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(Received for publication, April 25,
1995; and in revised form, June 5, 1995) From the
In the yeast Saccharomyces cerevisiae, the major
membrane phospholipid phosphatidylcholine is synthesized by the
CDP-diacylglycerol and CDP-choline pathways. We examined the regulation
of phosphatidylcholine synthesis by CTP. The cellular concentration of
CTP was elevated (2.4-fold) by overexpressing CTP synthetase, the
enzyme responsible for the synthesis of CTP. The overexpression of CTP
synthetase resulted in a 2-fold increase in the utilization of the
CDP-choline pathway for phosphatidylcholine synthesis. The increase in
CDP-choline pathway usage was not due to an increase in the expression
of any of the enzymes in this pathway. CDP-choline, the product of the
phosphocholine cytidylyltransferase reaction, was the limiting
intermediate in the CDP-choline pathway. The apparent K
PC (
Figure 1:
Pathways for
the biosynthesis of PC in S. cerevisiae. The pathways shown
for the biosynthesis of PC include the relevant steps discussed in the
text. The indicated reactions are catalyzed by the following enzymes: 1, CTP synthetase; 2, CDP-DG synthase; 3, PS
synthase; 4, PS decarboxylase; 5, phospholipid N-methyltransferases; 6, PI synthase; 7,
choline kinase; 8, phosphocholine cytidylyltransferase; 9, cholinephosphotransferase; and 10, PA phosphatase.
The CDP-DG pathway is indicated by the boxed area. A more
comprehensive phospholipid biosynthetic pathway that includes the steps
for the synthesis of phosphatidylglycerol and cardiolipin may be found
in (2) . CDP-Etn, CDP-ethanolamine; CDP-Cho,
CDP-choline; SL, sphingolipids; PIPs,
phosphoinositides; DG,
diacylglycerol.
Mutants defective in PS
synthase (cho1/pss mutants(18, 19) ), PS
decarboxylase (psd1,psd2 double
mutants(20, 21) ), or the phospholipid N-methyltransferases (pem1/cho2,pem2/opi3 double
mutants(22, 23, 24, 25) ) require
choline for growth in order to synthesize PC via the CDP-choline
pathway. The mutants defective in PS synthase (18, 19) and PS decarboxylase (20, 21) can also synthesize PC if they are
supplemented with ethanolamine. The ethanolamine is used for PE
synthesis via the CDP-ethanolamine pathway(26) . The PE is
subsequently methylated by the phospholipid N-methyltransferases to form PC (Fig.1). Overall,
these results led to the notion that the CDP-choline pathway was an
auxiliary or salvage pathway in S.
cerevisiae(2, 3, 4) . However, recent
studies have shown that the CDP-choline pathway in S. cerevisiae is not simply a salvage pathway for PC synthesis. In fact, the
CDP-choline pathway contributes to PC synthesis even when wild-type
cells are grown in the absence of choline(27, 28) . CTP plays an essential role in the synthesis of PC and all membrane
phospholipids in S. cerevisiae. CTP is the direct precursor of
the activated, energy-rich phospholipid pathway intermediates CDP-DG (29) , CDP-choline(26) , and CDP-ethanolamine (26) (Fig.1). CDP-DG is the source of the phosphatidyl
moiety of PS, PE, and PC synthesized by the CDP-DG pathway as well as
PI, phosphatidylglycerol, and cardiolipin (2, 4) .
CDP-choline and CDP-ethanolamine are the sources of the hydrophilic
head groups of PC and PE synthesized by the CDP-choline and
CDP-ethanolamine pathways, respectively(2, 4) . In
this work, we examined the effect of CTP on phospholipid synthesis. We
show that an elevation in the cellular concentration of CTP results in
an increase in the utilization of the CDP-choline pathway for the
synthesis of PC. The mechanism for this regulation includes the supply
of CTP for the phosphocholine cytidylyltransferase reaction in the
CDP-choline pathway and the inhibition of the PS synthase reaction in
the CDP-DG pathway.
Northern blot and immunoblot analyses were used to determine the
amounts of CTP synthetase mRNA and protein in cells bearing the URA7 gene on the multicopy and single copy plasmids. The
levels of CTP synthetase mRNA and protein found in cells bearing URA7 on the multicopy plasmid were 25- and 10-fold greater,
respectively, than those found in control cells (Fig.2A). Cells overexpressing CTP synthetase had a
2.4-fold higher cellular concentration of CTP when compared with the
control cells (Fig.2B). Similar results have been
reported for the overexpression of CTP synthetase protein and the
cellular concentration of CTP in cells bearing URA7 on a
multicopy plasmid when compared with wild-type cells(41) . The
cellular concentrations of UTP were not affected by the overexpression
of CTP synthetase (Fig.2B).
Figure 2:
Effect of URA7 overexpression on
CTP synthetase mRNA and protein levels and the cellular concentration
of UTP and CTP. Cells bearing URA7 on the multicopy (MC-URA7) and single copy centromeric (Cen-URA7)
plasmids were grown in complete synthetic medium to the exponential
phase of growth. A, CTP synthetase mRNA and protein were
determined by Northern blot and immunoblot analyses, respectively, as
described under ``Experimental Procedures.'' The amounts of
CTP synthetase mRNA and protein found in cells bearing URA7 on
the single copy plasmid were set at 1. B, nucleotides were
extracted and analyzed by high-performance liquid chromatography as
described in the text. The values reported for UTP and CTP were
determined from triplicate analyses with standard deviations of
±12 and ±8%, respectively, from a minimum of two
independent growth studies.
Figure 3:
Effect of CTP synthetase overexpression on
phospholipid composition and PC synthesized via the CDP-DG and
CDP-choline pathways. Cells bearing URA7 on the multicopy (MC-URA7) and single copy centromeric (Cen-URA7)
plasmids were grown in complete synthetic medium to the exponential
phase of growth. The steady-state compositions of phospholipids (A) and PC (B) were determined by labeling cells for
five to six generations with
Radiolabeled choline
was incorporated into PC (Fig.3B). This confirmed that
the CDP-choline pathway contributed to PC synthesis when cells were
grown in the absence of supplemented choline. The data shown in Fig.3B are plotted as the ratio of the cpm of
Figure 4:
Effect of CTP synthetase overexpression on
the CDP-choline pathway intermediates. Cells bearing URA7 on
the multicopy (MC-URA7) and single copy centromeric (Cen-URA7) plasmids were grown in complete synthetic medium to
the exponential phase of growth. The steady-state composition of the
CDP-choline pathway intermediates was determined by labeling cells for
five to six generations with
[methyl-
Figure 5:
Effect of CTP synthetase overexpression on
the enzyme activities of the CDP-DG and CDP-choline pathways. Cells
bearing URA7 on the multicopy (MC-URA7) and single
copy centromeric (Cen-URA7) plasmids were grown in complete
synthetic medium to the exponential phase of growth. Cell extracts were
prepared and used for the measurement of the indicated CDP-DG (A) and CDP-choline (B) pathway enzymes. The relative
activity (percent) was calculated by normalizing the specific activity
of each enzyme from cells bearing URA7 on the multicopy
plasmid to cells bearing URA7 on the single copy plasmid.
Enzyme activities were determined in triplicate with a standard
deviation of ±5% from a minimum of two independent growth
experiments. CDS, CDP-DG synthase; PSS, PS synthase; PSD, PS decarboxylase; PMT, phospholipid
methyltransferases; PIS, PI synthase; CK, choline
kinase; CCT, phosphocholine cytidylyltransferase; CPT, cholinephosphotransferase; PAP, PA
phosphatase.
We questioned whether the overexpression of CTP synthetase affected
the abundance of the mRNAs encoding for PS synthase,
cholinephosphotransferase, and PI synthase. Cells bearing the URA7 gene on the multicopy and single copy plasmids were grown to the
exponential phase of growth, and total RNA was extracted. The relative
abundance of the mRNAs from these cells was determined by Northern blot
analysis using CHO1, CPT1, and PIS probes.
The abundance of PS synthase, cholinephosphotransferase, and PI
synthase mRNAs was not affected by the overexpression of CTP synthetase
(data not shown).
Figure 6:
Effect of CTP on PS synthase activity. PS
synthase activity was measured in the absence and presence of CTP using
the following concentrations of MnCl
Figure 7:
Effect of CTP on cholinephosphotransferase
activity. Cholinephosphotransferase activity was measured in the
absence and presence of CTP using the indicated concentrations of
MgCl
The aim of this work was to examine the regulation of
phospholipid biosynthesis in S. cerevisiae by CTP. CTP is
essential for the biosynthesis of all membrane phospholipids in S.
cerevisiae whether they are synthesized via the CDP-DG or
CDP-choline pathway(1, 2, 3, 4) .
Our rationale was to elevate the cellular concentration of CTP by
overexpressing CTP synthetase. Given the pyrimidine biosynthetic
pathways in S. cerevisiae(62) , this was the most
straightforward way of elevating the cellular concentration of CTP. The
expression of the URA7 gene on a multicopy plasmid resulted in
an appreciable overexpression of CTP synthetase mRNA and protein when
compared with control cells. However, there was only a 2.4-fold
increase in the cellular concentration of CTP in these cells. The
discrepancy between the relatively high level of CTP synthetase
overexpression and the relatively low increase in the cellular
concentration of CTP can be explained by the inhibition of CTP
synthetase activity by CTP(41) . This regulation of CTP
synthetase activity by CTP inhibition could not be overcome by further
overexpression of the URA7 gene. Nevertheless, the 2.4-fold
elevation in the cellular concentration of CTP was enough to address
the regulation of phospholipid biosynthesis by CTP. The
overexpression of CTP synthetase did not have a significant effect on
the overall composition of the major membrane phospholipids. However,
the overexpression of CTP synthetase resulted in a 2-fold increase in
the utilization of the CDP-choline pathway for PC biosynthesis. This
increase in CDP-choline pathway usage was not due to an increase in the
expression of any of the enzyme activities in this pathway. Under the
growth conditions of our experiments (i.e. absence of
exogenous choline), the choline needed for PC synthesis via the
CDP-choline pathway was presumably derived from the turnover of PC
synthesized via the CDP-DG pathway(27, 28) . It is
unclear what the contribution of the CDP-choline pathway is relative to
the CDP-DG pathway when cells are grown in the absence of exogenous
choline. This is a difficult question to address and could not be
determined from the data presented here. The fact that the PI/PC
transfer protein (Sec14p) is essential in cells with a functional
CDP-choline pathway suggests that the CDP-choline pathway plays an
important role in PC synthesis(27, 68) . CDP-choline accounted for only 1% of the CDP-choline pathway
intermediates of control cells. In addition, the apparent K The K Mechanisms other than transcriptional regulation affected
the expression of PS synthase, cholinephosphotransferase, and PI
synthase activities in cells overexpressing CTP synthetase. In
addition, PS synthase and cholinephosphotransferase activities were
directly inhibited by CTP. The mechanism of PS synthase and
cholinephosphotransferase inhibition by CTP was the chelation of their
divalent metal cofactors. However, it is unclear whether this mechanism
of inhibition by CTP would be physiologically relevant. In contrast
to S. cerevisiae, the CDP-choline pathway is the main route of
PC synthesis in mammalian cells(71, 72) . In mammalian
cells(71) , as in S. cerevisiae, the phosphocholine
cytidylyltransferase reaction is the rate-limiting step in the
CDP-choline pathway. The elevation of CTP levels in poliovirus-infected
HeLa cells (73) and cytidine-supplemented neuron-related PC12
cells (74) also results in an increase in PC synthesis via the
CDP-choline pathway. In the poliovirus-infected HeLa cells, the
mechanism for the increase in the utilization of the CDP-choline for PC
synthesis is the stimulation of the phosphocholine cytidylyltransferase
reaction by CTP (73) . In addition to CTP, the supply of
other phospholipid pathway intermediates has been shown to play a role
in the regulation of phospholipid biosynthesis in S.
cerevisiae. The cellular concentration of ATP plays a role in the
proportional synthesis of triacylglycerols and phospholipids (64) and also the synthesis of
phosphoinositides(75, 76) . Elevated ATP levels favor
phospholipid synthesis at the expense of triacylglycerols, whereas
reduced ATP levels have the opposite effect. These effects have been
attributed in part to the regulation of PA phosphatase activity by the
cellular concentration of ATP(64) . Similarly, high levels of
ATP favor the synthesis of PI 4-phosphate and PI 4,5-bisphosphate,
whereas low ATP levels have the opposite effect. These effects are due
to the regulation of membrane-associated PI 4-kinase activity by the
cellular concentrations of ATP and ADP(77) . The synthesis of
phosphoinositides in S. cerevisiae is also regulated by the
cellular concentrations of CDP-DG through the control of
membrane-associated PI 4-kinase activity(78) . As a final
example, the cellular concentration of inositol regulates the
partitioning of CDP-DG between PI and PS through the regulation of PI
synthase and PS synthase activities(79) . In summary, we
have shown that CTP plays a role in the regulation of the pathways by
which PC is synthesized. This regulation includes the supply of CTP for
the phosphocholine cytidylyltransferase reaction in the CDP-choline
pathway and the inhibition of the PS synthase reaction in the CDP-DG
pathway. These studies further underscore the complexity of the
biochemical mechanisms that regulate phospholipid biosynthesis in S. cerevisiae.
Volume 270,
Number 32,
Issue of August 11, pp. 18774-18780, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
of CTP (1.4 mM) for
phosphocholine cytidylyltransferase was 2-fold higher than the cellular
concentration of CTP (0.7 mM) in control cells. This provided
an explanation of why the overexpression of CTP synthetase caused an
increase in the cellular concentration of CDP-choline.
Phosphatidylserine synthase activity was reduced in cells
overexpressing CTP synthetase. This was not due to a transcriptional
repression mechanism. Instead, the decrease in phosphatidylserine
synthase activity was due, at least in part, to a direct inhibition of
activity by CTP. These results show that CTP plays a role in the
regulation of the pathways by which phosphatidylcholine is synthesized.
This regulation includes the supply of CTP for the phosphocholine
cytidylyltransferase reaction in the CDP-choline pathway and the
inhibition of the phosphatidylserine synthase reaction in the
CDP-diacylglycerol pathway.
)is the essential end product of phospholipid
biosynthesis and the major membrane phospholipid found in the yeast Saccharomyces
cerevisiae(1, 2, 3, 4) . There
are two pathways by which PC is synthesized in S. cerevisiae,
the CDP-DG pathway and the CDP-choline pathway (1, 2, 3, 4) (Fig.1). The
CDP-DG pathway is primarily used by wild-type cells when they are grown
in the absence of
choline(1, 2, 3, 4) . However, the
CDP-choline pathway becomes more important for PC synthesis when the
enzymes in the CDP-DG pathway are repressed or
defective(2, 3, 4) . The CDP-DG pathway
enzymes CDP-DG synthase(5, 6) , PS
synthase(7, 8, 9, 10) , and PS
decarboxylase (11, 12, 13) and the two
phospholipid N-methyltransferases(7, 11, 14, 15, 16, 17) are
repressed when wild-type cells are supplemented with inositol plus
choline. The repression of these enzymes is absolutely dependent on
inositol, a PI precursor that plays a major role in the coordinate
regulation of phospholipid biosynthesis in S.
cerevisiae(2, 3, 4) . Under these growth
conditions, the exogenous choline is used to synthesize PC via the
CDP-choline pathway(2, 4) .
Materials
All chemicals were reagent grade. Growth medium supplies were
purchased from Difco. Nucleotides, choline, phosphocholine,
CDP-choline, and bovine serum albumin were obtained from Sigma.
Phospholipids were purchased from Avanti Polar Lipids, Inc. and Sigma.
Radiochemicals, EN
HANCE, and GeneScreen hybridization
transfer membrane were purchased from DuPont NEN. Scintillation
counting supplies were from National Diagnostics, Inc. Restriction
endonucleases and the random primer NEBlot kit were purchased from New
England Biolabs Inc. Protein assay reagent, electrophoresis reagents,
and immunochemical reagents were purchased from Bio-Rad. Silica Gel 60
thin-layer chromatography plates and high-performance thin-layer
chromatography plates were from EM Science.Methods
Strains, Plasmids, and Recombinant DNA
Techniques
CTP synthetase was expressed in S. cerevisiae strain OK8 (MAT
leu2 trp1 ura3 ura7
::TRP1
ura8) (30) bearing the URA7 gene (31) on
the multicopy plasmid pFL44S (32) or the single copy
centromeric plasmid pFL38(32) . Strain OK8 has mutations in
both the URA7 and URA8 genes, which are two duplicate
genes encoding for CTP synthetase(30, 31) . PS
synthase was partially purified from S. cerevisiae mutant
strain VAL2C (MATaleu2-3 leu2-112 ade6 cho1)
bearing plasmid YEpCHO1(33) . Plasmid YEpCHO1 contains the structural gene for PS synthase and directs the
overexpression of the enzyme(33) . Cholinephosphotransferase
was isolated from S. cerevisiae mutant strain HJ091 (MAT leu2-3 leu2-112 his3-1 ura3-52 trp1-289
cpt1::LEU2 ept1-1) (34) bearing CPT1 on
the multicopy plasmid pCM26CPT. Plasmid pCM26CPT was constructed by
subcloning CPT1 from plasmid pRH150 (35) using the SacI and SalI sites in the multiple cloning site to
the multicopy plasmid pRS426(36) . Plasmid pCM26CPT contains
the structural gene for cholinephosphotransferase and directs the
overexpression of activity. All DNA manipulations were performed
according to standard methods(37) . Plasmid maintenance and
amplifications were performed in Escherichia coli strain
DH5.Growth Conditions
Cells were grown in complete
synthetic medium (38) containing 2% glucose and appropriate
supplements at 30 °C to the exponential phase of growth (1-2
10
cells/ml). To label total phospholipids and PC,
cells were grown for five to six generations in the presence of P
(4 µCi/ml) and
[methyl-H]choline (0.4 µCi/ml),
respectively. To label the CDP-choline pathway intermediates, cells
were grown for five to six generations in the presence of
[methyl-H]choline (10 µCi/ml). Cell
numbers were determined by microscopic examination with a
hemocytometer.Preparation of RNA and Northern Blot
Analysis
Total RNA was extracted from cells using hot phenol as
described by Schmitt et al.(39) . The RNA was
separated by electrophoresis under denaturing conditions using a 1%
formaldehyde-agarose gel(40) . Following electrophoresis, RNA
was transferred to GeneScreen and probed with a radiolabeled fragment
of the URA7, CHO1, PIS, or CPT1 gene. The URA7 probe was a 1.6-kilobase fragment isolated
from YEp352URA7(41) by EcoRI and HindIII digestion. The CHO1 probe was a 1.0-kilobase
fragment isolated from pAS103 (42) by HindIII and SacI digestion. The PIS probe was a 1.6-kilobase
fragment isolated from pPI514 (43) by HindIII
digestion. The CPT1 probe was a 1.1-kilobase fragment isolated
from pRH150 (35) by NcoI digestion. The probes were
labeled with [-P]dCTP by the random priming
reaction using a NEBlot kit. Prehybridization and hybridization of
blots were carried out at 60 °C in modified Church buffer (44) as recommended by United States Biochemical Corp.
Ribosomal subunit L32 mRNA (45) was used as a constitutive
standard and loading control.
Preparation of Enzymes
Cells were disrupted with
glass beads with a Mini-Bead-Beater (Biospec Products, Inc.) in 50
mM Tris-HCl buffer (pH 7.5) containing 1 mM disodium
EDTA, 0.3 M sucrose, and 10 mM 2-mercaptoethanol(7) . Glass beads and cell debris were
removed by centrifugation at 1500 g for 5 min. The
supernatant (cell extract) was used for enzyme assays and
immunoblotting of CTP synthetase. PS synthase was partially purified
from the cell extract through the Triton X-100 solubilization step as
described by Bae-Lee and Carman(46) . Microsome-associated
cholinephosphotransferase was isolated from the cell extract as
described by McMaster and Bell(47) .
Immunoblotting of CTP Synthetase
Immunoblot assays
were performed with IgG anti-CTP synthetase antibodies (41) as
described previously(48) . The density of the CTP synthetase
bands on immunoblots was quantitated by scanning densitometry.
Immunoblot signals were in the linear range of detectability.Extraction and Analysis of UTP and CTP
Cells
bearing URA7 on the multicopy and single copy plasmids were
grown to the exponential phase of growth. Cellular nucleotides were
extracted (31) , and UTP and CTP were analyzed by
high-performance liquid chromatography as described by
Ozier-Kalogeropoulos et al.(30) .Analysis of Phospholipids
Membranes were prepared
from cell extracts(49) , and phospholipids were extracted by
the method of Bligh and Dyer(50) . The chloroform phase was
neutralized and dried in vacuo, and the residue was dissolved
in chloroform. Phospholipids were analyzed by one-dimensional
thin-layer chromatography on highperformance silica gel thin-layer
plates using the solvent system methyl acetate, isopropyl alcohol,
chloroform, methanol, 0.25% KCl (25:25:28:10:7) as described by Knoll et al.(51) . The positions of the labeled
phospholipids on chromatograms were determined by autoradiography and
compared with standard phospholipids after exposure to iodine vapor.
The amount of each labeled phospholipid was determined by liquid
scintillation counting of the corresponding spots on chromatograms.Analysis of CDP-choline Pathway
Intermediates
Choline, phosphocholine, and CDP-choline were
obtained from whole cells after lipid extraction(50) . The
aqueous phase was neutralized and dried in vacuo, and the
residue was dissolved in deionized water. Samples were subjected to
centrifugation at 12,000 g for 3 min to remove
insoluble material. The CDP-choline pathway intermediates were
separated by thin-layer chromatography with Silica Gel 60 plates using
the solvent system methanol, 0.5% sodium chloride/ammonia (50:50:1) as
described by Teegarden et al.(52) . The positions of
the labeled intermediates on chromatograms were determined by
fluorography and compared with standards. The amount of each labeled
compound was determined by liquid scintillation counting.
Enzyme Assays
All assays were conducted at 30
°C. The activities of CDP-DG synthase(53) , PS
synthase(54) , PS decarboxylase (55) , the phospholipid N-methyltransferases(14) , PI synthase(56) ,
choline kinase(57) , phosphocholine cytidylyltransferase (58) , cholinephosphotransferase(59) , and PA
phosphatase (60) were measured as described previously. All
assays were linear with time and protein concentration. All assays were
conducted in triplicate with an average standard deviation of
±5%. One unit of enzymatic activity is defined as the amount of
enzyme that catalyzes the formation of 1 nmol of product/min. Specific
activity is defined as units/mg of protein. Protein concentration was
determined by the method of Bradford (61) using bovine serum
albumin as the standard.
Effect of CTP Synthetase Overexpression on the Cellular
Concentration of CTP
The aim of this work was to examine the
regulation of phospholipid biosynthesis in S. cerevisiae by
CTP. Cellular CTP levels are not easily manipulated in S.
cerevisiae through the exogenous supply of cytidine or cytosine.
Cytidine is converted to cytosine, which is then converted to CTP
through the pyrimidine biosynthetic pathway (cytosine uracil
UMP
UDP
CTP)(62) . Since the pathway for
CTP synthesis is indirect, cytidine or cytosine supplementation was not
the best way of elevating the cellular concentration of CTP. Our
approach to elevate the cellular concentration of CTP was to
overexpress the enzyme CTP synthetase. CTP synthetase catalyzes the
final step in the pyrimidine biosynthetic pathway(31) . CTP
synthetase is encoded by two duplicate genes named URA7(31) and URA8(30) . Neither one of the URA7 and URA8 genes is essential provided that cells
possess one functional gene encoding for the
enzyme(30, 31) . The URA7 gene was expressed
in strain OK8 on a multicopy plasmid or a single copy centromeric
plasmid. The expression of the URA7 gene on the centromeric
plasmid was used as a control. The growth of strain OK8 bearing either
one of the plasmids was the same. This was an important consideration
in our studies because the regulation of phospholipid biosynthesis in S. cerevisiae is influenced by growth phase(63) .
Effect of CTP Synthetase Overexpression on Phospholipid
Composition
Cells were grown in synthetic medium in the absence
of inositol, choline, and ethanolamine. Under these growth conditions,
our studies were not complicated by the regulatory effects these
phospholipid precursors have on the expression of phospholipid
biosynthetic enzymes(2, 4) . Since cells utilize both
the CDP-DG and CDP-choline pathways in the absence of exogenous
choline(27, 28) , we determined the phospholipid
composition of membranes from cells labeled to steady state with both P
and
[methyl-H]choline. P
will be incorporated into phospholipids synthesized by either the
CDP-DG or CDP-choline pathway, whereas the labeled choline will only be
incorporated into PC synthesized via the CDP-choline pathway. The
concentration of choline added to the growth medium from the
radioactive label was 0.1 µM. This concentration was too
low to affect the rate of synthesis of PC by the CDP-choline
pathway(47) . The phospholipid composition of membranes
determined by P
labeling is shown in Fig.3A. The composition of the major membrane
phospholipids was not significantly affected by the overexpression of
CTP synthetase. There was, however, a 1.6-fold increase in the
percentage of label incorporated into CDP-DG.
P
(4 µCi/ml)
and [methyl-H]choline (0.4 µCi/ml).
The incorporation of P
and
[methyl-H]choline into total
phospholipids and PC was 1600-2000 and 10,000-12,000
cpm/10 cells, respectively. Phospholipids were extracted
and analyzed as described under ``Experimental Procedures.''
The values reported in A were determined from P
labeling. The values in B are
reported as the cpm of H incorporated into PC relative to
the cpm of P incorporated into PC. The percentages
reported for phospholipids were determined from duplicate analyses
with a standard deviation of ±10% from a minimum of two
independent growth studies. CDG,
CDPdiacylglycerol.
H incorporated into PC to the cpm of P
incorporated into PC. If the cellular concentration of CTP affected the
pathway by which PC was synthesized, the ratio of the labels found in
PC would change. Indeed, this ratio increased 2-fold for cells which
overexpressed CTP synthetase when compared with the control cells (Fig.3B). This indicated that the elevation in the
cellular concentration of CTP caused an increase in the utilization of
the CDP-choline pathway for PC synthesis.
Effect of CTP Synthetase Overexpression on CDP-choline
Pathway Intermediates
The intermediates of the CDP-choline
pathway include choline, phosphocholine, and CDP-choline (Fig.1). Cells were labeled with
[methyl-H]choline to steady state to
analyze the effect of CTP synthetase overexpression on the composition
of the CDP-choline pathway intermediates. In control cells, 1% of the
water-soluble label was incorporated into CDP-choline, whereas most of
the label was found in choline and phosphocholine (Fig.4).
Choline and phosphocholine were not affected by the overexpression of
CTP synthetase (Fig.4). However, the overexpression of CTP
synthetase caused a 1.4-fold increase in the steady-state concentration
of CDP-choline when compared with the control cells (Fig.4).
H]choline (10 µCi/ml). The
incorporation of [methyl-H]choline into
the CDP-choline pathway intermediates was 40,000-70,000
cpm/10 cells. The CDP-choline pathway intermediates were
extracted and analyzed as described under ``Experimental
Procedures.'' The percentages reported for choline,
phosphocholine, and CDP-choline were determined from triplicate
analyses with standard deviations of ±10, 10, and 15%,
respectively, from a minimum of two independent growth
studies.
Effect of CTP Synthetase Overexpression on Phospholipid
Biosynthetic Enzyme Activities
Steady-state labeling experiments
indicated that the increase in CTP synthetase expression affected the
pathways by which PC was synthesized. We questioned if phospholipid
biosynthetic enzyme activities were affected in cells that
overexpressed CTP synthetase. These enzyme activities included those
responsible for PC synthesis via the CDP-DG pathway (CDP-DG synthase,
PS synthase, PS decarboxylase, and the phospholipid N-methyltransferases) and the CDP-choline pathway (choline
kinase, phosphocholine cytidylyltransferase, and
cholinephosphotransferase). PI synthase and PA phosphatase activities
were also examined because their reactions play a role in the CDP-DG
and CDP-choline pathways(2, 4) . Cells were grown to
the exponential phase of growth, cell extracts were prepared, and the
activities of the enzymes were measured. The cell extracts were diluted
to lower the CTP concentration derived from cells to a concentration
that would not affect the enzymes directly. The overexpression of CTP
synthetase resulted in decreases in PS synthase and
cholinephosphotransferase activities of 41 and 23%, respectively (Fig.5). On the other hand, CTP synthetase overexpression
resulted in a 120% increase in PI synthase activity (Fig.5).
Effect of CTP on Phospholipid Biosynthetic Enzyme
Activities
We examined if CTP had a direct effect on the CDP-DG
and CDP-choline pathway enzymes using cell extracts prepared from
control cells. Enzyme assays were performed in the absence and presence
of 5 mM CTP. This analysis did not include CDP-DG synthase or
phosphocholine cytidylyltransferase since these enzymes use CTP as a
substrate. The only enzyme activities that were affected by CTP were PS
synthase, cholinephosphotransferase, and PA phosphatase. All three of
these enzymes were inhibited by CTP. The effects of CTP on PS synthase
and cholinephosphotransferase activities are described below. The
inhibition of PA phosphatase activity by CTP has been previously
reported(64) .Effect of CTP on PS Synthase Activity
A Triton
X-100-solubilized preparation of microsome-associated PS synthase was
used to examine the effect of CTP on activity. PS synthase activity is
absolutely dependent on either MnCl
(0.6 mM) or
MgCl (10 mM) as a cofactor(46) . The PS
synthase activity obtained with 0.6 mM MnCl was
2.7-fold greater than the activity obtained with 15 or 30 mM MgCl (Fig.6). These results were consistent
with those previously reported for a homogeneous preparation of the
enzyme(46) . CTP inhibited PS synthase activity in a
dose-dependent manner when 0.6 mM MnCl was used as
the cofactor (Fig.6). The CTP-mediated inhibition of activity
followed cooperative kinetics (Fig.6, inset). Analysis
of the data according to the Hill equation yielded an IC value for CTP of 2.1 mM and a Hill number of 4.3. The
inhibition of PS synthase activity by CTP using 15 mM
MgCl
as the cofactor followed saturation kinetics (Fig.6). PS synthase activity was less sensitive to inhibition
(IC = 5 mM) by CTP when MgCl
was the cofactor. At the point of maximum inhibition, the
remaining PS synthase activity was the same regardless of whether 0.6
or 15 mM MgCl was used as the cofactor (Fig.6). As the CTP concentration was increased in these
experiments, the free Mn and Mg
concentrations would decrease (65) below the optimum
concentrations required for PS synthase activity(46) . These
results were consistent with the conclusion that PS synthase was
inhibited by CTP by a chelation mechanism. Indeed, PS synthase activity
was not inhibited by CTP when the MgCl
concentration was
increased to 30 mM (Fig.6). The MnCl concentration could not be increased above 0.6 mM due to
the inhibition of the enzyme by MnCl(46) .
and MgCl as cofactors: 
, 0.6 mM MnCl; , 15
mM MgCl
; 
, 30 mM MgCl.
The inset is a replot of the CTP-mediated inhibition of PS
synthase activity using MnCl as the
cofactor.
Effect of CTP on Cholinephosphotransferase
Activity
Microsome-associated cholinephosphotransferase was used
to examine the effect of CTP on enzyme activity. The strain used to
isolate cholinephosphotransferase contained a mutation in the EPT1 gene, which is the structural gene for
ethanolaminephosphotransferase(66) .
Ethanolaminephosphotransferase also possesses cholinephosphotransferase
activity(59) . Thus, our studies were not complicated by the
presence of this second enzyme. Maximum cholinephosphotransferase
activity is dependent on 7.5-15 mM MgCl as a
cofactor. ()The addition of CTP to the assay system for
cholinephosphotransferase resulted in a dose-dependent inhibition of
activity (Fig.7). The enzyme was less sensitive to inhibition
by CTP when the MgCl concentration in the assay was
increased from 7.5 mM (IC = 5 mM)
to 30 mM (IC
= 9.5 mM). As
discussed above for the PS synthase reaction, these results indicated
that CTP inhibited cholinephosphotransferase activity by a chelation
mechanism. A similar conclusion was previously reported in a
preliminary study of the cholinephosphotransferase from S.
cerevisiae(67) .
as a cofactor.
of CTP (1.4 mM) for the
phosphocholine cytidylyltransferase reaction (69) was 2-fold
higher than the cellular concentration of CTP (0.7 mM) in
control cells. Taken together, these results were consistent with the
notion (47) that the phosphocholine cytidylyltransferase
reaction catalyzes the rate-limiting step in the CDP-choline pathway in S. cerevisiae. The overexpression of CTP synthetase brought
the cellular concentration of CTP (1.7 mM) up to the K
of CTP for the phosphocholine
cytidylyltransferase reaction. Thus, based on the kinetic constant for
CTP and its cellular concentration, one would expect that the increase
in the cellular concentration of CTP would cause an increase in
phosphocholine cytidylyltransferase activity in vivo. Indeed,
the overexpression of CTP synthetase resulted in an increase in the
cellular concentration of CDP-choline. This increase in the CDP-choline
concentration was consistent with the increased utilization of the
CDP-choline pathway for PC synthesis.
of CTP (1 mM) for the CDP-DG synthase reaction (70) was 1.4-fold higher than the cellular concentration of CTP
in control cells. The increase in the cellular concentration of CTP due
to CTP synthetase overexpression brought its concentration nearly
2-fold higher than the K
of CTP. As
discussed above for the phosphocholine cytidylyltransferase reaction,
an argument can be made based on the kinetic constant for CTP and its
cellular concentrations for the regulation of CDP-DG synthase activity
by CTP in vivo. Indeed, the overexpression of CTP synthetase
resulted in an increase in the concentration of CDP-DG. However, this
did not result in a greater utilization of the CDP-DG pathway for PC
synthesis. In contrast to the phosphocholine cytidylyltransferase
reaction, the synthesis of CDP-DG is not a rate-limiting step in the
CDP-DG pathway(79) . Moreover, PS synthase activity was reduced
in cells that overexpressed CTP synthetase. Since PS synthase plays a
major role in the regulation of the CDP-DG pathway for PC
biosynthesis(2, 3, 4) , the inhibition of
this enzyme would be expected to reduce the synthesis of PC via this
pathway.
We are grateful to Connie Clancey and William Dowhan
for performing the PS decarboxylase assays. We thank Susan A. Henry and
Satoshi Yamashita for providing the CHO1 and PIS clones, respectively, used in this study.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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