Effect of CTP Synthetase Regulation by CTP on Phospholipid Synthesis in Saccharomyces cerevisiae*

Darin B. OstranderDagger , Daniel J. O'BrienDagger , Jessica A. Gorman§, and George M. CarmanDagger

From the Dagger  Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick, New Jersey 08901 and the § Department of Microbial Molecular Biology, Pharmaceutical Research Institute, Bristol-Myers Squibb, Princeton, New Jersey 08543

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results & Discussion
References

CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) activity in Saccharomyces cerevisiae is allosterically regulated by CTP product inhibition. Amino acid residue Glu161 in the URA7-encoded and URA8-encoded CTP synthetases was identified as being involved in the regulation of these enzymes by CTP product inhibition. The specific activities of the URA7-encoded and URA8-encoded enzymes with a Glu161 right-arrow Lys (E161K) mutation were 2-fold greater when compared with the wild-type enzymes. The E161K mutant URA7-encoded and URA8-encoded CTP synthetases were less sensitive to CTP product inhibition with inhibitor constants for CTP of 8.4- and 5-fold greater, respectively, than those of their wild-type counterparts. Cells expressing the E161K mutant enzymes on a multicopy plasmid exhibited an increase in resistance to the pyrimidine poison and cancer therapeutic drug cyclopentenylcytosine and accumulated elevated (6-15-fold) levels of CTP when compared with cells expressing the wild-type enzymes. Cells expressing the E161K mutation in the URA7-encoded CTP synthetase exhibited an increase (1.5-fold) in the utilization of the Kennedy pathway for phosphatidylcholine synthesis when compared with control cells. Cells bearing the mutation also exhibited an increase in the synthesis of phosphatidylcholine (1.5-fold), phosphatidylethanolamine (1.3-fold), and phosphatidate (2-fold) and a decrease in the synthesis of phosphatidylserine (1.7-fold). These alterations were accompanied by an inositol excretion phenotype due to the misregulation of the INO1 gene. Moreover, cells bearing the E161K mutation exhibited an increase (1.6-fold) in the ratio of total neutral lipids to phospholipids, an increase in triacylglycerol (1.4-fold), free fatty acids (1.7-fold), and ergosterol ester (1.8-fold), and a decrease in diacylglycerol (1.3-fold) when compared with control cells. These data indicated that the regulation of CTP synthetase activity by CTP plays an important role in the regulation of phospholipid synthesis.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results & Discussion
References

CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) is a cytosolic-associated glutamine amidotransferase that catalyzes the ATP-dependent transfer of the amide nitrogen from glutamine to the C-4 position of UTP to form CTP (1, 2). This enzyme plays an essential role in the synthesis of all membrane phospholipids in eukaryotic cells (3, 4). Its reaction product CTP is the direct precursor of the activated, energy-rich phospholipid pathway intermediates CDP-DG1 (5), CDP-choline (6), and CDP-ethanolamine (6) (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 (3, 4). CDP-choline and CDP-ethanolamine are the sources of the hydrophilic head groups of PC and PE synthesized by the Kennedy pathways, respectively (3, 4). Our laboratory utilizes the yeast Saccharomyces cerevisiae as a model eukaryote to study the regulation of CTP synthetase and its impact on phospholipid metabolism.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 1.   Pathways for the biosynthesis of phospholipids in S. cerevisiae. The pathways shown for the biosynthesis of phospholipids include the relevant steps discussed in the text. The CDP-DG pathway is indicated by the boxed area. A more comprehensive phospholipid biosynthetic pathway which includes the intermediate steps in the pathway may be found in Ref. 53. The abbreviations used are: CDP-DG, CDP-diacylglycerol; CDP-Etn, CDP-ethanolamine; CDP-Cho, CDP-choline; PS, phosphatidylserine; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PG, phosphatidylglycerol; CL, cardiolipin; PI, phosphatidylinositol; SL, sphingolipids; PIPs, phosphoinositides; PA, phosphatidate; DG, diacylglycerol; TG, triacylglycerol.

CTP synthetase is encoded by the URA7 (7) and URA8 (8) genes in S. cerevisiae. Neither gene is essential provided that cells possess one functional gene encoding the enzyme (7, 8). Phenotypic analysis of ura7Delta and ura8Delta mutants (8) and the biochemical characterization of purified preparations of the URA7-encoded (9) and URA8-encoded (10) enzymes have shown that the two CTP synthetases are not functionally identical. Moreover, the URA7-encoded enzyme is more abundant than the URA8-encoded enzyme (11) and is responsible for the majority of the CTP synthesized in vivo (8). The overexpression of the URA7-encoded CTP synthetase causes a 2-fold increase in the utilization of the Kennedy pathway for PC synthesis (11). This has been attributed to an increase in the substrate availability of CTP for the phosphocholine cytidylyltransferase reaction in the Kennedy pathway and the inhibition of PS synthase activity by CTP in the CDP-DG pathway (11).

The URA7-encoded (9) and URA8-encoded (10) CTP synthetases are allosterically regulated by CTP product inhibition. CTP inhibits activity by increasing the positive cooperativity of these enzymes for UTP (9, 10). This regulation controls the cellular concentration of CTP in growing S. cerevisiae cells (7, 9, 11). In mammalian cells, inhibition of CTP synthetase by CTP plays an important role in the balance of the pyrimidine nucleoside triphosphate pools (12). A number of mammalian mutant cell lines possess CTP synthetase activity which is insensitive to inhibition by CTP. As a result, these cell lines display complex phenotypes which include increased intracellular pools of CTP and dCTP (13, 14), resistance to nucleotide analog drugs used in cancer chemotherapy (15-18), and an increased rate of spontaneous mutations (14-16). In addition, elevated CTP synthetase activity is a common property of leukemic cells (19) and rapidly growing tumors found in liver (20), colon (21), and lung (22). These findings underscore the importance of studies to understand the regulation of CTP synthetase activity by CTP product inhibition.

In this work we identified amino acid residue Glu161 in the URA7-encoded and URA8-encoded CTP synthetases to be involved in CTP product inhibition of the enzyme. Cells carrying a Glu161 right-arrow Lys (E161K) mutation in the CTP synthetases exhibited increased resistance to the pyrimidine poison and cancer therapeutic drug CPEC (23, 24) and accumulated high levels of CTP. The E161K mutation in the URA7-encoded CTP synthetase caused alterations in the synthesis of phospholipids by the CDP-DG and Kennedy pathways and in the proportional synthesis of phospholipids and neutral lipids.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results & Discussion
References

Materials

Growth medium supplies were purchased from Difco. Restriction endonucleases, modifying enzymes, and recombinant Vent DNA polymerase with 5'- and 3'-exonuclease activity and the DNA size ladder used for agarose gel electrophoresis were purchased from New England Biolabs. PCR and sequencing primers were prepared commercially by Genosys Biotechnologies, Inc. The Prism DyeDeoxy DNA sequencing kit was obtained from Applied Biosystems. Avian myeloblastosis virus reverse transcriptase was purchased from Life Technologies, Inc. Nucleotides, choline, phosphocholine, CDP-choline, 5-fluoroorotic acid, and bovine serum albumin were purchased from Sigma. The beta -galactosidase activity kit was from CLONTECH. Protein assay reagent, electrophoresis reagents, and immunochemical reagents were purchased from Bio-Rad. Lipids were purchased from Avanti Polar Lipids and Sigma. Radiochemicals and EN3HANCE were purchased from NEN Life Science Products. Scintillation counting supplies were from National Diagnostics. High performance thin layer chromatography and Silica Gel 60 thin layer chromatography plates were from EM Science. CPEC was a gift from Grant M. Hatch (University of Manitoba, Canada).

Methods

Strains, Plasmids, Oligonucleotides, and Growth Conditions-- The strains, plasmids, and oligonucleotides used in this work are listed in Tables I, II, and III respectively. Methods for growth and analysis of yeast were performed as described previously (25, 26). Yeast cultures were grown in YEPD medium (1% yeast extract, 2% peptone, 2% glucose) or in complete synthetic medium minus inositol (27) containing 2% glucose at 30 °C. The appropriate amino acid of complete synthetic medium was omitted for selection purposes, and inositol (50 µM) was added to the growth medium where indicated. Escherichia coli strain HB 101 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 HB 101 carrying plasmids. Media were supplemented with either 2% (yeast) or 1.5% (E. coli) agar for growth on plates. Yeast cell numbers in liquid media were determined spectrophotometrically at an absorbance of 540 nm. The sensitivity of yeast strains to CPEC (10 µg) was determined by a standard radial diffusion assay. The Opi (over production of inositol) phenotype (28) of yeast strains was examined on complete synthetic medium (minus inositol) by using growth of an inositol auxotrophic indicator strain MC13 (ino1) (27) as described by McGee et al. (29).

                              
View this table:
[in this window]
[in a new window]
 
Table I
Strains used in this work

                              
View this table:
[in this window]
[in a new window]
 
Table II
Plasmids used in this work

                              
View this table:
[in this window]
[in a new window]
 
Table III
Oligonucleotides used in this work

DNA Manipulations, Amplification of DNA by PCR, DNA Sequencing, and Quantitative Reverse Transcriptase-PCR-- Plasmid maintenance and amplifications were performed in E. coli strain HB 101. Plasmid and genomic DNA preparation, restriction enzyme digestion, and DNA ligations were performed by standard methods (26). Transformation of yeast (30, 31) and E. coli (26) was performed as described previously. Conditions for the amplification of DNA by PCR were optimized as described previously (32). The annealing temperature for PCRs was 52 °C, and extension time was 1 min at 72 °C. PCRs were routinely run for a total of 35 cycles. DNA sequencing reactions were performed with the Prism DyeDeoxy Terminator Cycle sequencing kit and analyzed with an automated DNA sequencer. Total RNA was extracted from cells (33), converted to cDNA by treatment with reverse transcriptase, and then amplified by PCR (34). The primers used for URA7 mRNA (Ura7-A and Ura7-B) produced a 1.7-kb product, and the primers used for URA8 mRNA (Ura8-A and Ura8-B) produced a 0.87-kb product.

Construction of Plasmids-- In order to construct a URA8 disruption vector, the 5' (primers Ura8-C and Ura8-D) and 3' (primers Ura8-E and Ura8-F) noncoding regions of the URA8 gene were isolated by PCR using DNA from strain W303-1A as the template. These fragments were digested with BglII and EcoRI and with BamHI and SalI, respectively, and ligated into plasmid pNKY51 to form plasmid pDO183. In order to construct a URA7 expression vector, a 2.49-kb fragment containing the URA7 gene was released from plasmid pFL44S-URA7 by digestion with BamHI and PstI. This fragment was ligated into plasmid YEpLac195 that was digested with the same restriction enzymes to form plasmid pDO134. In order to construct a URA7 disruption vector, a 1.4-kb DNA fragment containing the URA7Delta ::TRP1 allele (7) was obtained from strain OK8 (8) by PCR using primers Ura7-C and Ura7-D. This fragment was blunt-end ligated into pBlueScript II digested with EcoRV to form plasmid pDO163. In order to clone the URA7 open reading frame for mutagenesis experiments, a 1.81-kb DNA fragment containing the URA7 open reading frame was obtained by PCR (primers Ura7-E and Ura7-F) using DNA from strain W303-1A as the template. This fragment was digested with NotI and PstI and ligated into pBlueScript II that was digested with the same restriction enzymes to form plasmid pDO178. A 1.76-kb DNA fragment containing the URA8 open reading frame was obtained by PCR (primers Ura8-G and Ura8-H) using DNA from strain W303-1A as the template. This fragment was blunt-end ligated into pBlueScript II digested with EcoRV to form plasmid pDO179. The primary sequences of the cloned open reading frames of URA7 and URA8 in plasmids pDO178 and pDO179 were verified by DNA sequencing.

The URA7E161K (primer Ura7-G and complement) and URA7H233K (primer Ura7-H and complement) and URA8E161K (primer Ura8-I and complement) and URA8H233K (primer Ura8-J and complement) mutations were constructed by PCR using plasmids pDO178 and pDO179, respectively, as templates. The primers for the E161K mutations incorporated an ApaLI restriction site, and the primers for the H233K mutations incorporated an ApaI restriction site. These silent restriction sites were used to identify plasmids with the correct mutations. The URA7E161K,H233K and URA8E161K,H233K mutants were constructed with the appropriate primers for the H233K mutations using the URA7E161K and URA8E161K mutants, respectively, as templates. The mutated genes were completely sequenced to verify that no additional unwanted mutations were made.

The wild-type and mutant alleles of URA7 and URA8 were subcloned into an expression shuttle vector containing the ADH1 promoter. The ADH1 promoter was isolated from plasmid pDB20 by digestion with BamHI and PstI. This fragment was ligated into plasmid YEpLac181 digested with the same restriction enzymes to form plasmid pDO104. A pair of annealed oligonucleotides (Mcs-A and Mcs-B), which contains additional restriction sites, was ligated into plasmid pDO104 that was digested with NotI and PstI to form plasmid pDO105. The wild-type and mutant alleles of URA7 were released from plasmid pDO178 by digestion with NotI and PstI, and the wild-type and mutant alleles of URA8 were released from plasmid pDO179 by digestion with NotI and XbaI. These fragments were then ligated into plasmid pDO105, digested with NotI and PstI and NotI and XbaI, respectively, to form the expression shuttle vectors pDO169-pDO176.

Construction of the ura7Delta ura8Delta Double Mutant-- A ura7Delta ura8Delta double mutant was constructed and used as the host strain for the expression of the wild-type and mutant alleles of the URA7-encoded and URA8-encoded CTP synthetases. A ura8Delta mutant was constructed first. A 4.6-kb SspI fragment of plasmid pDO183, which contained the URA8Delta ::HisG/URA3/HisG cassette, was used to transform strain SGY157 to uracil prototrophy. HisG is a 1100-base pair DNA sequence from Salmonella typhimurium which is utilized in direct repeats flanking URA3 to increase the frequency of homologous recombination resulting in the loss of URA3. Colonies were subsequently plated onto media containing 5-fluoroorotic acid, and uracil auxotrophs were recovered. This step selected for the ura8Delta ::HisG recombination. One of the ura8Delta mutants was designated strain SDO159. Strain SDO159 was transformed with plasmid pDO134, which contains a wild-type URA7 allele. This plasmid was maintained by growth of the strain on plates without uracil. This step was necessary because a ura7Delta ura8Delta mutant is not viable (8). Strain SDO159 bearing plasmid pDO134 was then transformed to tryptophan prototrophy with a 1.34-kb fragment of plasmid pDO163 that was digested with DraI and NaeI. One of the ura7Delta ura8Delta double mutants was designated strain SDO195. Both mutations were confirmed by Southern blot analysis.

Strain SDO195, bearing plasmid pDO134, was transformed to leucine prototrophy with the ADH1 expression vectors containing the wild-type and mutant alleles of URA7 and URA8. Plasmid pDO134 was subsequently selected against with 5-fluoroorotic acid by plasmid shuffle (35). Cells were then examined to verify that they regained uracil auxotrophy. The presence of the ADH1 expression vectors containing the wild-type and mutant alleles of URA7 and URA8 was verified by re-isolation in E. coli and restriction analysis.

Preparation of Enzymes-- The URA7-encoded (9) and URA8-encoded (10) CTP synthetases were partially purified through the ammonium sulfate fractionation step as described previously. Cell extracts for beta -galactosidase assays were prepared by cell disruption with glass beads (9) using the Z buffer described by Guarente (36).

Enzyme Assays and Protein Determination-- CTP synthetase activity was determined by measuring the conversion of UTP to CTP (molar extinction coefficients of 182 and 1520 M-1 cm-1, respectively) by following the increase in absorbance at 291 nm on a recording spectrophotometer (2). The standard reaction mixture contained 50 mM Tris-HCl, pH 8.0, 2 mM UTP, 2 mM ATP, 2 mM L-glutamine, 0.1 mM GTP, 10 mM MgCl2, 10 mM 2-mercaptoethanol, and an appropriate dilution of enzyme protein in a total volume of 0.2 ml. Enzyme assays were performed in triplicate with an average standard deviation of ± 3%. All assays were linear with time and protein concentration. A unit of CTP synthetase activity was defined as the amount of enzyme that catalyzed the formation of 1 nmol of product/min. beta -Galactosidase activity was measured by a fluorescent assay using methylumbelliferyl galactoside as substrate according to the instructions of the manufacturer. A unit of beta -galactosidase activity was defined as the amount of enzyme that catalyzed the hydrolysis of 1 pmol of substrate/min. Protein was determined by the method of Bradford (37) using bovine serum albumin as the standard.

Immunoblotting of CTP Synthetase-- Immunoblot assays were performed with IgG anti-URA7-encoded (9) and IgG anti-URA8-encoded (10) CTP synthetase antibodies as described previously (38). The density of the URA7-encoded and URA8-encoded CTP synthetase bands on immunoblots was quantified by scanning densitometry. Immunoblot signals were in the linear range of detectability.

Extraction and Mass Analysis of Nucleotides-- Cells bearing the wild-type and mutant URA7-encoded and URA8-encoded CTP synthetases were grown to the exponential phase of growth. Cellular nucleotides were extracted (7) and were analyzed by high performance liquid chromatography (8).

Labeling and Analysis of Phospholipids and Neutral Lipids-- Labeling of phospholipids and neutral lipids with 32Pi, [methyl-3H]choline, and [2-14C]acetate were performed as described previously (11, 39-42). Lipids were extracted from labeled cells by the method of Bligh and Dyer (43) as described previously (42). Phospholipids were analyzed by two-dimensional thin layer chromatography on high performance silica gel thin layer chromatography plates using chloroform/methanol/glacial acetic acid (65:25:10, v/v) as the solvent for dimension one and chloroform/methanol/88% formic acid (65:25:10, v/v) as the solvent for dimension two (44). Neutral lipids were analyzed by one-dimensional thin layer chromatography on high performance silica gel thin layer plates using the solvent system hexane/diethyl ether/glacial acetic acid (80:20:2) (45). The 32P-labeled phospholipids were visualized by autoradiography, and the 14C-labeled neutral lipids were visualized by fluorography using EN3HANCE. The position of the labeled lipids on chromatography plates were compared with standard lipids after exposure to iodine vapor. The amount of each labeled lipid was determined by liquid scintillation counting of the corresponding spots on the chromatograms.

Labeling and Analysis of Kennedy Pathway Intermediates-- Labeling of the Kennedy (CDP-choline) pathway intermediates with [methyl-3H]choline was performed as described previously (11). Choline, phosphocholine, and CDP-choline were obtained from whole cells after lipid extraction (43). 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 Kennedy 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. (46). The positions of the labeled intermediates on chromatograms were determined by fluorography using EN3HANCE and compared with standards. The amount of each labeled compound was determined by liquid scintillation counting.

    RESULTS AND DISCUSSION
Top
Abstract
Introduction
Procedures
Results & Discussion
References

Construction and Characterization of the URA7-encoded and URA8-encoded CTP Synthetase E161K and H233K Mutants-- CTP synthetase mutants defective in CTP product inhibition have been isolated from Chinese hamster ovary cells (47). Sequence analysis of the CTP synthetase gene from these mutants revealed that most of the mutations were clustered within a stretch of 14 amino acids (47). The most frequent mutations in the clustered region of the enzyme were glutamate to lysine and histidine to lysine (47). The clustered sites are in a highly conserved region of the CTP synthetases from human cells (48), E. coli (49), Chlamydia trachomatis (50), Bacillus subtilis (51), and S. cerevisiae (7, 8). Based on this information, we hypothesized that the two amino acids that are most frequently mutated in Chinese hamster ovary cells (47) would be involved in the regulation of the S. cerevisiae CTP synthetases by CTP. These amino acids correspond to Glu161 and His233 in the URA7-encoded and URA8-encoded CTP synthetases.

The codons for Glu161 and His233 in the URA7-encoded and URA8-encoded CTP synthetases were changed to lysine codons by site-directed mutagenesis. The mutations were made individually and in combination for each of the CTP synthetase enzymes. The open reading frames of each of the mutated genes were subcloned behind the constitutive ADH1 promoter (52) on a multicopy plasmid. We used the ADH1 promoter for enzyme expression to obviate regulation mediated by the native URA7 and URA8 promoters. The mutant enzymes were separately expressed on a multicopy plasmid in a ura7Delta ura8Delta double mutant to examine the effects of the URA7-encoded and URA8-encoded CTP synthetase mutations on phospholipid synthesis. A multicopy plasmid was chosen to accentuate the effects of the mutations. The effects of the mutations in cells were compared with cells expressing the wild-type enzymes on a multicopy plasmid.

Cells bearing multicopy plasmids containing the wild-type and mutant alleles of the URA7 and URA8 genes exhibited growth rates similar to parent wild-type (SGY157) cells when grown vegetatively at 30 °C in liquid YEPD and complete synthetic media. However, cells bearing the E161K mutation in the URA7-encoded CTP synthetase entered the stationary phase of growth at a lower cell density (1-2 × 108 cells/ml) than that (2-5 × 108 cells/ml) of cells expressing the wild-type enzyme. No morphological differences were observed in the cells bearing the mutations in the URA7-encoded and URA8-encoded CTP synthetases. Quantitative reverse transcriptase-PCR analysis showed that there were no differences in the mRNA levels between strains containing the wild-type and mutant alleles of the URA7 and URA8 genes on the multicopy plasmid. Immunoblot analysis of cell extracts prepared from cells bearing multicopy plasmids with the wild-type and mutant alleles showed that there were no differences in the CTP synthetase protein levels expressed. As expected, the CTP synthetase mRNA (about 30-fold) and protein (6-7-fold) levels in all of the strains were elevated when compared with the mRNA and protein levels found in the parent wild-type strain SGY157. Therefore, the mutations in the URA7 and URA8 genes did not affect the functional expression of these genes.

Effect of CPEC on the Growth of Cells Bearing the E161K and H233K Mutations in the URA7-encoded and URA8-encoded CTP Synthetases-- The effect of CPEC on the growth of cells bearing the E161K and H233K mutations in the URA7-encoded and URA8-encoded CTP synthetases was examined by a radial diffusion assay. CPEC is a carbocyclic analogue of cytidine where the ribofuranose moiety is substituted by a cyclopentenyl ring (23). In mammalian cells, CPEC is rapidly phosphorylated to CPEC triphosphate, a specific and potent inhibitor of CTP synthetase activity (23). This compound would be expected to inhibit CTP synthetase activity, reduce the cellular levels of CTP, and thus inhibit growth (23). Indeed, CPEC inhibited S. cerevisiae cells bearing the multicopy plasmids with the wild-type alleles of the URA7 and URA8 genes (Fig. 2). This growth inhibition was the same as that observed with parent wild-type (SGY157) cells that did not contain a plasmid. On the other hand, cells bearing plasmids with the E161K mutation showed a dramatic decrease in sensitivity to CPEC (Fig. 2). Cells bearing the H233K mutation exhibited a less dramatic decrease in CPEC sensitivity when compared with cells containing the E161K mutation (Fig. 2). The CPEC resistance of cells bearing the E161K,H233K double mutation was similar to cells bearing the E161K mutation alone.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 2.   Effect of CPEC on the growth of cells bearing the E161K and H233K mutations in the URA7-encoded and URA8-encoded CTP synthetases. Cells expressing the indicated URA7-encoded (A) and URA8-encoded (B) wild-type and mutant CTP synthetases were seeded onto separate agar plates containing complete synthetic medium. 10 µg of CPEC was placed into a 3-mm hole in the center of each agar plate. The plates were incubated for 24 h, and the diameter of the zone of growth inhibition around the hole was measured. The values reported were the average of four separate experiments (S.D. ± 1 mm). WT, wild type.

Effect of the E161K and H233K Mutations in the URA7-encoded and URA8-encoded CTP Synthetases on the Inhibition of Activity by CTP-- We directly examined the hypothesis that the E161K and H233K mutations in the CTP synthetase enzymes affected the regulation of activity by CTP product inhibition. CTP synthetase was partially purified from cells bearing the wild-type and mutant alleles of the URA7 and URA8 genes and assayed for activity in the absence and presence of CTP. When measured in the absence of CTP, the specific activities of the E161K mutant enzymes were about 2-fold higher than the wild-type enzymes (Fig. 3, A and B). As described previously (9, 10), the wild-type URA7- and URA8-encoded CTP synthetase activities were inhibited by CTP in a dose-dependent manner (Fig. 3, C and D). The CTP synthetase activities of the E161K mutant enzymes were less sensitive to CTP inhibition (Fig. 3, C and D). The IC50 values for CTP of the URA7-encoded and URA8-encoded E161K mutant enzymes were 8.4- and 5-fold greater, respectively, than those of their wild-type counterparts (Table IV). Moreover, the apparent inhibitor constants for CTP of the mutant enzymes were within the physiological range of CTP (Table IV). The URA7-encoded E161K mutant enzyme was more resistant to CTP product inhibition when compared with the URA8-encoded E161K mutant enzyme (Fig. 3, C and D). The specific activities of the H233K mutant URA7-encoded and URA8-encoded enzymes and their sensitivities to CTP inhibition were similar to that of the wild-type enzymes (Fig. 3). The specific activities and patterns of CTP inhibition of the E161K,H233K double mutant enzymes were similar to that of the single E161K mutant (Fig. 3).


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 3.   Effect of the E161K and H233K mutations in the URA7-encoded and URA8-encoded CTP synthetases on the inhibition of activity by CTP. Cells expressing the indicated URA7-encoded (A and C) and URA8-encoded (B and D) wild-type and mutant CTP synthetases were grown in complete synthetic medium to the exponential phase of growth. The CTP synthetase enzymes were partially purified and assayed for activity in the absence (A and B) and presence (C and D) of the indicated concentrations of CTP as described under "Experimental Procedures." WT, wild type.

                              
View this table:
[in this window]
[in a new window]
 
Table IV
Inhibitor constants for CTP of mutant CTP synthetases and the cellular concentration of CTP in cells bearing the mutant CTP synthetases

Effect of the E161K and H233K Mutations in the URA7-encoded and URA8-encoded CTP Synthetases on the Cellular Concentrations of Nucleotides-- The effect of the E161K and H233K mutations in the URA7-encoded and URA8-encoded CTP synthetases on the cellular concentration of nucleotide triphosphates was examined. Cells were grown to the exponential phase of growth, and nucleotides were extracted and then analyzed by high performance liquid chromatography. The CTP levels contributed by the wild-type URA7-encoded CTP synthetase were greater than those contributed by the URA8-encoded enzyme (Fig. 4 and Table IV). Cells expressing the E161K mutation in the URA7-encoded (6-fold) and URA8-encoded (15-fold) CTP synthetases had elevated cellular concentrations of CTP when compared with cells that overexpressed their wild-type counterpart enzymes (Fig. 4 and Table IV). The CTP concentration in cells bearing the E161K mutation in the URA7-encoded enzyme was 36-fold greater than the CTP concentration in the parent wild-type strain SGY157 (Table IV). Although the H233K mutation in the URA7-encoded CTP synthetase did not have a significant effect on the cellular CTP concentration, the H233K mutation in the URA8-encoded enzyme resulted in an increase (5-fold) in CTP concentration (Fig. 4 and Table IV). The CTP concentration in cells bearing the E161K,H233K double mutation in URA7-encoded enzyme was not significantly different from cells bearing the E161K mutation alone (Fig. 4A). On the other hand, the CTP concentration in cells bearing the double mutation in the URA8-encoded enzyme was greater (1.3-fold) than that of cells bearing the E161K mutation alone (Fig. 4B and Table IV). The E161K and H233K mutations in both the URA7-encoded and URA8-encoded enzymes did not have a significant effect on the cellular concentrations of UTP, ATP, and GTP (Fig. 4).


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of the E161K and H233K mutations in the URA7-encoded and URA8-encoded CTP synthetases on the cellular concentrations of nucleotides. Cells expressing the indicated URA7-encoded (A) and URA8-encoded (B) wild-type and mutant CTP synthetases were grown in complete synthetic medium to the exponential phase of growth. Nucleotides were extracted and analyzed by high performance liquid chromatography as described under "Experimental Procedures." The values reported were the average of four separate experiments ± S.D. WT, wild type.

The differences in the effects of the E161K and H233K mutations in the URA7-encoded and URA8-encoded CTP synthetase on activity and cellular CTP levels further support the hypothesis (8, 10) that these enzymes are regulated differentially in vivo. Owing to the fact that the E161K mutation in the URA7-encoded CTP synthetase had the major effect on CTP synthetase regulation by CTP, the other mutants of the URA7-encoded and URA8-encoded CTP synthetases were not examined further in this study.

Effect of the E161K Mutation in the URA7-encoded CTP Synthetase on the Synthesis and Composition of Phospholipids-- The effect of the E161K mutation in the URA7-encoded CTP synthetase on the synthesis and steady-state composition of phospholipids was examined. Cells were grown in complete synthetic medium minus inositol and choline to obviate the regulatory effects these compounds have on phospholipid metabolism (4, 53-55). In the absence of exogenous choline, wild-type S. cerevisiae cells synthesize phospholipids by both the CDP-DG and Kennedy pathways (11, 56, 57). We examined the synthesis and composition of phospholipids by labeling cells with both 32Pi and [methyl-3H]choline. 32Pi will be incorporated into phospholipids synthesized by both the CDP-DG and Kennedy pathways, whereas the labeled choline will only be incorporated into PC synthesized via the Kennedy pathway. The concentration of choline added to the growth medium from the radioactive label was 0.1 µM, a concentration too low to affect the rate of synthesis of PC by the Kennedy pathway (58). Phospholipid synthesis was followed by pulse labeling. The amount of label incorporated into each phospholipid represented the relative rates of synthesis during the pulse. Cells overexpressing the E161K mutation in the URA7-encoded CTP synthetase showed an increase in the synthesis of PC (1.5-fold), PE (1.3-fold), and PA (2-fold), and a decrease in the synthesis of PS (1.7-fold) when compared with cells overexpressing the wild-type enzyme (Fig. 5A). The effect of the E161K mutation on the steady-state phospholipid composition is shown in Fig. 5C. The major effect of the E161K mutation on composition was an increase in PC (1.3-fold) and a decrease in PS (1.3-fold).


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of the E161K mutation in the URA7-encoded CTP synthetase on the synthesis and steady-state composition of phospholipids. Cells expressing either the wild-type URA7-encoded or the mutant URA7E161K-encoded CTP synthetases were grown to the exponential (1 × 107 cells/ml) phase of growth. For pulse labeling of phospholipids (A) and PC (B), cells were incubated with 32Pi (5 µCi/ml) and [methyl-3H]choline (0.5 µCi/ml) for 30 min. The incorporation of 32Pi and [methyl-3H]choline into total phospholipids and PC were approximately 1,000 cpm/107 cells and 5,000 cpm/107 cells, respectively. The steady-state composition of phospholipids (C) and PC (D) were determined by labeling cells for five to six generations with 32Pi (5 µCi/ml) and [methyl-3H]choline (0.5 µCi/ml). The incorporation of 32Pi and [methyl-3H]choline into total phospholipids and PC were about 10,000 cpm/107 cells and 2,000 cpm/107 cells, respectively. Phospholipids were extracted and analyzed as described under "Experimental Procedures." The values reported in A and C were determined from 32Pi labeling. The values in B and D were reported as the cpm of 3H incorporated into PC relative to the cpm of 32P incorporated into PC. The percentages shown for phospholipids were normalized to the total 32Pi-labeled chloroform-soluble fraction which included sphingolipids and other unidentified phospholipids. The values reported were the average of four separate experiments ± S.D. The abbreviations used are: PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; PA, phosphatidate; WT, wild type.

Radiolabeled choline was incorporated into PC during the pulse labeling and steady-state labeling experiments indicating that PC was synthesized via the Kennedy pathway. The data shown in Fig. 5, B and D, are plotted as the ratio of the cpm of 3H incorporated into PC to the cpm of 32P incorporated into PC. This allowed us to determine if the E161K mutation in the CTP synthetase affected the pathways by which cells synthesized PC (11). The E161K mutation in the URA7-encoded CTP synthetase caused an increase in this ratio in both the pulse labeling (1.7-fold) and steady-state labeling (1.5-fold) of PC when compared with cells overexpressing the wild-type enzyme. These results indicated that cells expressing the E161K mutation had an increase in the utilization of the Kennedy pathway for PC synthesis when compared with cells expressing the wild-type enzyme. This increased utilization of the Kennedy pathway is an underestimate since the 32Pi label incorporated into PC occurred by both the Kennedy and CDP-DG pathways. The decrease in the synthesis and steady-state content of PS was consistent with a decrease in the utilization of the CDP-DG pathway.

Cells were pulse-labeled and labeled to steady state with [methyl-3H]choline to analyze the Kennedy pathway intermediates choline, phosphocholine, and CDP-choline. The E161K mutation in the URA7-encoded CTP synthetase caused a small decrease in the synthesis of phosphocholine (1.2-fold) and an increase in the synthesis of CDP-choline (1.5-fold) when compared with the control cells (Fig. 6A). The E161K mutation had a more dramatic effect on the steady-state composition of the Kennedy pathway intermediates (Fig. 6B). The mutation caused a 1.2-fold decrease in the amount of phosphocholine and a 2.3-fold increase in the amount of CDP-choline when compared with the control cells. The increase in the synthesis and steady-state amounts of CDP-choline, the rate-limiting Kennedy pathway intermediate (58, 59), was consistent with the increased utilization of the Kennedy pathway for PC synthesis in the E161K mutant. An increase in the utilization of the Kennedy pathway is caused by the overexpression of the wild-type URA7-encoded CTP synthetase (11). However, the effects of the E161K mutation in CTP synthetase on Kennedy pathway utilization were over and above those effects brought about by the overexpression of the wild-type enzyme. The increase in utilization of the Kennedy pathway and in the amount of CDP-choline in cells bearing the E161K mutant CTP synthetase were 3.2- and 3-fold greater, respectively, when compared with cells expressing the wild-type URA7-encoded CTP synthetase from a single copy plasmid (11). Moreover, the overexpression of the wild-type CTP synthetase does not cause changes in phospholipid composition (11).


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 6.   Effect of the E161K mutation in the URA7-encoded CTP synthetase on the synthesis and steady-state composition of Kennedy pathway intermediates. Cells expressing either the wild-type URA7-encoded or the mutant URA7E161K-encoded CTP synthetases were grown to the exponential (1 × 107 cells/ml) phase of growth. For pulse labeling of the Kennedy pathway intermediates (A), cells were incubated with [methyl-3H]choline (5 µCi/ml) for 30 min. The incorporation of [methyl-3H]choline into the Kennedy pathway intermediates was about 20,000 cpm/107 cells. The steady-state composition of the Kennedy pathway intermediates (B) was determined by labeling cells for five to six generations with [methyl-3H]choline (5 µCi/ml). The incorporation of [methyl-3H]choline into the Kennedy pathway intermediates was about 50,000 cpm/107 cells. The Kennedy pathway intermediates were extracted and analyzed as described under "Experimental Procedures." The values reported were the average of three separate experiments ± S.D. WT, wild type.

Effect of the E161K Mutation in the URA7-encoded CTP Synthetase on the Regulation of INO1 Expression-- An Opi (inositol excretion) phenotype (28) is characteristic of defects in the regulation of phospholipid metabolism in S. cerevisiae (4, 53-55). The Opi phenotype was examined for cells bearing the E161K mutation in the URA7-encoded CTP synthetase using growth of an ino1 mutant as an indicator of the phenotype. Cells expressing the E161K mutant enzyme exhibited an Opi phenotype, whereas cells expressing the wild-type enzyme did not (Fig. 7A). Parent wild-type (SGY157) cells did not exhibit an Opi phenotype. The Opi phenotype of the E161K mutant was not as great as that exhibited by the inositol excreting opi1 mutant (28), which was used as a positive control (Fig. 7A).


View larger version (35K):
[in this window]
[in a new window]
 
Fig. 7.   Effect of the E161K mutation in the URA7-encoded CTP synthetase on the expression of the INO1 gene. A, the inositol-requiring ino1 mutant was streaked beside a patch of cells expressing either the wild-type or the E161K mutant CTP synthetase or a patch of opi1 mutant cells grown on agar plates containing complete synthetic medium minus inositol. The plates were incubated for 72 h at 30 °C. B, cells expressing either the wild-type URA7-encoded or mutant URA7E161K-encoded CTP synthetases were transformed with plasmid pJH359. This plasmid carries a fully regulated INO1-CYC1-lacI'Z construct (81). Cells were grown in complete synthetic medium in the absence (-I) and presence (+I) of 50 µM inositol. Cells were harvested at the exponential phase of growth; cell extracts were prepared, and beta -galactosidase activity was measured as described under "Experimental Procedures." The values reported were determined from duplicate assays (S.D. ± 3%) from a minimum of two independent growth studies. The abbreviations used are: WT, wild type; OP1, opi1 mutant cells.

The Opi phenotype is the result of the derepression of the INO1 gene encoding inositol-1-phosphate synthase (53, 55). The expression of the INO1 mRNA in cells bearing the E161K mutation in the URA7-encoded CTP synthetase was examined by using the INO1-CYC1-lacI'Z reporter construct. Cells bearing the reporter construct were grown in complete synthetic medium minus inositol; cell extracts were prepared, and beta -galactosidase activity was measured. The beta -galactosidase activity in cells expressing the E161K mutant was 2.4-fold greater than the activity in cells expressing the wild-type enzyme (Fig. 7B). The level of beta -galactosidase activity in parent wild-type (SGY157) cells was the same as that of cells overexpressing the wild-type enzyme. Thus the Opi phenotype of cells bearing the E161K mutation was due to the derepression of the INO1 gene. The addition of inositol to wild-type cells results in a repression of INO1 mRNA (60). We examined the effect of inositol supplementation on INO1 expression in cells bearing the E161K mutation in URA7-encoded CTP synthetase. The addition of inositol to the growth medium resulted in a 3-fold decrease in beta -galactosidase activity in cells bearing the E161K mutation. However, the beta -galactosidase activity in the repressed E161K mutant was not as low as the activity in the control cells supplemented with inositol (Fig. 7B). Cells bearing the E161K mutation were also analyzed for INO1 mRNA by reverse transcriptase-PCR. This analysis confirmed the results described above using the INO1-CYC1-lacI'Z reporter construct. Overall, these results showed that the E161K mutation in CTP synthetase affected the normal regulation of the INO1 gene.


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 8.   Effect of the E161K mutation in the URA7-encoded CTP synthetase on the synthesis and composition of lipids. Cells expressing either the wild-type URA7-encoded or the mutant URA7E161K-encoded CTP synthetases were grown to the exponential (1 × 107 cells/ml) phase of growth. For pulse labeling of total lipids (A and B), cells were incubated with [2-14C]acetate (1 µCi/ml) for 30 min. The incorporation of [2-14C]acetate into total lipids during the pulse was approximately 25,000 cpm/107 cells. The steady-state composition of total lipids (C and D) was determined by labeling cells for five to six generations with [2-14C]acetate (1 µCi/ml). The incorporation of [2-14C]acetate into total lipids during steady-state labeling was approximately 30,000 cpm/107 cells. Total lipids were extracted and analyzed as described under "Experimental Procedures." The percentages shown for total phospholipids and neutral lipids were normalized to the total 14C-labeled chloroform-soluble fraction which included sphingolipids and other unidentified lipids. The values reported were the average of three separate experiments ± S.D. The abbreviations used are: PL, total phospholipids; NL, total neutral lipids; TG, triacylglycerol; DG, diacylglycerol; FA, fatty acid; Erg, ergosterol; ErgE, ergosterol ester; WT, wild type.

Effect of the E161K Mutation in the URA7-encoded CTP Synthetase on Neutral Lipid Synthesis and Composition-- We examined the effect of the E161K mutation in the URA7-encoded CTP synthetase on the synthesis of total phospholipids and neutral lipids by pulse labeling with [2-14C]acetate. The E161K mutation did not have a significant effect on the relative synthesis of total neutral lipids and total phospholipids when compared with cells expressing the wild-type enzyme (Fig. 8A). The mutation also had little effect on the synthesis of the major neutral lipid compounds (Fig. 8B). On the other hand, the E161K mutation had a dramatic effect on the steady-state composition of lipids. The amount of total neutral lipids increased 1.3-fold, whereas the total amount of phospholipids decreased 1.3-fold in the E161K mutant when compared with control cells (Fig. 8C). The ratio of total neutral lipids to phospholipids (1.45) in cells bearing the E161K mutation was 1.6-fold greater than the ratio of total neutral lipids to phospholipids (0.93) in cells expressing the wild-type enzyme. Moreover, the E161K mutation caused increases in triacylglycerol (1.4-fold), free fatty acids (1.7-fold), and ergosterol ester (1.8-fold) and caused a decrease in diacylglycerol (1.3-fold) when compared with control cells (Fig. 8D).

Concluding Discussion-- To gain insight into the regulation of S. cerevisiae CTP synthetase activity by CTP product inhibition and its impact on phospholipid synthesis, we constructed mutant forms of the enzyme that were defective in CTP product inhibition. The E161K mutation had the major effect on the regulation of the URA7-encoded and URA8-encoded CTP synthetases by CTP. Cells bearing the E161K mutation were resistant to CPEC and accumulated elevated levels of CTP. The major consequence of the E161K mutation in the URA7-encoded CTP synthetase on phospholipid synthesis was an increase in the utilization of the Kennedy pathway. The mechanism for this regulation may be attributed to an increase in the substrate availability of CTP for the phosphocholine cytidylyltransferase reaction (11). The CDP-DG pathway is primarily used by wild-type S. cerevisiae when they are grown in the absence of choline (53, 54, 61, 62). However, the Kennedy pathway contributes to PC synthesis even when wild-type cells are grown in the absence of choline (11, 56, 57, 63). The choline required is derived from the phospholipase D-mediated turnover of PC synthesized by the CDP-DG pathway (63, 64). The Kennedy pathway becomes important for PC synthesis when the enzymes in the CDP-DG pathway are defective. Mutants defective in the synthesis of PS (39, 40), PE (65, 66), or PC (67-70) require choline for growth in order to synthesize PC via the Kennedy pathway.

Although the synthesis of PC by the Kennedy pathway is essential when the CDP-DG pathway is defective, there are circumstances when the Kennedy pathway is detrimental to S. cerevisiae. Bankaitis and co-workers (56, 71) have shown that the synthesis of PC via the Kennedy pathway is lethal in the absence of a functional PI/PC transfer protein (Sec14p). Sec14p activity is essential for viability and vesicle budding from the Golgi complex (72, 73). Yet this essential function can be obviated by mutations in the Kennedy pathway (71). Data suggest that Sec14p may function to down-regulate the Kennedy pathway by inhibiting phosphocholine cytidylyltransferase activity (74) and/or by preventing consumption of the DG used for PC synthesis via the Kennedy pathway (75).

The activation of the Kennedy pathway in response to the E161K mutation in CTP synthetase was not lethal but was accompanied by alterations in the synthesis of the major membrane phospholipids. These changes included significant increases in the synthesis of PC and PA and a decrease in the synthesis of PS. The decrease in PS synthesis is consistent with the conclusion that the E161K mutation caused a decrease in PC synthesis via the CDP-DG pathway. The mechanism for this regulation may be attributed to a direct inhibition of PS synthase activity by CTP (11). The increase in PA synthesis may be the result of a decrease in the utilization of the CDP-DG pathway. Defects in the synthesis of PC via the CDP-DG pathway are accompanied by the misregulation of the INO1 gene and an Opi phenotype (53-55, 76-78). Indeed, cells bearing the E161K mutation in CTP synthetase exhibited an Opi phenotype due to the misregulation of the INO1 gene. These observations further support the hypothesis that the misregulation of the pathways for PC synthesis generates a signal for the misregulation of the INO1 gene (63, 64, 78). Recent data suggest that this signaling molecule is PA (64). As indicated above, PA synthesis was elevated in cells bearing the E161K mutation in CTP synthetase. The synthesis of PC is coordinately regulated with the synthesis of PI (53, 54, 78). The derepression of the INO1 gene plays a role in this regulation to provide inositol for PI synthesis (53, 54, 78). PI and its derivative molecules (polyphosphoinositides and sphingolipids) are essential to the growth and viability of S. cerevisiae (53, 54, 78).

The E161K mutation in CTP synthetase also caused an increase in total neutral lipid content at the expense of total phospholipids as well as increases in triacylglycerols, free fatty acids, and ergosterol esters. These alterations, which occurred in exponential phase cells, were reminiscent of changes that occur in wild-type cells when they enter the stationary phase of growth (41, 79, 80). The effects of the E161K mutation on cellular CTP levels and on phospholipid synthesis may have created a stress-like condition that accounted for cells bearing the mutation to enter the stationary phase of growth at a lower cell density when compared with cells expressing the wild-type enzyme.

In this study we focused on the effect of CTP synthetase regulation by CTP on phospholipid synthesis. CTP synthetase activity is also essential for the synthesis of RNA, DNA, and sialoglycoproteins. Therefore alterations in the metabolism of these macromolecules in response to the E161K mutation in CTP synthetase could also have an influence on the regulation of phospholipid synthesis. Additional studies will be required to address this question. Clearly, the regulation of CTP synthetase activity by CTP would be expected to play a major role in cell growth and physiology.

    ACKNOWLEDGEMENTS

We thank Dr. Grant M. Hatch for a gift of CPEC, Subbarao Mantha for assistance with the high performance liquid chromatography of nucleotides, and Susan A. Henry for plasmid pJH359.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This work was supported in part by U. S. Public Health Service, National Institutes of Health Grant GM-50679 (to G. M. C.). This is New Jersey Agricultural Experiment Station Publication D-10581-2-98.

To whom correspondence and reprint requests should be addressed: Dept. of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers University, 65 Dudley Rd., New Brunswick, NJ 08901. Tel.: 732-932-9611 (ext. 217); Fax: 732-932-6776; E-mail: carman{at}aesop.rutgers.edu.

1 The abbreviations used are: CDP-DG, CDP-diacylglycerol; CPEC, cyclopentenylcytosine; PA, phosphatidate; PC, phosphatidylcholine; PCR, polymerase chain reaction; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results & Discussion
References

  1. Liberman, I. (1956) J. Biol. Chem. 222, 765-775[Free Full Text]
  2. Long, C. W., and Pardee, A. B. (1967) J. Biol. Chem. 242, 4715-4721[Abstract/Free Full Text]
  3. Vance, D. E. (1996) in Biochemistry of Lipids, Lipoproteins and Membranes (Vance, D. E., and Vance, J., eds), pp. 153-181, Elsevier Science Publishers B.V., Amsterdam
  4. Carman, G. M., and Zeimetz, G. M. (1996) J. Biol. Chem. 271, 13293-13296[Free Full Text]
  5. Carter, J. R., and Kennedy, E. P. (1966) J. Lipid Res. 7, 678-683[Abstract/Free Full Text]
  6. Kennedy, E. P., and Weiss, S. B. (1956) J. Biol. Chem. 222, 193-214[Free Full Text]
  7. Ozier-Kalogeropoulos, O., Fasiolo, F., Adeline, M.-T., Collin, J., and Lacroute, F. (1991) Mol. Gen. Genet. 231, 7-16[Medline] [Order article via Infotrieve]
  8. Ozier-Kalogeropoulos, O., Adeline, M.-T., Yang, W.-L., Carman, G. M., and Lacroute, F. (1994) Mol. Gen. Genet. 242, 431-439[Medline] [Order article via Infotrieve]
  9. Yang, W.-L., McDonough, V. M., Ozier-Kalogeropoulos, O., Adeline, M.-T., Flocco, M. T., and Carman, G. M. (1994) Biochemistry 33, 10785-10793[Medline] [Order article via Infotrieve]
  10. Nadkarni, A. K., McDonough, V. M., Yang, W.-L., Stukey, J. E., Ozier-Kalogeropoulos, O., and Carman, G. M. (1995) J. Biol. Chem. 270, 24982-24988[Abstract/Free Full Text]
  11. McDonough, V. M., Buxeda, R. J., Bruno, M. E. C., Ozier-Kalogeropoulos, O., Adeline, M.-T., McMaster, C. R., Bell, R. M., and Carman, G. M. (1995) J. Biol. Chem. 270, 18774-18780[Abstract/Free Full Text]
  12. Aronow, B., and Ullman, B. (1987) J. Biol. Chem. 262, 5106-5112[Abstract/Free Full Text]
  13. Robert de Saint Vincent, B., and Buttin, G. (1980) Biochim. Biophys. Acta 610, 352-359
  14. Meuth, M., L'Heureux-Huard, N., and Trudel, M. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 6505-6509[Abstract]
  15. Aronow, B., Watts, T., Lassetter, J., Washtien, W., and Ullman, B. (1984) J. Biol. Chem. 259, 9035-9043[Abstract/Free Full Text]
  16. Chu, E. H. Y., McLaren, J. D., Li, I.-C., and Lamb, B. (1984) Biochem. Genet. 22, 701-715[Medline] [Order article via Infotrieve]
  17. Kaufman, E. R. (1986) Mutat. Res. 161, 19-27[Medline] [Order article via Infotrieve]
  18. Meuth, M., Goncalves, O., and Thom, P. (1982) Somatic Cell Genet. 8, 423-432[Medline] [Order article via Infotrieve]
  19. van den Berg, A. A., van Lenthe, H., Busch, S., de Korte, D., Roos, D., van Kuilenburg, A. B. P., and van Gennip, A. H. (1993) Eur. J. Biochem. 216, 161-167[Abstract]
  20. Kizaki, H., Williams, J. C., Morris, H. P., and Weber, G. (1980) Cancer Res. 40, 3921-3927[Medline] [Order article via Infotrieve]
  21. Weber, G., Lui, M. S., Takeda, E., and Denton, J. E. (1980) Life Sci. 27, 793-799[Medline] [Order article via Infotrieve]
  22. Weber, G., Olah, E., Lui, M. S., and Tzeng, D. (1979) Adv. Enzyme Regul. 17, 1-21
  23. Kang, G. J., Cooney, D. A., Moyer, J. D., Kelley, J. A., Kim, H.-Y., Marquez, V. E., and Johns, D. G. (1989) J. Biol. Chem. 264, 713-718[Abstract/Free Full Text]
  24. Zhang, H., Cooney, D. A., Zhang, M. H., Ahluwalia, G., Ford, H., Jr., and Johns, D. G. (1993) Cancer Res. 53, 5714-5720[Abstract]
  25. Rose, M. D., Winston, F., and Heiter, P. (1990) Methods in Yeast Genetics: A Laboratory Course Manual, pp. 1-198, Cold Spring Harbor LaboratoryCold Spring HarborNY
  26. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  27. Culbertson, M. R., and Henry, S. A. (1975) Genetics 80, 23-40[Abstract/Free Full Text]
  28. Greenberg, M., Reiner, B., and Henry, S. A. (1982) Genetics 100, 19-33[Abstract/Free Full Text]
  29. McGee, T. P., Skinner, H. B., and Bankaitis, V. A. (1994) J. Bacteriol. 176, 6861-6868[Abstract]
  30. Ito, H., Yasuki, F., Murata, K., and Kimura, A. (1983) J. Bacteriol. 153, 163-168[Medline] [Order article via Infotrieve]
  31. Schiestl, R. H., and Gietz, R. D. (1989) Curr. Genet. 16, 339-346[Medline] [Order article via Infotrieve]
  32. Innis, M. A., and Gelfand, D. H. (1990) in PCR Protocols: A Guide to Methods and Applications (Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. J., eds), pp. 3-12, Academic Press, Inc., San Diego, CA
  33. Schmitt, M. E., Brown, T. A., and Trumpower, B. L. (1990) Nucleic Acids Res. 18, 3091-3092[Medline] [Order article via Infotrieve]
  34. Kawasaki, E. S. (1990) in PCR Protocols: A Guide to Methods and Applications (Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. J., eds), pp. 21-27, Academic Press, Inc., San Diego, CA
  35. Sikorski, R. S., and Boeke, J. D. (1991) Methods Enzymol. 194, 302-318[Medline] [Order article via Infotrieve]
  36. Guarente, L. (1983) Methods Enzymol. 101, 181-191[Medline] [Order article via Infotrieve]
  37. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve]
  38. Haid, A., and Suissa, M. (1983) Methods Enzymol. 96, 192-205[Medline] [Order article via Infotrieve]
  39. Atkinson, K., Fogel, S., and Henry, S. A. (1980) J. Biol. Chem. 255, 6653-6661[Abstract/Free Full Text]
  40. 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]
  41. Homann, M. J., Poole, M. A., Gaynor, P. M., Ho, C.-T., and Carman, G. M. (1987) J. Bacteriol. 169, 533-539[Medline] [Order article via Infotrieve]
  42. Morlock, K. R., Lin, Y.-P., and Carman, G. M. (1988) J. Bacteriol. 170, 3561-3566[Medline] [Order article via Infotrieve]
  43. Bligh, E. G., and Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37, 911-917
  44. Esko, J. D., and Raetz, C. R. H. (1980) J. Biol. Chem. 255, 4474-4480[Free Full Text]
  45. Henderson, R. J., and Tocher, D. R. (1992) in Lipid Analysis (Hamilton, R. J., and Hamilton, S., eds), pp. 65-111, IRL Press at Oxford University Press, Oxford
  46. Teegarden, D., Taparowsky, E. J., and Kent, C. (1990) J. Biol. Chem. 265, 6042-6047[Abstract/Free Full Text]
  47. Whelan, J., Phear, G., Yamauchi, M., and Meuth, M. (1993) Nat. Genet. 3, 317-321[Medline] [Order article via Infotrieve]
  48. Yamauchi, M., Yamauchi, N., and Meuth, M. (1990) EMBO J. 9, 2095-2099[Abstract]
  49. Weng, M., Makaroff, C. A., and Zalkin, H. (1986) J. Biol. Chem. 261, 5568-5574[Abstract/Free Full Text]
  50. Tipples, G., and McClarty, G. (1995) J. Biol. Chem. 270, 7908-7914[Abstract/Free Full Text]
  51. Trach, K., Chapman, J. W., Piggot, P., Lecoq, D., and Hoch, J. A. (1988) J. Bacteriol. 170, 4194-4208[Medline] [Order article via Infotrieve]
  52. Becker, D. M., Fikes, J. D., and Guarente, L. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 1968-1972[Abstract]
  53. 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
  54. Carman, G. M., and Henry, S. A. (1989) Annu. Rev. Biochem. 58, 635-669[CrossRef][Medline] [Order article via Infotrieve]
  55. Greenberg, M. L., and Lopes, J. M. (1996) Microbiol. Rev. 60, 1-20[Free Full Text]
  56. McGee, T. P., Skinner, H. B., Whitters, E. A., Henry, S. A., and Bankaitis, V. A. (1994) J. Cell Biol. 124, 273-287[Abstract]
  57. McMaster, C. R., and Bell, R. M. (1994) J. Biol. Chem. 269, 28010-28016[Abstract/Free Full Text]
  58. McMaster, C. R., and Bell, R. M. (1994) J. Biol. Chem. 269, 14776-14783[Abstract/Free Full Text]
  59. 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
  60. Hirsch, J. P., and Henry, S. A. (1986) Mol. Cell. Biol. 6, 3320-3328[Medline] [Order article via Infotrieve]
  61. 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
  62. Carman, G. M. (1989) in Phosphatidylcholine Metabolism (Vance, D. E., ed), pp. 165-183, CRC Press, Inc., Baca Raton, FL
  63. Patton-Vogt, J. L., Griac, P., Sreenivas, A., Bruno, V., Dowd, S., Swede, M. J., and Henry, S. A. (1997) J. Biol. Chem. 272, 20873-20883[Abstract/Free Full Text]
  64. Sreenivas, A., Patton-Vogt, J. L., Bruno, V., Griac, P., and Henry, S. A. (1998) J. Biol. Chem. 273, 16635-16638[Abstract/Free Full Text]
  65. Trotter, P. J., and Voelker, D. R. (1995) J. Biol. Chem. 270, 6062-6070[Abstract/Free Full Text]
  66. Trotter, P. J., Pedretti, J., Yates, R., and Voelker, D. R. (1995) J. Biol. Chem. 270, 6071-6080[Abstract/Free Full Text]
  67. Kodaki, T., and Yamashita, S. (1987) J. Biol. Chem. 262, 15428-15435[Abstract/Free Full Text]
  68. Kodaki, T., and Yamashita, S. (1989) Eur. J. Biochem. 185, 243-251[Abstract]
  69. Summers, E. F., Letts, V. A., McGraw, P., and Henry, S. A. (1988) Genetics 120, 909-922[Abstract/Free Full Text]
  70. McGraw, P., and Henry, S. A. (1989) Genetics 122, 317-330[Abstract/Free Full Text]
  71. Cleves, A. E., McGee, T. P., Whitters, E. A., Champion, K. M., Aitkin, J. R., Dowhan, W., Goebl, M., and Bankaitis, V. A. (1991) Cell 64, 789-800[Medline] [Order article via Infotrieve]
  72. Bankaitis, V. A., Malehorn, D. E., Emr, S. D., and Greene, R. (1989) J. Cell Biol. 108, 1271-1281[Abstract]
  73. Sha, B., Phillips, S. E., Bankaitis, V. A., and Luo, M. (1998) Nature 391, 506-510[CrossRef][Medline] [Order article via Infotrieve]
  74. Skinner, H. B., McGee, T. P., McMaster, C. R., Fry, M. R., Bell, R. M., and Bankaitis, V. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 112-116[Abstract]
  75. Kearns, B. G., McGee, T. P., Mayinger, P., Gedvilaite, A., Phillips, S. E., Kagiwada, S., and Bankaitis, V. A. (1997) Nature 387, 101-105[CrossRef][Medline] [Order article via Infotrieve]
  76. Shen, H., and Dowhan, W. (1996) J. Biol. Chem. 271, 29043-29048[Abstract/Free Full Text]
  77. Shen, H., and Dowhan, W. (1997) J. Biol. Chem. 272, 11215-11220[Abstract/Free Full Text]
  78. Henry, S. A., and Patton-Vogt, J. L. (1998) Prog. Nucleic Acid Res. 61, 133-179[Medline] [Order article via Infotrieve]
  79. Taylor, F. R., and Parks, L. W. (1979) Biochim. Biophys. Acta 575, 204-214[Medline] [Order article via Infotrieve]
  80. Taylor, F. R., and Parks, L. W. (1978) J. Bacteriol. 136, 531-537[Medline] [Order article via Infotrieve]
  81. Lopes, J. M., Hirsch, J. P., Chorgo, P. A., Schulze, K. L., and Henry, S. A. (1991) Nucleic Acids Res. 19, 1687-1693[Abstract]
  82. Thomas, B., and Rothstein, R. (1989) Cell 56, 619-630[Medline] [Order article via Infotrieve]
  83. Sclafani, R. A., and Fangman, W. L. (1994) Proc. Natl. Acad. Sci. U. S. A. 81, 5821-5825
  84. Alani, E., Cao, L., and Kleckner, N. (1987) Genetics 116, 541-545[Abstract/Free Full Text]
  85. Gietz, R. D., and Sugino, A. (1988) Gene (Amst.) 74, 527-534[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.