(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 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.
PC ()is the essential end product of phospholipid
biosynthesis and the major membrane phospholipid found in the yeast Saccharomyces
cerevisiae(1, 2, 3, 4) . There
are two pathways by which PC is synthesized in S. cerevisiae,
the CDP-DG pathway and the CDP-choline pathway (1, 2, 3, 4) (Fig.1). The
CDP-DG pathway is primarily used by wild-type cells when they are grown
in the absence of
choline(1, 2, 3, 4) . However, the
CDP-choline pathway becomes more important for PC synthesis when the
enzymes in the CDP-DG pathway are repressed or
defective(2, 3, 4) . The CDP-DG pathway
enzymes CDP-DG synthase(5, 6) , PS
synthase(7, 8, 9, 10) , and PS
decarboxylase (11, 12, 13) and the two
phospholipid N-methyltransferases(7, 11, 14, 15, 16, 17) are
repressed when wild-type cells are supplemented with inositol plus
choline. The repression of these enzymes is absolutely dependent on
inositol, a PI precursor that plays a major role in the coordinate
regulation of phospholipid biosynthesis in S.
cerevisiae(2, 3, 4) . Under these growth
conditions, the exogenous choline is used to synthesize PC via the
CDP-choline pathway(2, 4) .
Figure 1: Pathways for the biosynthesis of PC in S. cerevisiae. The pathways shown for the biosynthesis of PC include the relevant steps discussed in the text. The indicated reactions are catalyzed by the following enzymes: 1, CTP synthetase; 2, CDP-DG synthase; 3, PS synthase; 4, PS decarboxylase; 5, phospholipid N-methyltransferases; 6, PI synthase; 7, choline kinase; 8, phosphocholine cytidylyltransferase; 9, cholinephosphotransferase; and 10, PA phosphatase. The CDP-DG pathway is indicated by the boxed area. A more comprehensive phospholipid biosynthetic pathway that includes the steps for the synthesis of phosphatidylglycerol and cardiolipin may be found in (2) . CDP-Etn, CDP-ethanolamine; CDP-Cho, CDP-choline; SL, sphingolipids; PIPs, phosphoinositides; DG, diacylglycerol.
Mutants defective in PS synthase (cho1/pss mutants(18, 19) ), PS decarboxylase (psd1,psd2 double mutants(20, 21) ), or the phospholipid N-methyltransferases (pem1/cho2,pem2/opi3 double mutants(22, 23, 24, 25) ) require choline for growth in order to synthesize PC via the CDP-choline pathway. The mutants defective in PS synthase (18, 19) and PS decarboxylase (20, 21) can also synthesize PC if they are supplemented with ethanolamine. The ethanolamine is used for PE synthesis via the CDP-ethanolamine pathway(26) . The PE is subsequently methylated by the phospholipid N-methyltransferases to form PC (Fig.1). Overall, these results led to the notion that the CDP-choline pathway was an auxiliary or salvage pathway in S. cerevisiae(2, 3, 4) . However, recent studies have shown that the CDP-choline pathway in S. cerevisiae is not simply a salvage pathway for PC synthesis. In fact, the CDP-choline pathway contributes to PC synthesis even when wild-type cells are grown in the absence of choline(27, 28) .
CTP plays an essential role in the synthesis of PC and all membrane phospholipids in S. cerevisiae. CTP is the direct precursor of the activated, energy-rich phospholipid pathway intermediates CDP-DG (29) , CDP-choline(26) , and CDP-ethanolamine (26) (Fig.1). CDP-DG is the source of the phosphatidyl moiety of PS, PE, and PC synthesized by the CDP-DG pathway as well as PI, phosphatidylglycerol, and cardiolipin (2, 4) . CDP-choline and CDP-ethanolamine are the sources of the hydrophilic head groups of PC and PE synthesized by the CDP-choline and CDP-ethanolamine pathways, respectively(2, 4) . In this work, we examined the effect of CTP on phospholipid synthesis. We show that an elevation in the cellular concentration of CTP results in an increase in the utilization of the CDP-choline pathway for the synthesis of PC. The mechanism for this regulation includes the supply of CTP for the phosphocholine cytidylyltransferase reaction in the CDP-choline pathway and the inhibition of the PS synthase reaction in the CDP-DG pathway.
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 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.
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 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.
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-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.
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 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.
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 as a cofactor.
The aim of this work was to examine the regulation of phospholipid biosynthesis in S. cerevisiae by CTP. CTP is essential for the biosynthesis of all membrane phospholipids in S. cerevisiae whether they are synthesized via the CDP-DG or CDP-choline pathway(1, 2, 3, 4) . Our rationale was to elevate the cellular concentration of CTP by overexpressing CTP synthetase. Given the pyrimidine biosynthetic pathways in S. cerevisiae(62) , this was the most straightforward way of elevating the cellular concentration of CTP. The expression of the URA7 gene on a multicopy plasmid resulted in an appreciable overexpression of CTP synthetase mRNA and protein when compared with control cells. However, there was only a 2.4-fold increase in the cellular concentration of CTP in these cells. The discrepancy between the relatively high level of CTP synthetase overexpression and the relatively low increase in the cellular concentration of CTP can be explained by the inhibition of CTP synthetase activity by CTP(41) . This regulation of CTP synthetase activity by CTP inhibition could not be overcome by further overexpression of the URA7 gene. Nevertheless, the 2.4-fold elevation in the cellular concentration of CTP was enough to address the regulation of phospholipid biosynthesis by CTP.
The overexpression of CTP synthetase did not have a significant effect on the overall composition of the major membrane phospholipids. However, the overexpression of CTP synthetase resulted in a 2-fold increase in the utilization of the CDP-choline pathway for PC biosynthesis. This increase in CDP-choline pathway usage was not due to an increase in the expression of any of the enzyme activities in this pathway. Under the growth conditions of our experiments (i.e. absence of exogenous choline), the choline needed for PC synthesis via the CDP-choline pathway was presumably derived from the turnover of PC synthesized via the CDP-DG pathway(27, 28) . It is unclear what the contribution of the CDP-choline pathway is relative to the CDP-DG pathway when cells are grown in the absence of exogenous choline. This is a difficult question to address and could not be determined from the data presented here. The fact that the PI/PC transfer protein (Sec14p) is essential in cells with a functional CDP-choline pathway suggests that the CDP-choline pathway plays an important role in PC synthesis(27, 68) .
CDP-choline accounted for only 1% of the CDP-choline pathway
intermediates of control cells. In addition, the apparent K of CTP (1.4 mM) for the
phosphocholine cytidylyltransferase reaction (69) was 2-fold
higher than the cellular concentration of CTP (0.7 mM) in
control cells. Taken together, these results were consistent with the
notion (47) that the phosphocholine cytidylyltransferase
reaction catalyzes the rate-limiting step in the CDP-choline pathway in S. cerevisiae. The overexpression of CTP synthetase brought
the cellular concentration of CTP (1.7 mM) up to the K
of CTP for the phosphocholine
cytidylyltransferase reaction. Thus, based on the kinetic constant for
CTP and its cellular concentration, one would expect that the increase
in the cellular concentration of CTP would cause an increase in
phosphocholine cytidylyltransferase activity in vivo. Indeed,
the overexpression of CTP synthetase resulted in an increase in the
cellular concentration of CDP-choline. This increase in the CDP-choline
concentration was consistent with the increased utilization of the
CDP-choline pathway for PC synthesis.
The K of CTP (1 mM) for the CDP-DG synthase reaction (70) was 1.4-fold higher than the cellular concentration of CTP
in control cells. The increase in the cellular concentration of CTP due
to CTP synthetase overexpression brought its concentration nearly
2-fold higher than the K
of CTP. As
discussed above for the phosphocholine cytidylyltransferase reaction,
an argument can be made based on the kinetic constant for CTP and its
cellular concentrations for the regulation of CDP-DG synthase activity
by CTP in vivo. Indeed, the overexpression of CTP synthetase
resulted in an increase in the concentration of CDP-DG. However, this
did not result in a greater utilization of the CDP-DG pathway for PC
synthesis. In contrast to the phosphocholine cytidylyltransferase
reaction, the synthesis of CDP-DG is not a rate-limiting step in the
CDP-DG pathway(79) . Moreover, PS synthase activity was reduced
in cells that overexpressed CTP synthetase. Since PS synthase plays a
major role in the regulation of the CDP-DG pathway for PC
biosynthesis(2, 3, 4) , the inhibition of
this enzyme would be expected to reduce the synthesis of PC via this
pathway.
Mechanisms other than transcriptional regulation affected the expression of PS synthase, cholinephosphotransferase, and PI synthase activities in cells overexpressing CTP synthetase. In addition, PS synthase and cholinephosphotransferase activities were directly inhibited by CTP. The mechanism of PS synthase and cholinephosphotransferase inhibition by CTP was the chelation of their divalent metal cofactors. However, it is unclear whether this mechanism of inhibition by CTP would be physiologically relevant.
In contrast to S. cerevisiae, the CDP-choline pathway is the main route of PC synthesis in mammalian cells(71, 72) . In mammalian cells(71) , as in S. cerevisiae, the phosphocholine cytidylyltransferase reaction is the rate-limiting step in the CDP-choline pathway. The elevation of CTP levels in poliovirus-infected HeLa cells (73) and cytidine-supplemented neuron-related PC12 cells (74) also results in an increase in PC synthesis via the CDP-choline pathway. In the poliovirus-infected HeLa cells, the mechanism for the increase in the utilization of the CDP-choline for PC synthesis is the stimulation of the phosphocholine cytidylyltransferase reaction by CTP (73) .
In addition to CTP, the supply of other phospholipid pathway intermediates has been shown to play a role in the regulation of phospholipid biosynthesis in S. cerevisiae. The cellular concentration of ATP plays a role in the proportional synthesis of triacylglycerols and phospholipids (64) and also the synthesis of phosphoinositides(75, 76) . Elevated ATP levels favor phospholipid synthesis at the expense of triacylglycerols, whereas reduced ATP levels have the opposite effect. These effects have been attributed in part to the regulation of PA phosphatase activity by the cellular concentration of ATP(64) . Similarly, high levels of ATP favor the synthesis of PI 4-phosphate and PI 4,5-bisphosphate, whereas low ATP levels have the opposite effect. These effects are due to the regulation of membrane-associated PI 4-kinase activity by the cellular concentrations of ATP and ADP(77) . The synthesis of phosphoinositides in S. cerevisiae is also regulated by the cellular concentrations of CDP-DG through the control of membrane-associated PI 4-kinase activity(78) . As a final example, the cellular concentration of inositol regulates the partitioning of CDP-DG between PI and PS through the regulation of PI synthase and PS synthase activities(79) .
In summary, we have shown that CTP plays a role in the regulation of the pathways by which PC is synthesized. This regulation includes the supply of CTP for the phosphocholine cytidylyltransferase reaction in the CDP-choline pathway and the inhibition of the PS synthase reaction in the CDP-DG pathway. These studies further underscore the complexity of the biochemical mechanisms that regulate phospholipid biosynthesis in S. cerevisiae.