©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of Phospholipid Biosynthesis in Saccharomyces cerevisiae by CTP (*)

(Received for publication, April 25, 1995; and in revised form, June 5, 1995)

Virginia M. McDonough (1) Rosa J. Buxeda (1) Maria E. C. Bruno (1) Odile Ozier-Kalogeropoulos (2) (3),  (§),   Marie-Thérèse Adeline (3)(§),   Christopher R. McMaster (4) Robert M. Bell (4) George M. Carman (1)(¶)

From the (1)Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick, New Jersey 08903, the (2)Centre de Génétique Moléculaire du CNRS, Laboratoire Propre Associéà l'Université Pierre et Marie Curie and the (3)Institut de Chimie des Substances Naturelles, 91198 Gil-sur-Yvette Cedex, France, and the (4)Department of Molecular Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In the yeast Saccharomyces cerevisiae, the major membrane phospholipid phosphatidylcholine is synthesized by the CDP-diacylglycerol and CDP-choline pathways. We examined the regulation of phosphatidylcholine synthesis by CTP. The cellular concentration of CTP was elevated (2.4-fold) by overexpressing CTP synthetase, the enzyme responsible for the synthesis of CTP. The overexpression of CTP synthetase resulted in a 2-fold increase in the utilization of the CDP-choline pathway for phosphatidylcholine synthesis. The increase in CDP-choline pathway usage was not due to an increase in the expression of any of the enzymes in this pathway. CDP-choline, the product of the phosphocholine cytidylyltransferase reaction, was the limiting intermediate in the CDP-choline pathway. The apparent K of CTP (1.4 mM) for phosphocholine cytidylyltransferase was 2-fold higher than the cellular concentration of CTP (0.7 mM) in control cells. This provided an explanation of why the overexpression of CTP synthetase caused an increase in the cellular concentration of CDP-choline. Phosphatidylserine synthase activity was reduced in cells overexpressing CTP synthetase. This was not due to a transcriptional repression mechanism. Instead, the decrease in phosphatidylserine synthase activity was due, at least in part, to a direct inhibition of activity by CTP. These results show that CTP plays a role in the regulation of the pathways by which phosphatidylcholine is synthesized. This regulation includes the supply of CTP for the phosphocholine cytidylyltransferase reaction in the CDP-choline pathway and the inhibition of the phosphatidylserine synthase reaction in the CDP-diacylglycerol pathway.


INTRODUCTION

PC (^1)is the essential end product of phospholipid biosynthesis and the major membrane phospholipid found in the yeast Saccharomyces cerevisiae(1, 2, 3, 4) . There are two pathways by which PC is synthesized in S. cerevisiae, the CDP-DG pathway and the CDP-choline pathway (1, 2, 3, 4) (Fig.1). The CDP-DG pathway is primarily used by wild-type cells when they are grown in the absence of choline(1, 2, 3, 4) . However, the CDP-choline pathway becomes more important for PC synthesis when the enzymes in the CDP-DG pathway are repressed or defective(2, 3, 4) . The CDP-DG pathway enzymes CDP-DG synthase(5, 6) , PS synthase(7, 8, 9, 10) , and PS decarboxylase (11, 12, 13) and the two phospholipid N-methyltransferases(7, 11, 14, 15, 16, 17) are repressed when wild-type cells are supplemented with inositol plus choline. The repression of these enzymes is absolutely dependent on inositol, a PI precursor that plays a major role in the coordinate regulation of phospholipid biosynthesis in S. cerevisiae(2, 3, 4) . Under these growth conditions, the exogenous choline is used to synthesize PC via the CDP-choline pathway(2, 4) .


Figure 1: Pathways for the biosynthesis of PC in S. cerevisiae. The pathways shown for the biosynthesis of PC include the relevant steps discussed in the text. The indicated reactions are catalyzed by the following enzymes: 1, CTP synthetase; 2, CDP-DG synthase; 3, PS synthase; 4, PS decarboxylase; 5, phospholipid N-methyltransferases; 6, PI synthase; 7, choline kinase; 8, phosphocholine cytidylyltransferase; 9, cholinephosphotransferase; and 10, PA phosphatase. The CDP-DG pathway is indicated by the boxed area. A more comprehensive phospholipid biosynthetic pathway that includes the steps for the synthesis of phosphatidylglycerol and cardiolipin may be found in (2) . CDP-Etn, CDP-ethanolamine; CDP-Cho, CDP-choline; SL, sphingolipids; PIPs, phosphoinositides; DG, diacylglycerol.



Mutants defective in PS synthase (cho1/pss mutants(18, 19) ), PS decarboxylase (psd1,psd2 double mutants(20, 21) ), or the phospholipid N-methyltransferases (pem1/cho2,pem2/opi3 double mutants(22, 23, 24, 25) ) require choline for growth in order to synthesize PC via the CDP-choline pathway. The mutants defective in PS synthase (18, 19) and PS decarboxylase (20, 21) can also synthesize PC if they are supplemented with ethanolamine. The ethanolamine is used for PE synthesis via the CDP-ethanolamine pathway(26) . The PE is subsequently methylated by the phospholipid N-methyltransferases to form PC (Fig.1). Overall, these results led to the notion that the CDP-choline pathway was an auxiliary or salvage pathway in S. cerevisiae(2, 3, 4) . However, recent studies have shown that the CDP-choline pathway in S. cerevisiae is not simply a salvage pathway for PC synthesis. In fact, the CDP-choline pathway contributes to PC synthesis even when wild-type cells are grown in the absence of choline(27, 28) .

CTP plays an essential role in the synthesis of PC and all membrane phospholipids in S. cerevisiae. CTP is the direct precursor of the activated, energy-rich phospholipid pathway intermediates CDP-DG (29) , CDP-choline(26) , and CDP-ethanolamine (26) (Fig.1). CDP-DG is the source of the phosphatidyl moiety of PS, PE, and PC synthesized by the CDP-DG pathway as well as PI, phosphatidylglycerol, and cardiolipin (2, 4) . CDP-choline and CDP-ethanolamine are the sources of the hydrophilic head groups of PC and PE synthesized by the CDP-choline and CDP-ethanolamine pathways, respectively(2, 4) . In this work, we examined the effect of CTP on phospholipid synthesis. We show that an elevation in the cellular concentration of CTP results in an increase in the utilization of the CDP-choline pathway for the synthesis of PC. The mechanism for this regulation includes the supply of CTP for the phosphocholine cytidylyltransferase reaction in the CDP-choline pathway and the inhibition of the PS synthase reaction in the CDP-DG pathway.


EXPERIMENTAL PROCEDURES

Materials

All chemicals were reagent grade. Growth medium supplies were purchased from Difco. Nucleotides, choline, phosphocholine, CDP-choline, and bovine serum albumin were obtained from Sigma. Phospholipids were purchased from Avanti Polar Lipids, Inc. and Sigma. Radiochemicals, EN^3HANCE, and GeneScreen hybridization transfer membrane were purchased from DuPont NEN. Scintillation counting supplies were from National Diagnostics, Inc. Restriction endonucleases and the random primer NEBlot kit were purchased from New England Biolabs Inc. Protein assay reagent, electrophoresis reagents, and immunochemical reagents were purchased from Bio-Rad. Silica Gel 60 thin-layer chromatography plates and high-performance thin-layer chromatography plates were from EM Science.

Methods

Strains, Plasmids, and Recombinant DNA Techniques

CTP synthetase was expressed in S. cerevisiae strain OK8 (MATalpha leu2 trp1 ura3 ura7Delta::TRP1 ura8) (30) bearing the URA7 gene (31) on the multicopy plasmid pFL44S (32) or the single copy centromeric plasmid pFL38(32) . Strain OK8 has mutations in both the URA7 and URA8 genes, which are two duplicate genes encoding for CTP synthetase(30, 31) . PS synthase was partially purified from S. cerevisiae mutant strain VAL2C (MATaleu2-3 leu2-112 ade6 cho1) bearing plasmid YEpCHO1(33) . Plasmid YEpCHO1 contains the structural gene for PS synthase and directs the overexpression of the enzyme(33) . Cholinephosphotransferase was isolated from S. cerevisiae mutant strain HJ091 (MATalpha leu2-3 leu2-112 his3-1 ura3-52 trp1-289 cpt1::LEU2 ept1-1) (34) bearing CPT1 on the multicopy plasmid pCM26CPT. Plasmid pCM26CPT was constructed by subcloning CPT1 from plasmid pRH150 (35) using the SacI and SalI sites in the multiple cloning site to the multicopy plasmid pRS426(36) . Plasmid pCM26CPT contains the structural gene for cholinephosphotransferase and directs the overexpression of activity. All DNA manipulations were performed according to standard methods(37) . Plasmid maintenance and amplifications were performed in Escherichia coli strain DH5alpha.

Growth Conditions

Cells were grown in complete synthetic medium (38) containing 2% glucose and appropriate supplements at 30 °C to the exponential phase of growth (1-2 10^7 cells/ml). To label total phospholipids and PC, cells were grown for five to six generations in the presence of P(i) (4 µCi/ml) and [methyl-^3H]choline (0.4 µCi/ml), respectively. To label the CDP-choline pathway intermediates, cells were grown for five to six generations in the presence of [methyl-^3H]choline (10 µCi/ml). Cell numbers were determined by microscopic examination with a hemocytometer.

Preparation of RNA and Northern Blot Analysis

Total RNA was extracted from cells using hot phenol as described by Schmitt et al.(39) . The RNA was separated by electrophoresis under denaturing conditions using a 1% formaldehyde-agarose gel(40) . Following electrophoresis, RNA was transferred to GeneScreen and probed with a radiolabeled fragment of the URA7, CHO1, PIS, or CPT1 gene. The URA7 probe was a 1.6-kilobase fragment isolated from YEp352URA7(41) by EcoRI and HindIII digestion. The CHO1 probe was a 1.0-kilobase fragment isolated from pAS103 (42) by HindIII and SacI digestion. The PIS probe was a 1.6-kilobase fragment isolated from pPI514 (43) by HindIII digestion. The CPT1 probe was a 1.1-kilobase fragment isolated from pRH150 (35) by NcoI digestion. The probes were labeled with [alpha-P]dCTP by the random priming reaction using a NEBlot kit. Prehybridization and hybridization of blots were carried out at 60 °C in modified Church buffer (44) as recommended by United States Biochemical Corp. Ribosomal subunit L32 mRNA (45) was used as a constitutive standard and loading control.

Preparation of Enzymes

Cells were disrupted with glass beads with a Mini-Bead-Beater (Biospec Products, Inc.) in 50 mM Tris-HCl buffer (pH 7.5) containing 1 mM disodium EDTA, 0.3 M sucrose, and 10 mM 2-mercaptoethanol(7) . Glass beads and cell debris were removed by centrifugation at 1500 g for 5 min. The supernatant (cell extract) was used for enzyme assays and immunoblotting of CTP synthetase. PS synthase was partially purified from the cell extract through the Triton X-100 solubilization step as described by Bae-Lee and Carman(46) . Microsome-associated cholinephosphotransferase was isolated from the cell extract as described by McMaster and Bell(47) .

Immunoblotting of CTP Synthetase

Immunoblot assays were performed with IgG anti-CTP synthetase antibodies (41) as described previously(48) . The density of the CTP synthetase bands on immunoblots was quantitated by scanning densitometry. Immunoblot signals were in the linear range of detectability.

Extraction and Analysis of UTP and CTP

Cells bearing URA7 on the multicopy and single copy plasmids were grown to the exponential phase of growth. Cellular nucleotides were extracted (31) , and UTP and CTP were analyzed by high-performance liquid chromatography as described by Ozier-Kalogeropoulos et al.(30) .

Analysis of Phospholipids

Membranes were prepared from cell extracts(49) , and phospholipids were extracted by the method of Bligh and Dyer(50) . The chloroform phase was neutralized and dried in vacuo, and the residue was dissolved in chloroform. Phospholipids were analyzed by one-dimensional thin-layer chromatography on highperformance silica gel thin-layer plates using the solvent system methyl acetate, isopropyl alcohol, chloroform, methanol, 0.25% KCl (25:25:28:10:7) as described by Knoll et al.(51) . The positions of the labeled phospholipids on chromatograms were determined by autoradiography and compared with standard phospholipids after exposure to iodine vapor. The amount of each labeled phospholipid was determined by liquid scintillation counting of the corresponding spots on chromatograms.

Analysis of CDP-choline Pathway Intermediates

Choline, phosphocholine, and CDP-choline were obtained from whole cells after lipid extraction(50) . The aqueous phase was neutralized and dried in vacuo, and the residue was dissolved in deionized water. Samples were subjected to centrifugation at 12,000 g for 3 min to remove insoluble material. The CDP-choline pathway intermediates were separated by thin-layer chromatography with Silica Gel 60 plates using the solvent system methanol, 0.5% sodium chloride/ammonia (50:50:1) as described by Teegarden et al.(52) . The positions of the labeled intermediates on chromatograms were determined by fluorography and compared with standards. The amount of each labeled compound was determined by liquid scintillation counting.

Enzyme Assays

All assays were conducted at 30 °C. The activities of CDP-DG synthase(53) , PS synthase(54) , PS decarboxylase (55) , the phospholipid N-methyltransferases(14) , PI synthase(56) , choline kinase(57) , phosphocholine cytidylyltransferase (58) , cholinephosphotransferase(59) , and PA phosphatase (60) were measured as described previously. All assays were linear with time and protein concentration. All assays were conducted in triplicate with an average standard deviation of ±5%. One unit of enzymatic activity is defined as the amount of enzyme that catalyzes the formation of 1 nmol of product/min. Specific activity is defined as units/mg of protein. Protein concentration was determined by the method of Bradford (61) using bovine serum albumin as the standard.


RESULTS

Effect of CTP Synthetase Overexpression on the Cellular Concentration of CTP

The aim of this work was to examine the regulation of phospholipid biosynthesis in S. cerevisiae by CTP. Cellular CTP levels are not easily manipulated in S. cerevisiae through the exogenous supply of cytidine or cytosine. Cytidine is converted to cytosine, which is then converted to CTP through the pyrimidine biosynthetic pathway (cytosine uracil UMP UDP CTP)(62) . Since the pathway for CTP synthesis is indirect, cytidine or cytosine supplementation was not the best way of elevating the cellular concentration of CTP. Our approach to elevate the cellular concentration of CTP was to overexpress the enzyme CTP synthetase. CTP synthetase catalyzes the final step in the pyrimidine biosynthetic pathway(31) . CTP synthetase is encoded by two duplicate genes named URA7(31) and URA8(30) . Neither one of the URA7 and URA8 genes is essential provided that cells possess one functional gene encoding for the enzyme(30, 31) . The URA7 gene was expressed in strain OK8 on a multicopy plasmid or a single copy centromeric plasmid. The expression of the URA7 gene on the centromeric plasmid was used as a control. The growth of strain OK8 bearing either one of the plasmids was the same. This was an important consideration in our studies because the regulation of phospholipid biosynthesis in S. cerevisiae is influenced by growth phase(63) .

Northern blot and immunoblot analyses were used to determine the amounts of CTP synthetase mRNA and protein in cells bearing the URA7 gene on the multicopy and single copy plasmids. The levels of CTP synthetase mRNA and protein found in cells bearing URA7 on the multicopy plasmid were 25- and 10-fold greater, respectively, than those found in control cells (Fig.2A). Cells overexpressing CTP synthetase had a 2.4-fold higher cellular concentration of CTP when compared with the control cells (Fig.2B). Similar results have been reported for the overexpression of CTP synthetase protein and the cellular concentration of CTP in cells bearing URA7 on a multicopy plasmid when compared with wild-type cells(41) . The cellular concentrations of UTP were not affected by the overexpression of CTP synthetase (Fig.2B).


Figure 2: Effect of URA7 overexpression on CTP synthetase mRNA and protein levels and the cellular concentration of UTP and CTP. Cells bearing URA7 on the multicopy (MC-URA7) and single copy centromeric (Cen-URA7) plasmids were grown in complete synthetic medium to the exponential phase of growth. A, CTP synthetase mRNA and protein were determined by Northern blot and immunoblot analyses, respectively, as described under ``Experimental Procedures.'' The amounts of CTP synthetase mRNA and protein found in cells bearing URA7 on the single copy plasmid were set at 1. B, nucleotides were extracted and analyzed by high-performance liquid chromatography as described in the text. The values reported for UTP and CTP were determined from triplicate analyses with standard deviations of ±12 and ±8%, respectively, from a minimum of two independent growth studies.



Effect of CTP Synthetase Overexpression on Phospholipid Composition

Cells were grown in synthetic medium in the absence of inositol, choline, and ethanolamine. Under these growth conditions, our studies were not complicated by the regulatory effects these phospholipid precursors have on the expression of phospholipid biosynthetic enzymes(2, 4) . Since cells utilize both the CDP-DG and CDP-choline pathways in the absence of exogenous choline(27, 28) , we determined the phospholipid composition of membranes from cells labeled to steady state with both P(i) and [methyl-^3H]choline. P(i) will be incorporated into phospholipids synthesized by either the CDP-DG or CDP-choline pathway, whereas the labeled choline will only be incorporated into PC synthesized via the CDP-choline pathway. The concentration of choline added to the growth medium from the radioactive label was 0.1 µM. This concentration was too low to affect the rate of synthesis of PC by the CDP-choline pathway(47) . The phospholipid composition of membranes determined by P(i) labeling is shown in Fig.3A. The composition of the major membrane phospholipids was not significantly affected by the overexpression of CTP synthetase. There was, however, a 1.6-fold increase in the percentage of label incorporated into CDP-DG.


Figure 3: Effect of CTP synthetase overexpression on phospholipid composition and PC synthesized via the CDP-DG and CDP-choline pathways. Cells bearing URA7 on the multicopy (MC-URA7) and single copy centromeric (Cen-URA7) plasmids were grown in complete synthetic medium to the exponential phase of growth. The steady-state compositions of phospholipids (A) and PC (B) were determined by labeling cells for five to six generations with P(i) (4 µCi/ml) and [methyl-^3H]choline (0.4 µCi/ml). The incorporation of P(i) and [methyl-^3H]choline into total phospholipids and PC was 1600-2000 and 10,000-12,000 cpm/10^7 cells, respectively. Phospholipids were extracted and analyzed as described under ``Experimental Procedures.'' The values reported in A were determined from P(i) labeling. The values in B are reported as the cpm of ^3H incorporated into PC relative to the cpm of P incorporated into PC. The percentages reported for phospholipids were determined from duplicate analyses with a standard deviation of ±10% from a minimum of two independent growth studies. CDG, CDPdiacylglycerol.



Radiolabeled choline was incorporated into PC (Fig.3B). This confirmed that the CDP-choline pathway contributed to PC synthesis when cells were grown in the absence of supplemented choline. The data shown in Fig.3B are plotted as the ratio of the cpm of ^3H incorporated into PC to the cpm of P incorporated into PC. If the cellular concentration of CTP affected the pathway by which PC was synthesized, the ratio of the labels found in PC would change. Indeed, this ratio increased 2-fold for cells which overexpressed CTP synthetase when compared with the control cells (Fig.3B). This indicated that the elevation in the cellular concentration of CTP caused an increase in the utilization of the CDP-choline pathway for PC synthesis.

Effect of CTP Synthetase Overexpression on CDP-choline Pathway Intermediates

The intermediates of the CDP-choline pathway include choline, phosphocholine, and CDP-choline (Fig.1). Cells were labeled with [methyl-^3H]choline to steady state to analyze the effect of CTP synthetase overexpression on the composition of the CDP-choline pathway intermediates. In control cells, 1% of the water-soluble label was incorporated into CDP-choline, whereas most of the label was found in choline and phosphocholine (Fig.4). Choline and phosphocholine were not affected by the overexpression of CTP synthetase (Fig.4). However, the overexpression of CTP synthetase caused a 1.4-fold increase in the steady-state concentration of CDP-choline when compared with the control cells (Fig.4).


Figure 4: Effect of CTP synthetase overexpression on the CDP-choline pathway intermediates. Cells bearing URA7 on the multicopy (MC-URA7) and single copy centromeric (Cen-URA7) plasmids were grown in complete synthetic medium to the exponential phase of growth. The steady-state composition of the CDP-choline pathway intermediates was determined by labeling cells for five to six generations with [methyl-^3H]choline (10 µCi/ml). The incorporation of [methyl-^3H]choline into the CDP-choline pathway intermediates was 40,000-70,000 cpm/10^7 cells. The CDP-choline pathway intermediates were extracted and analyzed as described under ``Experimental Procedures.'' The percentages reported for choline, phosphocholine, and CDP-choline were determined from triplicate analyses with standard deviations of ±10, 10, and 15%, respectively, from a minimum of two independent growth studies.



Effect of CTP Synthetase Overexpression on Phospholipid Biosynthetic Enzyme Activities

Steady-state labeling experiments indicated that the increase in CTP synthetase expression affected the pathways by which PC was synthesized. We questioned if phospholipid biosynthetic enzyme activities were affected in cells that overexpressed CTP synthetase. These enzyme activities included those responsible for PC synthesis via the CDP-DG pathway (CDP-DG synthase, PS synthase, PS decarboxylase, and the phospholipid N-methyltransferases) and the CDP-choline pathway (choline kinase, phosphocholine cytidylyltransferase, and cholinephosphotransferase). PI synthase and PA phosphatase activities were also examined because their reactions play a role in the CDP-DG and CDP-choline pathways(2, 4) . Cells were grown to the exponential phase of growth, cell extracts were prepared, and the activities of the enzymes were measured. The cell extracts were diluted to lower the CTP concentration derived from cells to a concentration that would not affect the enzymes directly. The overexpression of CTP synthetase resulted in decreases in PS synthase and cholinephosphotransferase activities of 41 and 23%, respectively (Fig.5). On the other hand, CTP synthetase overexpression resulted in a 120% increase in PI synthase activity (Fig.5).


Figure 5: Effect of CTP synthetase overexpression on the enzyme activities of the CDP-DG and CDP-choline pathways. Cells bearing URA7 on the multicopy (MC-URA7) and single copy centromeric (Cen-URA7) plasmids were grown in complete synthetic medium to the exponential phase of growth. Cell extracts were prepared and used for the measurement of the indicated CDP-DG (A) and CDP-choline (B) pathway enzymes. The relative activity (percent) was calculated by normalizing the specific activity of each enzyme from cells bearing URA7 on the multicopy plasmid to cells bearing URA7 on the single copy plasmid. Enzyme activities were determined in triplicate with a standard deviation of ±5% from a minimum of two independent growth experiments. CDS, CDP-DG synthase; PSS, PS synthase; PSD, PS decarboxylase; PMT, phospholipid methyltransferases; PIS, PI synthase; CK, choline kinase; CCT, phosphocholine cytidylyltransferase; CPT, cholinephosphotransferase; PAP, PA phosphatase.



We questioned whether the overexpression of CTP synthetase affected the abundance of the mRNAs encoding for PS synthase, cholinephosphotransferase, and PI synthase. Cells bearing the URA7 gene on the multicopy and single copy plasmids were grown to the exponential phase of growth, and total RNA was extracted. The relative abundance of the mRNAs from these cells was determined by Northern blot analysis using CHO1, CPT1, and PIS probes. The abundance of PS synthase, cholinephosphotransferase, and PI synthase mRNAs was not affected by the overexpression of CTP synthetase (data not shown).

Effect of CTP on Phospholipid Biosynthetic Enzyme Activities

We examined if CTP had a direct effect on the CDP-DG and CDP-choline pathway enzymes using cell extracts prepared from control cells. Enzyme assays were performed in the absence and presence of 5 mM CTP. This analysis did not include CDP-DG synthase or phosphocholine cytidylyltransferase since these enzymes use CTP as a substrate. The only enzyme activities that were affected by CTP were PS synthase, cholinephosphotransferase, and PA phosphatase. All three of these enzymes were inhibited by CTP. The effects of CTP on PS synthase and cholinephosphotransferase activities are described below. The inhibition of PA phosphatase activity by CTP has been previously reported(64) .

Effect of CTP on PS Synthase Activity

A Triton X-100-solubilized preparation of microsome-associated PS synthase was used to examine the effect of CTP on activity. PS synthase activity is absolutely dependent on either MnCl(2) (0.6 mM) or MgCl(2) (10 mM) as a cofactor(46) . The PS synthase activity obtained with 0.6 mM MnCl(2) was 2.7-fold greater than the activity obtained with 15 or 30 mM MgCl(2) (Fig.6). These results were consistent with those previously reported for a homogeneous preparation of the enzyme(46) . CTP inhibited PS synthase activity in a dose-dependent manner when 0.6 mM MnCl(2) was used as the cofactor (Fig.6). The CTP-mediated inhibition of activity followed cooperative kinetics (Fig.6, inset). Analysis of the data according to the Hill equation yielded an IC value for CTP of 2.1 mM and a Hill number of 4.3. The inhibition of PS synthase activity by CTP using 15 mM MgCl(2) as the cofactor followed saturation kinetics (Fig.6). PS synthase activity was less sensitive to inhibition (IC = 5 mM) by CTP when MgCl(2) was the cofactor. At the point of maximum inhibition, the remaining PS synthase activity was the same regardless of whether 0.6 or 15 mM MgCl(2) was used as the cofactor (Fig.6). As the CTP concentration was increased in these experiments, the free Mn and Mg concentrations would decrease (65) below the optimum concentrations required for PS synthase activity(46) . These results were consistent with the conclusion that PS synthase was inhibited by CTP by a chelation mechanism. Indeed, PS synthase activity was not inhibited by CTP when the MgCl(2) concentration was increased to 30 mM (Fig.6). The MnCl(2) concentration could not be increased above 0.6 mM due to the inhibition of the enzyme by MnCl(2)(46) .


Figure 6: Effect of CTP on PS synthase activity. PS synthase activity was measured in the absence and presence of CTP using the following concentrations of MnCl(2) and MgCl(2) as cofactors: bullet, 0.6 mM MnCl(2); , 15 mM MgCl(2); black square, 30 mM MgCl(2). The inset is a replot of the CTP-mediated inhibition of PS synthase activity using MnCl(2) as the cofactor.



Effect of CTP on Cholinephosphotransferase Activity

Microsome-associated cholinephosphotransferase was used to examine the effect of CTP on enzyme activity. The strain used to isolate cholinephosphotransferase contained a mutation in the EPT1 gene, which is the structural gene for ethanolaminephosphotransferase(66) . Ethanolaminephosphotransferase also possesses cholinephosphotransferase activity(59) . Thus, our studies were not complicated by the presence of this second enzyme. Maximum cholinephosphotransferase activity is dependent on 7.5-15 mM MgCl(2) as a cofactor. (^2)The addition of CTP to the assay system for cholinephosphotransferase resulted in a dose-dependent inhibition of activity (Fig.7). The enzyme was less sensitive to inhibition by CTP when the MgCl(2) concentration in the assay was increased from 7.5 mM (IC = 5 mM) to 30 mM (IC = 9.5 mM). As discussed above for the PS synthase reaction, these results indicated that CTP inhibited cholinephosphotransferase activity by a chelation mechanism. A similar conclusion was previously reported in a preliminary study of the cholinephosphotransferase from S. cerevisiae(67) .


Figure 7: Effect of CTP on cholinephosphotransferase activity. Cholinephosphotransferase activity was measured in the absence and presence of CTP using the indicated concentrations of MgCl(2) as a cofactor.




DISCUSSION

The aim of this work was to examine the regulation of phospholipid biosynthesis in S. cerevisiae by CTP. CTP is essential for the biosynthesis of all membrane phospholipids in S. cerevisiae whether they are synthesized via the CDP-DG or CDP-choline pathway(1, 2, 3, 4) . Our rationale was to elevate the cellular concentration of CTP by overexpressing CTP synthetase. Given the pyrimidine biosynthetic pathways in S. cerevisiae(62) , this was the most straightforward way of elevating the cellular concentration of CTP. The expression of the URA7 gene on a multicopy plasmid resulted in an appreciable overexpression of CTP synthetase mRNA and protein when compared with control cells. However, there was only a 2.4-fold increase in the cellular concentration of CTP in these cells. The discrepancy between the relatively high level of CTP synthetase overexpression and the relatively low increase in the cellular concentration of CTP can be explained by the inhibition of CTP synthetase activity by CTP(41) . This regulation of CTP synthetase activity by CTP inhibition could not be overcome by further overexpression of the URA7 gene. Nevertheless, the 2.4-fold elevation in the cellular concentration of CTP was enough to address the regulation of phospholipid biosynthesis by CTP.

The overexpression of CTP synthetase did not have a significant effect on the overall composition of the major membrane phospholipids. However, the overexpression of CTP synthetase resulted in a 2-fold increase in the utilization of the CDP-choline pathway for PC biosynthesis. This increase in CDP-choline pathway usage was not due to an increase in the expression of any of the enzyme activities in this pathway. Under the growth conditions of our experiments (i.e. absence of exogenous choline), the choline needed for PC synthesis via the CDP-choline pathway was presumably derived from the turnover of PC synthesized via the CDP-DG pathway(27, 28) . It is unclear what the contribution of the CDP-choline pathway is relative to the CDP-DG pathway when cells are grown in the absence of exogenous choline. This is a difficult question to address and could not be determined from the data presented here. The fact that the PI/PC transfer protein (Sec14p) is essential in cells with a functional CDP-choline pathway suggests that the CDP-choline pathway plays an important role in PC synthesis(27, 68) .

CDP-choline accounted for only 1% of the CDP-choline pathway intermediates of control cells. In addition, the apparent K of CTP (1.4 mM) for the phosphocholine cytidylyltransferase reaction (69) was 2-fold higher than the cellular concentration of CTP (0.7 mM) in control cells. Taken together, these results were consistent with the notion (47) that the phosphocholine cytidylyltransferase reaction catalyzes the rate-limiting step in the CDP-choline pathway in S. cerevisiae. The overexpression of CTP synthetase brought the cellular concentration of CTP (1.7 mM) up to the K of CTP for the phosphocholine cytidylyltransferase reaction. Thus, based on the kinetic constant for CTP and its cellular concentration, one would expect that the increase in the cellular concentration of CTP would cause an increase in phosphocholine cytidylyltransferase activity in vivo. Indeed, the overexpression of CTP synthetase resulted in an increase in the cellular concentration of CDP-choline. This increase in the CDP-choline concentration was consistent with the increased utilization of the CDP-choline pathway for PC synthesis.

The K of CTP (1 mM) for the CDP-DG synthase reaction (70) was 1.4-fold higher than the cellular concentration of CTP in control cells. The increase in the cellular concentration of CTP due to CTP synthetase overexpression brought its concentration nearly 2-fold higher than the K of CTP. As discussed above for the phosphocholine cytidylyltransferase reaction, an argument can be made based on the kinetic constant for CTP and its cellular concentrations for the regulation of CDP-DG synthase activity by CTP in vivo. Indeed, the overexpression of CTP synthetase resulted in an increase in the concentration of CDP-DG. However, this did not result in a greater utilization of the CDP-DG pathway for PC synthesis. In contrast to the phosphocholine cytidylyltransferase reaction, the synthesis of CDP-DG is not a rate-limiting step in the CDP-DG pathway(79) . Moreover, PS synthase activity was reduced in cells that overexpressed CTP synthetase. Since PS synthase plays a major role in the regulation of the CDP-DG pathway for PC biosynthesis(2, 3, 4) , the inhibition of this enzyme would be expected to reduce the synthesis of PC via this pathway.

Mechanisms other than transcriptional regulation affected the expression of PS synthase, cholinephosphotransferase, and PI synthase activities in cells overexpressing CTP synthetase. In addition, PS synthase and cholinephosphotransferase activities were directly inhibited by CTP. The mechanism of PS synthase and cholinephosphotransferase inhibition by CTP was the chelation of their divalent metal cofactors. However, it is unclear whether this mechanism of inhibition by CTP would be physiologically relevant.

In contrast to S. cerevisiae, the CDP-choline pathway is the main route of PC synthesis in mammalian cells(71, 72) . In mammalian cells(71) , as in S. cerevisiae, the phosphocholine cytidylyltransferase reaction is the rate-limiting step in the CDP-choline pathway. The elevation of CTP levels in poliovirus-infected HeLa cells (73) and cytidine-supplemented neuron-related PC12 cells (74) also results in an increase in PC synthesis via the CDP-choline pathway. In the poliovirus-infected HeLa cells, the mechanism for the increase in the utilization of the CDP-choline for PC synthesis is the stimulation of the phosphocholine cytidylyltransferase reaction by CTP (73) .

In addition to CTP, the supply of other phospholipid pathway intermediates has been shown to play a role in the regulation of phospholipid biosynthesis in S. cerevisiae. The cellular concentration of ATP plays a role in the proportional synthesis of triacylglycerols and phospholipids (64) and also the synthesis of phosphoinositides(75, 76) . Elevated ATP levels favor phospholipid synthesis at the expense of triacylglycerols, whereas reduced ATP levels have the opposite effect. These effects have been attributed in part to the regulation of PA phosphatase activity by the cellular concentration of ATP(64) . Similarly, high levels of ATP favor the synthesis of PI 4-phosphate and PI 4,5-bisphosphate, whereas low ATP levels have the opposite effect. These effects are due to the regulation of membrane-associated PI 4-kinase activity by the cellular concentrations of ATP and ADP(77) . The synthesis of phosphoinositides in S. cerevisiae is also regulated by the cellular concentrations of CDP-DG through the control of membrane-associated PI 4-kinase activity(78) . As a final example, the cellular concentration of inositol regulates the partitioning of CDP-DG between PI and PS through the regulation of PI synthase and PS synthase activities(79) .

In summary, we have shown that CTP plays a role in the regulation of the pathways by which PC is synthesized. This regulation includes the supply of CTP for the phosphocholine cytidylyltransferase reaction in the CDP-choline pathway and the inhibition of the PS synthase reaction in the CDP-DG pathway. These studies further underscore the complexity of the biochemical mechanisms that regulate phospholipid biosynthesis in S. cerevisiae.


FOOTNOTES

*
This work was supported by United States Public Health Service Grants GM-50679 (to G. M. C.) and GM-20015 (to R. M. B.) from the National Institutes of Health and by the Charles and Johanna Busch Memorial Fund (to G. M. C.). This is New Jersey Agricultural Experiment Station Publication D-10581-2-95. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Unité de Génétique Moléculaire des Levures, Dépt. des Biotechnologies, Inst. Pasteur, 25 rue du Docteur Roux, 75724 Paris, France.

To whom correspondence and reprint requests should be addressed. Tel: 908-932-9663; Fax: 908-932-6776; george{at}a1.caft1vax.rutgers.edu.

^1
The abbreviations used are: PC, phosphatidylcholine; PS, phosphatidylserine; PI, phosphatidylinositol; PE, phosphatidylethanolamine; PA, phosphatidate; CDP-DG, CDP-diacylglycerol.

^2
C. R. McMaster and R. M. Bell, unpublished data.


ACKNOWLEDGEMENTS

We are grateful to Connie Clancey and William Dowhan for performing the PS decarboxylase assays. We thank Susan A. Henry and Satoshi Yamashita for providing the CHO1 and PIS clones, respectively, used in this study.


REFERENCES

  1. Henry, S. A. (1982) in The Molecular Biology of the Yeast Saccharomyces: Metabolism and Gene Expression (Strathern, J. N., Jones, E. W., and Broach, J. R., eds) pp. 101-158, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  2. Carman, G. M., and Henry, S. A. (1989) Annu. Rev. Biochem. 58,635-669 [CrossRef][Medline] [Order article via Infotrieve]
  3. Carman, G. M. (1989) in Phosphatidylcholine Metabolism (Vance, D. E., ed) pp. 165-183, CRC Press, Inc., Baca Raton, FL
  4. Paltauf, F., Kohlwein, S. D., and Henry, S. A. (1992) in The Molecular and Cellular Biology of the Yeast Saccharomyces: Gene Expression (Jones, E. W., Pringle, J. R., and Broach, J. R., eds) pp. 415-500, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  5. Homann, M. J., Henry, S. A., and Carman, G. M. (1985) J. Bacteriol. 163,1265-1266 [Medline] [Order article via Infotrieve]
  6. Klig, L. S., Homann, M. J., Kohlwein, S., Kelley, M. J., Henry, S. A., and Carman, G. M. (1988) J. Bacteriol. 170,1878-1886 [Medline] [Order article via Infotrieve]
  7. Klig, L. S., Homann, M. J., Carman, G. M., and Henry, S. A. (1985) J. Bacteriol. 162,1135-1141 [Medline] [Order article via Infotrieve]
  8. Poole, M. A., Homann, M. J., Bae-Lee, M., and Carman, G. M. (1986) J. Bacteriol. 168,668-672 [Medline] [Order article via Infotrieve]
  9. Bailis, A. M., Poole, M. A., Carman, G. M., and Henry, S. A. (1987) Mol. Cell. Biol. 7,167-176 [Medline] [Order article via Infotrieve]
  10. Carson, M. A., Atkinson, K. D., and Waechter, C. J. (1982) J. Biol. Chem. 257,8115-8121 [Abstract/Free Full Text]
  11. Carson, M. A., Emala, M., Hogsten, P., and Waechter, C. J. (1984) J. Biol. Chem. 259,6267-6273 [Abstract/Free Full Text]
  12. Overmeyer, J. H., and Waechter, C. J. (1991) Arch. Biochem. Biophys. 290,511-516 [Medline] [Order article via Infotrieve]
  13. Lamping, E., Kohlwein, S. D., Henry, S. A., and Paltauf, F. (1991) J. Bacteriol. 173,6432-6437 [Medline] [Order article via Infotrieve]
  14. Gaynor, P. M., Gill, T., Toutenhoofd, S., Summers, E. F., McGraw, P., Homann, M. J., Henry, S. A., and Carman, G. M. (1991) Biochim. Biophys. Acta 1090,326-332 [Medline] [Order article via Infotrieve]
  15. Yamashita, S., Oshima, A., Nikawa, J., and Hosaka, K. (1982) Eur. J. Biochem. 128,589-595 [Abstract]
  16. Yamashita, S., and Oshima, A. (1980) Eur. J. Biochem. 104,611-616 [Abstract]
  17. Waechter, C. J., and Lester, R. L. (1973) Arch. Biochem. Biophys. 158,401-410 [Medline] [Order article via Infotrieve]
  18. Atkinson, K., Fogel, S., and Henry, S. A. (1980) J. Biol. Chem. 255,6653-6661 [Abstract/Free Full Text]
  19. Atkinson, K. D., Jensen, B., Kolat, A. I., Storm, E. M., Henry, S. A., and Fogel, S. (1980) J. Bacteriol. 141,558-564 [Medline] [Order article via Infotrieve]
  20. Trotter, P. J., and Voelker, D. R. (1995) J. Biol. Chem. 270,6062-6070 [Abstract/Free Full Text]
  21. Trotter, P. J., Pedretti, J., Yates, R., and Voelker, D. R. (1995) J. Biol. Chem. 270,6071-6080 [Abstract/Free Full Text]
  22. Kodaki, T., and Yamashita, S. (1987) J. Biol. Chem. 262,15428-15435 [Abstract/Free Full Text]
  23. Kodaki, T., and Yamashita, S. (1989) Eur. J. Biochem. 185,243-251 [Abstract]
  24. Summers, E. F., Letts, V. A., McGraw, P., and Henry, S. A. (1988) Genetics 120,909-922 [Abstract/Free Full Text]
  25. McGraw, P., and Henry, S. A. (1989) Genetics 122,317-330 [Abstract/Free Full Text]
  26. Kennedy, E. P., and Weiss, S. B. (1956) J. Biol. Chem. 222,193-214 [Free Full Text]
  27. McGee, T. P., Skinner, H. B., Whitters, E. A., Henry, S. A., and Bankaitis, V. A. (1994) J. Cell Biol. 124,273-287 [Abstract]
  28. McMaster, C. R., and Bell, R. M. (1994) J. Biol. Chem. 269,28010-28016 [Abstract/Free Full Text]
  29. Carter, J. R., and Kennedy, E. P. (1966) J. Lipid Res. 7,678-683 [Abstract/Free Full Text]
  30. Ozier-Kalogeropoulos, O., Adeline, M., Yang, W., Carman, G. M., and Lacroute, F. (1994) Mol. & Gen. Genet. 242,431-439
  31. Ozier-Kalogeropoulos, O., Fasiolo, F., Adeline, M., Collin, J., and Lacroute, F. (1991) Mol. & Gen. Genet. 231,7-16
  32. Bonneaud, N., Ozier-Kalogeropoulos, O., Li, G., Labouesse, M., Minvielle-Sebastia, L., and Lacroute, F. (1991) Yeast 7,609-615 [Medline] [Order article via Infotrieve]
  33. Letts, V. A., Klig, L. S., Bae-Lee, M., Carman, G. M., and Henry, S. A. (1983) Proc. Natl. Acad. Sci. U. S. A. 80,7279-7283 [Abstract]
  34. Hjelmstad, R. H., Morash, S. C., McMaster, C. R., and Bell, R. M. (1994) J. Biol. Chem. 269,20995-21002 [Abstract/Free Full Text]
  35. Hjelmstad, R. H., and Bell, R. M. (1987) J. Biol. Chem. 262,3909-3917 [Abstract/Free Full Text]
  36. Christianson, T. W., Sikorski, R. S., Dante, M., Shero, J. H., and Hieter, P. (1992) Gene (Amst.) 110,119-122 [CrossRef][Medline] [Order article via Infotrieve]
  37. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  38. Culbertson, M. R., and Henry, S. A. (1975) Genetics 80,23-40 [Abstract/Free Full Text]
  39. Schmitt, M. E., Brown, T. A., and Trumpower, B. L. (1990) Nucleic Acids Res. 18,3091-3092 [Medline] [Order article via Infotrieve]
  40. Selden, R. F. (1987) in Current Protocols in Molecular Biology (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K., eds) pp. 4.9.1-4.9.8, John Wiley & Sons, Inc., New York
  41. Yang, W., McDonough, V. M., Ozier-Kalogeropoulos, O., Adeline, M., Flocco, M. T., and Carman, G. M. (1994) Biochemistry 33,10785-10793 [Medline] [Order article via Infotrieve]
  42. Sperka-Gottlieb, C., Fasch, E.-V., Kuchler, K., Bailis, A. M., Henry, S. A., Paltauf, F., and Kohlwein, S. D. (1990) Yeast 6,331-343 [Medline] [Order article via Infotrieve]
  43. Nikawa, J., and Yamashita, S. (1984) Eur. J. Biochem. 143,251-256 [Abstract]
  44. Church, G. M., and Gelbert, W. (1984) Proc. Natl. Acad. Sci. U. S. A. 81,1991-1995 [Abstract]
  45. Eng, F. J., and Warner, J. R. (1991) Cell 65,797-804 [Medline] [Order article via Infotrieve]
  46. Bae-Lee, M., and Carman, G. M. (1984) J. Biol. Chem. 259,10857-10862 [Abstract/Free Full Text]
  47. McMaster, C. R., and Bell, R. M. (1994) J. Biol. Chem. 269,14776-14783 [Abstract/Free Full Text]
  48. Haid, A., and Suissa, M. (1983) Methods Enzymol. 96,192-205 [Medline] [Order article via Infotrieve]
  49. Fischl, A. S., and Carman, G. M. (1983) J. Bacteriol. 154,304-311 [Medline] [Order article via Infotrieve]
  50. Bligh, E. G., and Dyer, W. J. (1959) Can J. Biochem. Physiol. 37,911-917
  51. Knoll, L. J., Johnson, D. R., and Gordon, J. I. (1994) J. Biol. Chem. 269,16348-16356 [Abstract/Free Full Text]
  52. Teegarden, D., Taparowsky, E. J., and Kent, C. (1990) J. Biol. Chem. 265,6042-6047 [Abstract/Free Full Text]
  53. Carman, G. M., and Kelley, M. J. (1992) Methods Enzymol. 209,242-247 [Medline] [Order article via Infotrieve]
  54. Carman, G. M., and Bae-Lee, M. (1992) Methods Enzymol. 209,298-305 [Medline] [Order article via Infotrieve]
  55. Dowhan, W., and Li, Q.-X. (1992) Methods Enzymol. 209,348-359 [Medline] [Order article via Infotrieve]
  56. Carman, G. M., and Fischl, A. S. (1992) Methods Enzymol. 209,305-312 [Medline] [Order article via Infotrieve]
  57. Hosaka, K., Kodaki, T., and Yamashita, S. (1989) J. Biol. Chem. 264,2053-2059 [Abstract/Free Full Text]
  58. Hosaka, H., and Yamashita, S. (1980) J. Bacteriol. 143,176-181 [Medline] [Order article via Infotrieve]
  59. Hjelmstad, R. H., and Bell, R. M. (1991) J. Biol. Chem. 266,4357-4365 [Abstract/Free Full Text]
  60. Carman, G. M., and Lin, Y. (1991) Methods Enzymol. 197,548-553 [Medline] [Order article via Infotrieve]
  61. Bradford, M. M. (1976) Anal. Biochem. 72,248-254 [CrossRef][Medline] [Order article via Infotrieve]
  62. Jund, R., and Lacroute, F. (1970) J. Bacteriol. 102,607-615 [Medline] [Order article via Infotrieve]
  63. Homann, M. J., Poole, M. A., Gaynor, P. M., Ho, C., and Carman, G. M. (1987) J. Bacteriol. 169,533-539 [Medline] [Order article via Infotrieve]
  64. Wu, W.-I., and Carman, G. M. (1994) J. Biol. Chem. 269,29495-29501 [Abstract/Free Full Text]
  65. Storer, A. C., and Cornish-Bowden, A. (1976) Biochem. J. 159,1-5 [Medline] [Order article via Infotrieve]
  66. Hjelmstad, R. H., and Bell, R. M. (1988) J. Biol. Chem. 263,19748-19757 [Abstract/Free Full Text]
  67. Christiansen, K. (1979) Biochim. Biophys. Acta 574,448-460 [Medline] [Order article via Infotrieve]
  68. Cleves, A. E., McGee, T. P., Whitters, E. A., Champion, K. M., Aitken, J. R., Dowhan, W., Goebl, M., and Bankaitis, V. A. (1991) Cell 64,789-800 [Medline] [Order article via Infotrieve]
  69. Nikawa, J., Yonemura, K., and Yamashita, S. (1983) Eur. J. Biochem. 131,223-229 [Medline] [Order article via Infotrieve]
  70. Kelley, M. J., and Carman, G. M. (1987) J. Biol. Chem. 262,14563-14570 [Abstract/Free Full Text]
  71. Vance, D. E. (1991) in Biochemistry of Lipids, Lipoproteins, and Membranes (Vance, D. E., and Vance, J., eds) pp. 205-240, Elsevier Science Publishers B. V., Amsterdam
  72. Vance, D. E. (1989) in Phosphatidylcholine Metabolism (Vance, D. E., ed) pp. 225-239, CRC Press, Inc., Boca Raton, FL
  73. Vance, D. E., Trip, E. M., and Paddon, H. B. (1980) J. Biol. Chem. 255,1064-1069 [Free Full Text]
  74. Lopez G-Coviella, I., and Wurtman, R. J. (1992) J. Neurochem. 59,338-343 [Medline] [Order article via Infotrieve]
  75. Talwalkar, R. T., and Lester, R. L. (1973) Biochim. Biophys. Acta 306,412-421 [Medline] [Order article via Infotrieve]
  76. Patton, J. L., and Lester, R. L. (1992) Arch. Biochem. Biophys. 292,70-76 [Medline] [Order article via Infotrieve]
  77. Buxeda, R. J., Nickels, J. T., Jr., and Carman, G. M. (1993) J. Biol. Chem. 268,6248-6255 [Abstract/Free Full Text]
  78. Nickels, J. T., Jr., Buxeda, R. J., and Carman, G. M. (1994) J. Biol. Chem. 269,11018-11024 [Abstract/Free Full Text]
  79. Kelley, M. J., Bailis, A. M., Henry, S. A., and Carman, G. M. (1988) J. Biol. Chem. 263,18,078-18,085

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