaging analysis.

Analyses of Data

Statistical significance was determined by performing the Student’s t-test using SigmaPlot software. P values < 0.05 were taken as a significant difference.

RESULTS

Effect of the zrt1Δ zrt2Δ 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 zrt1Δ zrt2Δ 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 zrt1Δ zrt2Δ mutant grown in the presence of zinc was similar to that expressed in the wild type control 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.

FIG. 2
Effect of the zrt1Δ zrt2Δ mutations on the expression of PI synthase activity in response to zinc depletion

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 ~7-fold. As a further confirmation, an immunoblot experiment was performed using a cell extract from wild type cells that expressed the PIS1HA 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 nearly a 2-fold increase in the amount of the PI synthase protein when 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.

FIG. 3
Effect of zinc depletion on the expression of PI synthase protein and PIS1 mRNA

We next examined the level of PIS1 mRNA to determine if the increase in enzyme content was due to 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 when 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 β-galactosidase Activity in Cells Bearing the PPIS1-lacZ Reporter Gene

The analysis of PIS1 expression was facilitated by the use of plasmid pMA109 that bears a PPIS1-lacZ reporter gene where the expression levels of β-galactosidase activity are dependent on transcription driven by the PIS1 promoter (60). To further examine the effect of zinc depletion on the expression of the PIS1 gene, we measured β-galactosidase activity from wild type cells bearing plasmid pMA109 that were grown with various concentrations of zinc. Reduction for zinc in the growth medium resulted in a dose-dependent increase in β-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 μM zinc (Fig. 4). Concentrations of zinc above 1.5 μM did not result in a further reduction in β-galactosidase activity.

FIG. 4
Dose-dependent induction of β-galactosidase activity in cells bearing the PPIS1-lacZ reporter gene in response to zinc depletion

Effects of ino2Δ, ino4Δ, and opi1Δ 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 UASINO element in the CHO1 promoter and by the positive transcription factors Ino2p and Ino4p, and the negative transcription factor Opi1p (40). Owing to the fact that the PIS1 promoter contains a UASINO element (60) and that the synthesis of PI and PS is coordinately regulated in S. cerevisiae (3, 27, 27, 30, 31), we questioned whether the regulation of PI synthase expression by zinc depletion was mediated by Ino2p, Ino4p, and Opi1p. To address this question, PI synthase activity was measured in ino2Δ, ino4Δ, and opi1Δ 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 zap1Δ Mutation on the Regulation of PI synthase by Zinc Depletion

Zap1p is a positive transcription factor that is maximally expressed in zinc-deplete cells and repressed in zinc-replete cells (61). Zap1p directly regulates UASZRE-containing genes (e.g., ZRT1, ZRT2, ZRT3, ZRC1, FET4, DPP1) whose expression is induced by zinc depletion (43, 58, 6264). Inspection of the PIS1 promoter revealed that it contains sequences that bear resemblance to the consensus UASZRE (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 zap1Δ mutant bearing the PPIS1-lacZ reporter gene was grown in the presence and absence of zinc followed by the measurement of β-galactosidase activity. In contrast to wild type cells, zinc depletion did not result in the induction of β-galactosidase activity (Fig. 5A). In a second set of experiments, PI synthase protein and activity levels were measured in cell extracts derived from zap1Δ mutant cells grown in the presence and absence of zinc. Unlike wild type cells, the depletion of zinc from the growth medium of the zap1Δ 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.

FIG. 5
Effect of the zap1Δ mutation on the regulation of PI synthase by zinc depletion

Interactions of GST-Zap1p687–880 with Putative UASZRE Sites in the PIS1 Promoter

We sought evidence that Zap1p mediates the regulation of PIS1 expression in response to zinc depletion by direct interaction with the PIS1 promoter. The PIS1 promoter contains three putative UASZRE sites (UASZRE1, UASZRE2, and UASZRE3) with sequences that resemble the consensus UASZRE sequence for Zap1p binding (Fig. 6A). Electrophoretic mobility shift assays were performed with labeled oligonucleotides containing the putative UASZRE sites using recombinant GST-Zap1p687–880 purified from E. coli. Zap1p687–880 contains the UASZRE binding domain (amino acids 687–880) of Zap1p (65). Of the three probes, the oligonucleotide containing UASZRE3 showed the strongest interaction with GST-Zap1p687–880 (Fig. 6B). The interaction of GST-Zap1p687–880 with UASZRE1 was ~20-fold lower when compared with UASZRE3 whereas an interaction with UASZRE2 was hardly detectable (Fig. 6B). The interaction of GST-Zap1p687–880 with UASZRE3 was examined further using the same assay. The formation of the GST-Zap1p687–880-UASZRE3 complex was dependent on the concentration of GST-Zap1p687–880 (Fig. 7A). In addition, the unlabeled UASZRE3 probe competed with the labeled probe for binding to GST-Zap1p687–880 in a dose-dependent manner (Fig. 7B). Moreover, this interaction was abolished when the UASZRE3 sequence was mutated (M1) to a nonconsensus sequence (Fig. 7C). When the UASZRE3 sequence was mutated (M2) to the consensus UASZRE, the extent of interaction with GST-Zap1p687–880 was 10-fold greater than the interaction with the wild type UASZRE3 sequence (Fig. 7C).

FIG. 6
Interactions of GST-Zap1p687–880 with putative UASZRE sites in the PIS1 promoter
FIG. 7
Interactions of GST-Zap1p687–880 with UASZRE3

Effects of Mutations in the Putative UASZRE Elements in the PIS1 Promoter on the Zinc-mediated Regulation of PIS1 Expression

The effects of mutations in UASZRE1, UASZRE2, and UASZRE3 in the PIS1 promoter on the zinc-mediated regulation of PIS1 expression was examined. PPIS1-lacZ reporter genes were constructed with mutations in each of the three putative UASZRE elements. For each element, the core sequences were changed to the nonconsensus sequence of 5′-CAATTCCAATT-3′. Cells bearing the wild type or mutant PPIS1-lacZ reporter genes were grown in the presence and absence of zinc; cell extracts were prepared and assayed for β-galactosidase activity. The mutations in UASZRE3 in the reporter plasmid pPZM3 abolished the induction of β-galactosidase activity that was observed in zinc-depleted cells bearing the wild type PPIS1-lacZ reporter plasmid pMA109 (Fig. 8). Although the expression of the β-galactosidase activities found in cells bearing the reporter plasmids with mutations in UASZRE1 (pPZM1) and UASZRE2 (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 primarily mediated by the UASZRE3 sequence in its promoter.

FIG. 8
Effects of mutations in UASZRE1, UASZRE2, and UASZRE3 in the PIS1 promoter on the zinc-mediated regulation of β-galactosidase activity in cells bearing the PPIS1-lacZ reporter gene

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 that is attributed to elevated expression of PI synthase activity (40). Analysis of the zrt1Δ zrt2Δ 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 β-galactosidase activity driven by the PPIS1-lacZ reporter gene were elevated in zinc-depleted cells indicated that the increase in PI synthase activity was due to a transcriptional mechanism.

The zinc-mediated induction of the PPIS1-lacZ reporter gene, and PI synthase protein and activity was lost in zap1Δ 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 UASZRE found in the promoter of zinc-regulated genes to drive their transcription (58, 61, 6871). 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 transporters 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 UASZRE. However, three putative UASZRE 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 UASZRE sites and purified recombinant GST-Zap1p687–880 showed that UASZRE3 in the PIS1 promoter was required for GST-Zap1p687–880 binding in vitro. Moreover, mutations in UASZRE3 to a nonconsensus sequence abolished the GST-Zap1p687–880-DNA interactions in vitro and abolished the induction of PIS1 gene expression (as reflected in β-galactosidase activity) in response to zinc depletion. A genome-wide 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 (~ 2-fold) when compared with the > 10-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-Zap1p687–880 with the PIS1 promoter UASZRE3 sequence when compared with this sequence mutated to a consensus UASZRE sequence. Notwithstanding, the 2-fold induction of the PIS1 gene in response to zinc depletion correlated with the ~ 2-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 (~2- to 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 (25, 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 due to a repression mechanism that involves the positive transcription factors Ino2p and Ino4p, the negative transcription factor Opi1p, and a UASINO 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, 3032) (Fig. 1). The coordinate repression of the CDP-diacylglycerol pathway enzymes by inositol requires the ongoing synthesis of PC (73, 74), and is enhanced by the inclusion of choline in the growth medium (3, 27, 3032). The increased level of PI in response to inositol/choline supplementation is not due to 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 UASINO element in its promoter or the transcription factors Ino2p and Opi1p (60). The regulation of PI synthesis by inositol is due to a biochemical mechanism (28). Given the low intracellular levels of inositol and the relatively high Km 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. When 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 glycosyl PI anchors) that are essential to the growth and metabolism of this eukaryotic organism (3, 920).

Acknowledgments

We thank David Eide, Susan Henry, John Lopes, and Satoshi Yamashita for strains and plasmids used in this study. We also acknowledge helpful discussions with Avula Sreenivas and Michael Kersting.

Footnotes

*This work was supported in part by United States Public Health Service, National Institutes of Health Grant GM-28140.

1The abbreviations used are: PI, phosphatidylinositol; PS, phosphatidylserine; PE, phosphatidylethanolamine; PC, phosphatidylcholine; UASZRE, zinc-responsive element; UASINO, inositol-responsive element.

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