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Originally published In Press as doi:10.1074/jbc.M402047200 on March 17, 2004

J. Biol. Chem., Vol. 279, Issue 21, 21976-21983, May 21, 2004
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Regulation of Phospholipid Synthesis in Saccharomyces cerevisiae by Zinc*

Wendy M. Iwanyshyn, Gil-Soo Han, and George M. Carman{ddagger}

From the Department of Food Science, Rutgers University, New Brunswick, New Jersey 08901

Received for publication, February 24, 2004 , and in revised form, March 16, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Zinc is an essential nutrient required for the growth and metabolism of eukaryotic cells. In this work, we examined the effects of zinc depletion on the regulation of phospholipid synthesis in the yeast Saccharomyces cerevisiae. Zinc depletion resulted in a decrease in the activity levels of the CDP-diacylglycerol pathway enzymes phosphatidylserine synthase, phosphatidylserine decarboxylase, phosphatidylethanolamine methyltransferase, and phospholipid methyltransferase. In contrast, the activity of phosphatidylinositol synthase was elevated in response to zinc depletion. The level of Aut7p, a marker for the induction of autophagy, was also elevated in zinc-depleted cells. For the CHO1-encoded phosphatidylserine synthase, the reduction in activity in response to zinc depletion was controlled at the level of transcription. This regulation was mediated through the UASINO element and by the transcription factors Ino2p, Ino4p, and Opi1p that are responsible for the inositol-mediated regulation of UASINO-containing genes involved in phospholipid synthesis. Analysis of the cellular composition of the major membrane phospholipids showed that zinc depletion resulted in a 66% decrease in phosphatidylethanolamine and a 29% increase in phosphatidylinositol. A zrt1{Delta} zrt2{Delta} mutant (defective in the plasma membrane zinc transporters Zrt1p and Zrt2p) grown in the presence of zinc exhibited a phospholipid composition similar to that of wild type cells depleted for zinc. These results indicated that a decrease in the cytoplasmic levels of zinc was responsible for the alterations in phospholipid composition.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Phospholipids are amphipathic molecules that are major structural components of cellular membranes (1). In addition, phospholipids provide precursors for the synthesis of macromolecules (2–6), serve as molecular chaperons (7, 8), serve in protein modification for membrane association (9), and are reservoirs of second messengers (10). Thus, phospholipids are essential for vital cellular processes. The major phospholipids in the membranes of the model eukaryote Saccharomyces cerevisiae are PC,1 PE, PI, and PS (11–13). Mitochondrial membranes also contain phosphatidylglycerol and cardiolipin (11–13). PC, the most abundant phospholipid in S. cerevisiae, is synthesized by the CDP-DAG and Kennedy pathways (11–13) (Fig. 1). Under standard laboratory growth conditions, PC is primarily synthesized by the CDP-DAG pathway (13–15), but when cells are supplemented with choline, PC is primarily synthesized by the Kennedy pathway (16). Notwithstanding, both pathways contribute to the synthesis of PC whether or not choline is supplemented to the growth medium (17–22). The choline required for the Kennedy pathway in cells not supplemented with choline is derived from the turnover of PC synthesized by the CDP-DAG pathway (22, 23).



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  		FIG. 1.
Effect of zinc depletion on the expression of phospholipid biosynthetic enzyme activities. The pathways shown for the synthesis of phospholipids include the CDP-DAG and Kennedy pathways and the relevant steps discussed throughout this paper. A more detailed description of the pathway and phospholipid structures can be found in Ref. 12. Cell extracts were prepared from wild type (strain DY1457) cells grown in the presence and absence of zinc and used for the assay of CDP-DAG synthase (CDS), PS synthase (PSS), PS decarboxylase (PSD), PE methyltransferase (PEMT), phospholipid methyltransferase (PLMT), and PI synthase (PIS) activities as described under "Experimental Procedures." The specific activities (nmol/min/mg) of these enzymes from cells grown with zinc were 0.62 ± 0.04, 0.82 ± 0.08, 0.02 ± 0.001, 0.70 ± 0.003, 0.53 ± 0.002, and 1.05 + 0.07, respectively. Each data point represents the average of triplicate enzyme determinations from a minimum of two independent experiments ± S.D.

 
The regulation of phospholipid synthesis in S. cerevisiae is complex and is controlled by genetic and biochemical mechanisms (11, 13, 24, 25). The expression of several phospholipid biosynthetic enzymes is coordinately regulated by inositol supplementation and growth phase, and this regulation is mediated by regulatory proteins (e.g. Ino2p, Ino4p, and Opi1p) that control the rate of transcription (11–13, 24, 26). In addition to transcriptional control of gene expression, mRNA decay/stability contributes to the levels of some phospholipid biosynthetic enzymes (27). Posttranslational modification plays a role in the regulation of phospholipid synthesis through the phosphorylation of key enzymes by protein kinases A and C (28–32). Finally, the activities of several phospholipid biosynthetic enzymes are modulated by phospholipids (33–38), sphingolipids (39, 40), and nucleotides (20, 41, 42).

Recent data indicate that phospholipid metabolism in yeast is coordinately regulated with mechanisms that control zinc homeostasis (43). Zinc, an essential nutrient, is required for the growth and metabolism of eukaryotic cells (44). It is a cofactor for several hundred enzymes (44) and is a structural component for many transcription factors (45). Zinc deficiency in humans is manifested by defects in appetite, cognitive function, embryonic development, epithelial integrity, and immune function (46). In addition, zinc deficiency in rats is associated with oxidative damage to DNA, lipids, and proteins (47), as well as causing a decrease in the overall phospholipid content (48–50). Although zinc is an essential nutrient, it can be toxic when accumulated in excess amounts (44). The cytoplasmic levels of zinc in yeast are controlled by a variety of mechanisms, including cellular influx (51), efflux (52, 53), and chelation by metallothioneins (54). The cytoplasmic levels of zinc are largely controlled by high affinity and low affinity zinc transporters (Zrt1p and Zrt2p) in the plasma membrane (55, 56). Zrt1p and Zrt2p are induced when the extracellular concentration of zinc is low (55, 56). Cytoplasmic levels of zinc are controlled further by the vacuole membrane efflux transporter Zrt3p. Expression of the vacuole membrane-associated DPP1-encoded DGPP phosphatase (43, 57),2 a novel enzyme in yeast phospholipid metabolism (58), is also induced by zinc depletion (43). The induction of DGPP phosphatase expression in zinc-depleted cells correlates with diminished levels of the minor vacuole membrane phospholipids DGPP and PA (59). In addition to the changes in DGPP and PA, zinc depletion results in a reduction in the level of PE and an increase in the level of PI in the vacuole membrane (59). Analysis of dpp1{Delta} mutant cells depleted for zinc indicates that the alterations in the major vacuole membrane phospholipids are not dependent on the regulation of DPP1 expression (60). In this work, we showed that zinc depletion resulted in a decrease in the activities of CDP-DAG pathway enzymes and an increase in the activity of PI synthase. The level of Aut7p, a marker for the induction of autophagy, was also elevated in zinc-depleted cells. For the CHO1-encoded PS synthase, the enzyme that catalyzes the committed step in the CDP-DAG pathway, the reduction in activity was controlled at the level of transcription. This regulation was mediated through the UASINO element in the CHO1 promoter and by the transcription factors Ino2p, Ino4p, and Opi1p.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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 YeastmakerTM yeast transformation kit was obtained from Clontech. The plasmid DNA purification and DNA gel extraction kits were from Qiagen, Inc. ProbeQuant G-50 columns, polyvinylidene difluoride membranes, and the enhanced chemifluorescence Western blotting detection kit were purchased from Amersham Biosciences. The DNA size ladder used for agarose gel electrophoresis, Zeta Probe blotting membranes, protein assay reagents, electrophoretic reagents, immunochemical reagents, isopropyl-{beta}-D-thiogalactoside, protein molecular mass standards for SDS-PAGE, and acrylamide solutions were purchased from Bio-Rad. S-adenosylmethionine, ampicillin, aprotinin, benzamidine, bovine serum albumin, leupeptin, O-nitrophenyl {beta}-D-galactopyranoside, pepstatin, phenylmethylsulfonyl fluoride, and Triton X-100 were purchased from Sigma. Radiochemicals and scintillation counting supplies were purchased from PerkinElmer Life Sciences and National Diagnostics, respectively. Phospholipids were purchased from Avanti Polar Lipids. TLC plates were from EM science, and DE52 (DEAE-cellulose) was from Whatman. 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. Methods for the growth of yeast were performed as described previously (61, 62). Cells were grown at 30 °C in YEPD medium (1% yeast extract, 2% peptone, 2% glucose) or in synthetic complete medium containing 2% glucose. For selection of cells bearing plasmids, appropriate nutrients were omitted from synthetic complete medium. 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 1.4 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 x 106 cells/ml in medium containing 0 or 1.4 µM zinc sulfate, and grown for 24 h. Cultures were then diluted to 1 x 106 cells/ml and grown again in medium containing 0 or 1.4 µM zinc sulfate. This growth regimen with medium lacking zinc was used to deplete internal stores of zinc. Cells in liquid media were grown to the exponential phase (1–2 x 107 cells/ml), and cell numbers were determined spectrophotometrically at an absorbance of 600 nm. Plasmids were maintained and amplified in Escherichia coli strain DH5{alpha}, which was 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 cultures of DH5{alpha}-carrying plasmids. For growth on plates, yeast and bacterial media were supplemented with 2 and 1.5% agar, respectively. To prevent zinc contamination, glassware was washed with Liqui-Nox, rinsed with 0.1 mM EDTA, and then rinsed several times with deionized distilled water.


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  		TABLE I
Strains and plasmids used in this work

 
DNA Manipulations, RNA Isolation, and Northern Blot Analysis— Plasmid and genomic DNA were prepared according to standard protocols (62). Transformations of yeast (63) and E. coli (62) were performed as described previously. Total RNA was isolated from cells as described previously (64, 65). The RNA was resolved by agarose gel electrophoresis (66) and then transferred to Zeta Probe membranes by vacuum blotting. The CHO1 (20) probe was labeled with [{alpha}-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 the manufacturer's instructions. Radioactive images were acquired by phosphorimaging.

Preparation of Cell Extracts and Protein Determination—Cell extracts were prepared as described previously (67, 68). 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-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 x g for 10 min. Protein concentration was determined by the method of Bradford (69) 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. CDP-DAG synthase activity was measured with 50 mM Tris maleate buffer (pH 6.5), 20 mM MgCl2, 15 mM Triton X-100, 0.5 mM PA, and 1.0 mM [5-3H]CTP (70). PS synthase activity was measured with 50 mM Tris-HCl buffer (pH 8.0), 0.6 mM MnCl2, 4 mM Triton X-100, 0.2 mM CDP-DAG, and 0.5 mM [3-3H]serine (71). PS decarboxylase activity was measured with 50 mM Tris-HCl buffer (pH 7.2), 10 mM 2-mercaptoethanol, 5 mM EDTA, 2 mM Triton X-100, and 0.2 mM [3-3H]PS (72, 73). PE methyltransferase activity was measured with 50 mM Tris-HCl buffer (pH 9.0), 0.2 mM PE, and 0.5 mM [methyl-3H]S-adenosylmethionine (74). Phospholipid methyltransferase activity was measured with 50 mM Tris-HCl buffer (pH 7.5), 10 mM MgCl2, 0.2 mM phosphatidylmonomethylethanolamine, and 0.5 mM [methyl-3H]S-adenosylmethionine (74). PI synthase activity was measured with 50 mM Tris-HCl buffer (pH 8.0), 2 mM MnCl2, 2.4 mM Triton X-100, 0.2 mM CDP-DAG, and 0.5 mM [2-3H]inositol (75). {beta}-Galactosidase activity was measured with 100 mM sodium phosphate buffer (pH 7.0), 3 mM O-nitrophenyl {beta}-D-galactopyranoside, 1 mM MgCl2, and 100 mM 2-mercaptoethanol (76). All assays were linear with time and protein concentration. The average S.D. value of the assays was ±5%. A unit of phospholipid enzymatic activity was defined as the amount of enzyme that catalyzed the formation of 1 nmol of product/min. A unit of {beta}-galactosidase activity was defined in µmol of product/min. Specific activity was defined as units/mg of protein.

SDS-PAGE and Immunoblotting—SDS-PAGE (77) was routinely performed with 12% slab gels. Analysis of Aut7p was performed with a 14% slab gel containing 6 M urea. These conditions were used to identify the PE-modified form of Aut7p (Aut7p-PE) that migrates slightly faster than the unmodified form on SDS-polyacrylamide gels (78). Proteins were transferred from SDS-polyacrylamide gels to polyvinylidene difluoride membranes as described previously (79). Membranes were probed with a 1:500 dilution of anti-PS synthase (27), 1:1000 dilution of anti-Aut7p (80), or 1 µg/ml anti-DGPP phosphatase (38) antibodies. Goat anti-rabbit IgG alkaline phosphatase conjugate was used as a secondary antibody at a dilution of 1:5000. Immunoreactive proteins were detected using the enhanced chemifluorescence Western blotting detection kit, and fluorescent signals were acquired by fluoroimaging. Immunoblot signals were in the linear range of detectability.

Labeling and Analysis of Phospholipids—The steady state composition of phospholipids was determined by labeling cells with 32Pi for six generations as described previously (14, 15). Phospholipids were extracted from labeled cells (81) and then separated by DEAE-cellulose column chromatography followed by one-dimensional TLC on silica gel plates (38, 82). The identity of the labeled phospholipids on TLC plates was confirmed by comparison with standards after exposure to iodine vapor. Radiolabeled phospholipids were visualized by phosphorimaging analysis. The relative quantities of 32P-labeled phospholipids were analyzed using ImageQuant software.

Analyses of Data—Statistical significance was determined by performing Student's t test using SigmaPlot software. p values of <0.05 were taken as a significant difference.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of Zinc Depletion on the Expression of Phospholipid Biosynthetic Enzyme Activities—The effects of zinc depletion on the expression of several phospholipid biosynthetic enzyme activities were examined in wild type cells. In these experiments, cells were grown without inositol and choline supplementation to preclude the regulatory effects that these phospholipid precursors have on phospholipid synthesis (11–13, 24). Under these growth conditions, the major membrane phospholipids are primarily synthesized via the CDP-DAG pathway (13–15). In this pathway, PS, PE, and PC are synthesized from CDP-DAG via the reactions catalyzed by PS synthase (83–85), PS decarboxylase (86–88), PE methyltransferase (89, 90), and phospholipid methyltransferase (89, 91). Zinc-depleted cells showed reduced activity levels of the CDP-DAG pathway enzymes. The activities of PS synthase, PS decarboxylase, PE methyltransferase, and phospholipid methyltransferase were reduced by 50, 25, 50, and 36%, respectively, when compared with cells grown with zinc (Fig. 1). The activity of CDP-DAG synthase, which is responsible for the formation of CDP-DAG (92), was not significantly affected by zinc depletion (Fig. 1). On the other hand, the activity of PI synthase (93), which competes with PS synthase for the substrate CDP-DAG (94), was elevated by 2-fold in zinc-depleted cells (Fig. 1).

Effect of Zinc Depletion on the Expression of PS Synthase Protein and CHO1 mRNA—The expression of the PS synthase enzyme was examined to gain insight into the mechanism by which the phospholipid biosynthetic enzyme activities were regulated by zinc depletion. PS synthase was chosen as a representative enzyme because it catalyzes the committed step in the CDP-DAG pathway (Fig. 1), and its gene expression is coordinately regulated with other genes in the pathway (11–13, 24–26). The levels of the PS synthase protein (Cho1p) were compared by immunoblot analysis of cell extracts derived from exponential phase cells that were grown in the presence and absence of zinc. Zinc depletion resulted in a 46% decrease in the amount of the PS synthase protein when compared with cells grown with zinc (Fig. 2). This indicated that the decrease in PS synthase activity was a result of a decrease in enzyme level. We examined the level of CHO1 mRNA to determine whether the decrease in enzyme content was due to a decrease in gene expression. Northern blot analysis of total RNA isolated from exponential phase cells showed that the relative amount of CHO1 mRNA in zinc-depleted cells was 60% lower than that found in cells grown with zinc (Fig. 2). These results indicated that a transcriptional mechanism was responsible for the regulation of PS synthase in zinc-depleted cells.



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  		FIG. 2.
Effect of zinc depletion on the expression of PS synthase protein and CHO1 mRNA. Wild type (strain DY1457) cells were grown in the presence and absence of zinc. For analysis of the PS synthase protein (Cho1p), 10 µg of the cell extract was subjected to immunoblotting using a 1:500 dilution of anti-PS synthase antibodies. For analysis of CHO1 mRNA, 12.5 µg of total RNA was subjected to Northern blotting using the labeled CHO1 DNA probe. Ribosomal RNA was used as a loading control to determine the relative amount of CHO1 mRNA. The signals of PS synthase protein and CHO1 mRNA were quantified using ImageQuant software. The amounts of PS synthase protein and CHO1 mRNA found in cells grown with zinc were set at 100%. The data shown are from three independent experiments ± S.D.

 
Effect of the zap1{Delta} Mutation on the Regulation of PS Synthase by Zinc Depletion—The expression of many S. cerevisiae genes is both positively and negatively regulated by zinc depletion (95). Zap1p is a transcription factor (96) that directly regulates UASZRE-containing genes (e.g. ZRT1, ZRT2, ZRT3, ZRC1, FET4, and DPP1), whose expression is induced by zinc depletion (43, 95, 97–99). Although the CHO1 gene does not contain the UASZRE in its promoter and its expression was repressed by zinc depletion, we questioned whether this regulation was indirectly mediated by Zap1p. To address this question, PS synthase activity was measured in zap1{Delta} mutant cells that were grown in the presence and absence of zinc. Although PS synthase activity was slightly higher (25%) in the zap1{Delta} mutant when compared with the wild type control, the enzyme was regulated by zinc depletion as in wild type cells. The level of PS synthase activity in the mutant was reduced (50%) in response to zinc depletion (Fig. 3). These results indicated that the repression of PS synthase in zinc-depleted cells was not mediated by Zap1p.



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  		FIG. 3.
Effect of the zap1{Delta} mutation on the regulation of PS synthase by zinc depletion. Wild type (WT; strain DY1457) and zap1{Delta} mutant cells were grown in the presence and absence of zinc. Cell extracts were prepared and assayed for PS synthase activity. Each data point represents the average of triplicate enzyme determinations from a minimum of two independent experiments ± S.D.

 
Effects of the UASINO Element and the Transcription Factors Ino2p, Ino4p, and Opi1p on the Regulation of PS Synthase Expression by Zinc Depletion—A cis-acting element, UASINO, in the CHO1 promoter is required for maximum expression of the CHO1 gene in wild type cells grown in the absence of inositol (11, 24, 100). It contains a consensus-binding site (5'-CANNTG-3') for a heterodimer complex of the positive transcription factors Ino2p and Ino4p (11, 24, 100, 101). The role of UASINO in the regulation of CHO1 by zinc depletion was examined using a PCHO1-lacZ reporter gene (100) with mutations in the cis-acting element (27). In the control experiment with the wild type reporter gene, the {beta}-galactosidase activity from zinc-depleted cells was 40% less than that of cells grown with zinc (Fig. 4). The decrease in the reporter activity in response to zinc depletion was consistent with the results of the Northern blot analysis, indicating that transcription of CHO1 was repressed by zinc depletion. As described previously (27), the mutations in the UASINO element caused a 43% decrease in {beta}-galactosidase activity in wild type cells grown with zinc (Fig. 4). The {beta}-galactosidase activity driven by the mutant reporter gene was not significantly affected by zinc depletion (Fig. 4). These results indicated that the zinc-mediated regulation of CHO1 expression was dependent on the UASINO element in its promoter.



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  		FIG. 4.
Effect of UASINO mutations on the regulation of the CHO1 gene by zinc depletion. Wild type (strain DY1457) cells bearing the PCHO1-lacZ reporter plasmid pAB709 or pHC2 were grown in the presence and absence of zinc. Cell extracts were prepared and used for the assay of {beta}-galactosidase activity. Each data point represents the average of triplicate enzyme determinations from a minimum of two independent experiments ± S.D. Mutations in the UASINO element in plasmid pHC2 are underlined.

 
To further explore the role of the UASINO element in the regulation of CHO1 expression by zinc depletion, PS synthase activity was measured in the ino2{Delta} and ino4{Delta} mutants defective in the positive transcription factors Ino2p and Ino4p, respectively. Due to the fact that the ino2{Delta} and ino4{Delta} mutants are inositol auxotrophs (102), it was necessary to supplement their growth medium with inositol (75 µM). As described previously (103), the PS synthase activity in the ino2{Delta} and ino4{Delta} mutants grown in the presence of zinc was reduced (78%) when compared with the wild type control (Fig. 5). Zinc depletion did not affect the expression of PS synthase activity in these mutants (Fig. 5). The regulation of PS synthase by zinc depletion was also examined in the opi1{Delta} mutant defective in the negative transcription factor Opi1p. As described previously (67), the PS synthase activity in the opi1{Delta} mutant grown with zinc was elevated (34%) when compared with the control (Fig. 5). However, zinc depletion did not significantly affect the expression of PS synthase activity (Fig. 5). Overall, these findings were consistent with the conclusion that Ino2p, Ino4p, and Opi1p played a role in the regulation of CHO1 expression by zinc depletion.



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  		FIG. 5.
Effects of the ino2{Delta}, ino4{Delta}, and opi1{Delta} mutations on the regulation of PS synthase by zinc depletion. Wild type (WT, strain W303-1A), ino2{Delta}, ino4{Delta}, and opi1{Delta} mutant cells were grown in the presence and absence of zinc. The growth medium for the ino2{Delta} and ino4{Delta} mutants was supplemented with inositol (75 µM). Cell extracts were prepared and assayed for PS synthase activity. Each data point represents the average of triplicate enzyme determinations from a minimum of two independent experiments ± S.D.

 
Effect of Inositol on the Zinc-mediated Regulation of PS Synthase—The expression of the CHO1 gene in wild type S. cerevisiae is repressed by the addition of inositol to the growth medium (100, 104). We questioned whether the zinc-mediated regulation of PS synthase was affected by inositol supplementation. PS synthase activity was measured in cell extracts derived from inositol-supplemented cells that were grown in the presence and absence of zinc. As described previously (67, 103), the addition of inositol to zinc-containing growth medium resulted in a 57% reduction in PS synthase activity (Fig. 6). The reduction in PS synthase activity caused by inositol supplementation was similar to that caused by zinc depletion (Fig. 6). When inositol was present in the growth medium, zinc depletion had no further effect on the expression of PS synthase activity (Fig. 6). These results raised the suggestion that the same regulatory mechanism was responsible for the repression of PS synthase by zinc depletion and by inositol supplementation.



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  		FIG. 6.
Effect of inositol on the zinc-mediated regulation of PS synthase. Wild type (strain DY1457) cells were grown in the presence and absence of zinc in growth medium without and with 75 µM inositol. Cell extracts were prepared and assayed for PS synthase activity. Each data point represents the average of triplicate enzyme determinations from a minimum of two independent experiments ± S.D.

 
Effect of Zinc on the Expression of the INO1 Gene—The expression of the INO1 gene, which encodes inositol 3-phosphate synthase,3 is regulated by inositol supplementation (105–107). This regulation is mediated by the transcription factors Ino2p, Ino4p, and Opi1p and occurs through the UASINO element in the INO1 promoter (67, 101, 102, 108–115). Because the INO1 and CHO1 genes are coordinately regulated by inositol through the same mechanism, we questioned whether the INO1 gene was regulated in response to zinc depletion similar to that of CHO1. The regulation of INO1 expression was examined using a PINO1-lacZ reporter gene where the expression of {beta}-galactosidase activity is dependent on transcription driven by the INO1 promoter (116). The {beta}-galactosidase activity in cells bearing the reporter gene was reduced by 60% when zinc was depleted from the growth medium (Fig. 7). This result indicated that INO1 expression was also regulated by zinc depletion.



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  		FIG. 7.
Effect of zinc depletion on the expression of the INO1 gene. Wild type (strain W303-1A) cells bearing the PINO1-lacZ reporter plasmid pJH359-LEU2 were grown in the presence and absence of zinc. Cell extracts were prepared and used for the assay of {beta}-galactosidase activity. Each data point represents the average of triplicate enzyme determinations from a minimum of two independent experiments ± S.D.

 
Effect of Zinc Depletion on Phospholipid Composition in Wild Type and the zrt1{Delta} zrt2{Delta}, zrt3{Delta}, and cot1{Delta} zrc1{Delta} Mutants Defective in Zinc Transport—We examined the effects of zinc depletion on the cellular composition of the major membrane phospholipids. In wild type cells, zinc depletion caused a 66% decrease in the level of PE but a 29% increase in the level of PI (Fig. 8A). We also analyzed the phospholipid composition of the mutants (zrt1{Delta} zrt2{Delta}, zrt3{Delta}, and cot1{Delta} zrc1{Delta}) defective in zinc transporters that control the cytoplasmic levels of zinc (55, 56, 117, 118). The phospholipid composition of zrt1{Delta} zrt2{Delta} mutant cells grown in the presence of zinc was similar to that of wild type cells grown in the absence of zinc (Fig. 8, A and B). In particular, the amounts of PE and PI in zrt1{Delta} zrt2{Delta} mutant cells grown with zinc were reduced (66%) and elevated (28%), respectively, when compared with the amounts of PE and PI in the wild type cells grown with zinc (Fig. 8, A and B). Zinc depletion did not have a further effect on the phospholipid composition of the zrt1{Delta} zrt2{Delta} mutant with the exception of a 42% increase in PS (Fig. 8B). The phospholipid composition of the zrt3{Delta} (Fig. 8C) and the cot1{Delta} zrc1{Delta} (Fig. 8D) mutants grown in the presence of zinc was not significantly different from that of wild type cells grown with zinc. Like wild type cells, these mutants showed a decrease in PE and an increase in PI in response to zinc depletion (Fig. 8, C and D). These results indicated that the cytoplasmic levels of zinc that are controlled by the plasma membrane Zrt1p and Zrt2p zinc transporters were primarily responsible for regulating the cellular composition of phospholipids.



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  		FIG. 8.
Effect of zinc depletion on phospholipid composition. Wild type (WT; strain DY1457) (A), zrt1{Delta} zrt2{Delta} (B), zrt3{Delta} (C), and cot1{Delta} zrc1{Delta} (D) mutant cells were grown in the presence and absence of zinc. The steady-state composition of phospholipids was determined by labeling cells for six generations with 32Pi (5 µCi/ml). Phospholipids were extracted and analyzed as described under "Experimental Procedures." The percentages shown for phospholipids were normalized to the total 32Pi-labeled chloroform-soluble fraction, which included sphingolipids and other unidentified phospholipids. Each data point represents the average of three independent experiments ± S.D.

 
Effect of Zinc Depletion on the Expression of Aut7p—An increase in the utilization of PE may contribute to the decrease in PE content in response to zinc depletion. In addition to its role in membrane structure, PE is used directly for the modification of proteins to allow for their association with membranes (9). The Aut7p,4 which is essential for the process of autophagy (i.e. bulk import of cytosolic components into the vacuole for degradation in response to nutrient stress (119)), is modified with PE through covalent modification (78, 120, 121). When autophagy is induced, this modification occurs and is essential for the function of Aut7p in this process (121). Due to the fact that the level of Aut7p is low in growing cells but induced upon nutrient (e.g. nitrogen and carbon) starvation (80, 119), we examined the levels of Aut7p and Aut7p-PE in wild type cells depleted for zinc. Immunoblot analysis showed a significant induction of Aut7p and Aut7p-PE in zinc-depleted cells when compared with cells grown in the presence of zinc (Fig. 9). The specificity of detection for these proteins was confirmed by analysis of a cell extract derived from an aut7{Delta} mutant (Fig. 9). The induction of DGPP phosphatase (Dpp1p), a positive control for zinc depletion (43), was not affected by the aut7{Delta} mutation (Fig. 9).



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  		FIG. 9.
Effect of zinc depletion on the expression of Aut7p and DGPP phosphatase. Wild type (WT; strain SEY6210) and aut7{Delta} mutant cells were grown in the presence and absence of zinc. Cell extracts were prepared and subjected to SDS-PAGE using a 14% gel containing 6 M urea. Proteins were transferred to polyvinylidene difluoride membrane, followed by immunoblot analysis of Aut7p and DGPP phosphatase (Dpp1p) using anti-Aut7p antibodies and anti-Dpp1p antibodies, respectively. Portions of each immunoblot are shown, and the positions of Aut7p, Aut7p-PE, and Dpp1p are indicated.

 
We next questioned whether the induction of Aut7p in response to zinc depletion had an effect on the decrease in PE content. To address this question, aut7{Delta} mutant cells were grown in the presence and absence of zinc, and phospholipids were labeled to steady state with 32Pi. The effects of zinc depletion on the phospholipid composition of the aut7{Delta} mutant were similar to the effects that zinc depletion had on wild type cells (data not shown). Thus, the zinc-mediated decrease in cellular PE content was not due to the induction and modification of Aut7p.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this work, we studied the effects of zinc depletion on the regulation of phospholipid synthesis in S. cerevisiae. The activity levels of the CDP-DAG pathway enzymes PS synthase, PS decarboxylase, PE methyltransferase, and phospholipid methyltransferase were reduced in zinc-depleted cells. In contrast, the activity of PI synthase was elevated in response to zinc depletion. Zinc depletion resulted in alterations in the cellular levels of the major membrane phospholipids PE and PI. The decrease in PE content correlated with the decreases in the activities of PS synthase and PS decarboxylase, whereas the increase in PI content correlated with the increase in the activity of PI synthase. Thus, the regulation of phospholipid synthesis contributed to alterations in phospholipid composition. Although the activities of the phospholipid methyltransferase enzymes were reduced in zinc-depleted cells, this growth condition did not have a major affect on PC content. Enzymes in the CDP-choline pathway for PC synthesis might be activated to compensate for the decrease in activities of the CDP-DAG pathway enzymes.

The elevated expression of Aut7p is a marker for the induction of autophagy (80). The increased expression of Aut7p and Aut7p-PE in response to zinc depletion indicated that this stress condition induced autophagy in yeast. Although the decrease in PE content in response to zinc depletion correlated with the induction of Aut7p and Aut7p-PE, the induction of Aut7p was not a major cause for the reduction in PE content. Therefore, in addition to the decrease in PE synthesis, cumulative effects of PE utilization by additional metabolic processes (e.g. synthesis of glycosylphosphatidylinositol anchors (3)) may have contributed to the decrease in PE content in response to zinc depletion.

One of the most highly regulated CDP-DAG pathway enzymes in S. cerevisiae is the CHO1-encoded PS synthase (13, 25, 123). This enzyme is regulated by genetic and biochemical mechanisms that influence the synthesis of phospholipids via the CDP-DAG pathway as well as the synthesis of PI (13, 25, 123). The expression of CHO1 is repressed by water-soluble phospholipid precursors (67, 100, 103, 104) and in the stationary phase of growth (124, 125). The PS synthase enzyme is activated by DGPP, PA, PC, and PI but inhibited by cardiolipin, diacylglycerol, sphingoid bases, inositol, and CTP (20, 34, 38, 40, 126). The enzyme is also inhibited following its phosphorylation by cAMP-dependent protein kinase (28).

We showed in this study that PS synthase was regulated by zinc availability. Zinc depletion led to the repression of CHO1 transcription, resulting in decreased levels of the PS synthase protein and activity. A lack of the UASZRE in the promoter of the CHO1 gene indicated that the transcription factor Zap1p, a zinc-regulated transcription factor (96), does not directly regulate PS synthase expression in response to zinc depletion. Moreover, an indirect effect of Zap1p on the expression of PS synthase was ruled out because the zap1{Delta} mutation did not affect the zinc-mediated regulation of the enzyme. Instead, the expression of PS synthase by zinc was controlled through the UASINO element and by the transcription factors Ino2p, Ino4p, and Opi1p. These regulatory components play an important role in the inositol-mediated regulation of UASINO-containing genes involved in phospholipid synthesis (13, 24, 26, 67, 100, 103). When wild type cells are grown in the presence of zinc but in the absence of inositol, the Ino2p-Ino4p complex binds the UASINO element to drive transcription for maximum gene expression (13, 24, 26, 100). When cells are grown with zinc and supplemented with inositol, Opi1p represses transcription by binding to DNA-bound Ino2p (127). Like CHO1, the UASINO-containing INO1 gene was repressed by zinc depletion although inositol was absent from the growth medium. Additional studies will be required to verify that the reduced activity levels of the PS decarboxylase, PE methyltransferase, and phospholipid methyltransferase enzymes in zinc-depleted cells were mediated through the UASINO elements present (11, 24) in the promoters of PSD1, CHO2, and OPI3, respectively. Expression of the PIS1 gene encoding PI synthase is not regulated by inositol supplementation and growth phase (33, 124), and the gene does not contain the UASZRE in its promoter. Understanding the molecular basis for the elevated expression of PI synthase activity in response to zinc depletion is a subject of future studies.

It is unclear whether the low level of CHO1 expression in response to zinc depletion resulted from reduced activation by Ino2p-Ino4p and/or by increased repression by Opi1p. Ino2p and Ino4p are not zinc-containing proteins, and thus a reduction in their function was not likely to be a direct consequence of zinc depletion. The INO2 gene contains the UASINO element, and its expression is regulated by inositol supplementation (110). Thus, the reduced expression of CHO1 may be explained if INO2 was also repressed by zinc depletion. Data indicate that Opi1p plays the major role in the repression of UASINO-containing genes in response to inositol supplementation (128). It is unknown whether OPI1 expression is induced by zinc depletion. Opi1p function is regulated by phosphorylation (129, 130) and possibly by cytoplasmic membrane association and nuclear localization (131). It is unknown whether this regulation responds to zinc depletion.

There are other examples of UASINO-mediated regulation of phospholipid biosynthetic genes that occurs in the absence of inositol. The UASINO-containing genes and their enzyme products are repressed when cells enter the stationary phase of growth (124, 125, 132). Cells enter this growth phase and stop proliferating when essential nutrients (e.g. carbon and nitrogen) become limiting (133). In fact, the INO1 gene is repressed by nitrogen starvation, and this regulation is mediated through its UASINO element and the transcription factor Opi1p (134). Nutrient limitation is a common stress condition in both zinc-depleted and stationary phase cells. The common effect of zinc depletion and stationary phase on phospholipid composition was an increase in PI content (124). However, in contrast to the zinc-mediated effect on PE content, stationary phase has little effect on PE (124). Thus, whereas zinc depletion and stationary phase (124, 125, 132) regulated enzyme expression through the UASINO element, these stress conditions yielded different effects on phospholipid composition.

In summary, this work showed that the expression of several phospholipid biosynthetic enzyme activities was coordinately regulated by zinc depletion. The decrease in the level of PS synthase activity was due to decreased CHO1 mRNA and protein levels. This regulation occurred through the UASINO element and by the transcription factors Ino2p, Ino4p, and Opi1p. This work demonstrates that multiple signals, other than inositol, govern the UASINO-mediated regulation of phospholipid synthesis in S. cerevisiae.


   FOOTNOTES
 
* This work was supported in part by United States Public Health Service, National Institutes of Health, Grant GM-28140. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Food Science, Rutgers University, 65 Dudley Rd., New Brunswick, NJ 08901. Tel.: 732-932-9611 (ext. 217); E-mail: carman{at}aesop.rutgers.edu.

1 The abbreviations used are: PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; CDP-DAG, CDP-diacylglycerol; PA, phosphatidate; DGPP, diacylglycerol pyrophosphate. Back

2 The DGPP phosphatase enzyme catalyzes the dephosphorylation of the {beta}-phosphate from DGPP to form PA and then removes the phosphate from PA to form diacylglycerol (58). Back

3 Inositol 3-phosphate synthase is also referred to as inositol 1-phosphate synthase. Back

4 Aut7p is also referred to as Apg8p. Back


   ACKNOWLEDGMENTS
 
We thank Susan A. Henry, David J. Eide, and Daniel J. Klionsky for mutants and reagents used in this study. We also thank Avula Sreenivas for many helpful discussions.



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