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INTRODUCTION |
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.

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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.
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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 ura7
and
ura8
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
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.
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EXPERIMENTAL PROCEDURES |
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
-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).
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 URA7
::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 ura7
ura8
Double Mutant--
A
ura7
ura8
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 ura8
mutant was constructed first. A
4.6-kb SspI fragment of plasmid pDO183, which contained the
URA8
::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
ura8
::HisG recombination. One of the
ura8
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
ura7
ura8
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 ura7
ura8
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
-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.
-Galactosidase activity was measured by a
fluorescent assay using methylumbelliferyl galactoside as substrate
according to the instructions of the manufacturer. A unit of
-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.
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RESULTS AND DISCUSSION |
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 ura7
ura8
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.

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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.
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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).

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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.
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Table IV
Inhibitor constants for CTP of mutant CTP synthetases and the cellular
concentration of CTP in cells bearing the mutant CTP synthetases
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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).

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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.
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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).

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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.
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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).

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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.
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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).

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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
-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.
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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
-galactosidase activity was measured. The
-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
-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
-galactosidase
activity in cells bearing the E161K mutation. However, the
-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.

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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.
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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.
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.
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.