From the Departments of Biological Sciences and
§ Chemistry, Carnegie Mellon University,
Pittsburgh, Pennsylvania 15213
Received for publication, May 1, 2000, and in revised form, November 3, 2000
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ABSTRACT |
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The regulation of phosphatidylcholine degradation
as a function of the route of phosphatidylcholine (PC) synthesis and
changing environmental conditions has been investigated in the yeast
Saccharomyces cerevisiae. In the wild-type strains studied,
deacylation of phosphatidylcholine to glycerophosphocholine is induced
when choline is supplied to the culture medium and, also, when the
culture temperature is raised from 30 to 37 °C. In strains bearing
mutations in any of the genes encoding enzymes of the CDP-choline
pathway for phosphatidylcholine biosynthesis (CKI1,
choline kinase; CPT1, 1, 2-diacylglycerol choline
phosphotransferase; PCT1, CTP:phosphocholine
cytidylyltransferase), no induction of phosphatidylcholine turnover and
glycerophosphocholine production is seen in response to choline
availability or elevated temperature. In contrast, the induction of
phosphatidylcholine deacylation does occur in a strain bearing
mutations in genes encoding enzymes of the methylation pathway for
phosphatidylcholine biosynthesis (i.e. CHO2/PEM1 and
OPI3/PEM2). Whereas the synthesis of PC via CDP-choline is
accelerated when shifted from 30 to 37 °C, synthesis of PC via the
methylation pathway is largely unaffected by the temperature shift.
These results suggest that the deacylation of PC to GroPC requires an
active CDP-choline pathway for PC biosynthesis but not an active
methylation pathway. Furthermore, the data indicate that the synthesis
and turnover of CDP-choline-derived PC, but not methylation
pathway-derived PC, are accelerated by the stress of elevated temperature.
Two separate biosynthetic pathways lead to the production of
phosphatidylcholine (PC)1 in
Saccharomyces cerevisiae. The stepwise methylation of
phosphatidylethanolamine (PE) to PC occurs through the action of a pair
of methyltransferases encoded by the CHO2/PEM1
and OPI3/PEM2 genes (Fig.
1B). The CDP-choline pathway
for PC biosynthesis (in which CDP-choline is an intermediate) consists
of three reactions catalyzed by choline kinase (Cki1p) as follows: CTP,
phosphocholine cytidylyltransferase (Pct1p); and 1,2-diacylglycerol
choline phosphotransferase (Cpt1p), respectively (Fig. 1B).
The CDP-choline pathway requires free choline, which can be obtained
through exogenous sources or as a result of the phospholipase D
(PLD)-mediated hydrolysis of PC. As in yeast, hepatocytes synthesize PC
through both the methylation and the CDP-choline pathways, whereas
other mammalian cell types produce only minor amounts of PC through the
methylation of PE (1).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Potential routes of phosphatidylcholine
turnover and biosynthesis. A, the catabolites produced
upon hydrolysis of PC via PLD, phospholipase C (PLC),
phospholipase A1 and A2
(PLA1 and PLA2), and PLB.
FA, fatty acid; DAG, diacylglycerol.
B, PC can be synthesized by the methylation or the
CDP-choline pathway in S. cerevisiae. Dashed
arrows designate routes of PC turnover. The complete deacylation
of PC results in the generation of GroPC. PLD-mediated hydrolysis of PC
produces free choline that can be recycled via the CDP-choline pathway.
Gene designations for each metabolic step are in
italics next to the appropriate arrow.
PMME, phosphatidylmonomethylethanolamine;
PDME, phosphatidyldimethylethanolamine.
Although the regulation of PC biosynthesis, and phospholipid biosynthesis in general, has been well studied in S. cerevisiae (2), the regulation of phospholipid turnover has gathered much less attention. PC is known to be catabolized by B and D type phospholipases in S. cerevisiae. Phospholipase B (PLB) deacylates PC to produce glycerophosphocholine (GroPC) and free fatty acids, whereas PLD hydrolyzes PC to phosphatidic acid (PA) and choline (Fig. 1A). Three PLB genes (PLB1, PLB2, and PLB3) have been identified in Bakers' yeast (3, 4). Although Plb1p appears to be primarily responsible for the GroPC released into the culture medium (3, 4), the roles of the three PLB enzymes in intracellular GroPC production have not been analyzed previously. Only one PLD gene, PLD1 (SPO14), has been identified (5-7), although other PLD-encoding genes have been postulated (8, 9). Pld1p has been shown to hydrolyze PC in vivo, to be activated when Sec14p (the phosphatidylinositol (PI)/phosphatidylcholine (PC) transfer protein) is inactivated (10, 11), and to be required for sporulation (12).
The synthesis and turnover of PC are highly regulated in all eukaryotic cells. The importance of this regulation is illustrated by the many pathological and physiological events associated with alterations in PC metabolism. Blockage of the CDP-choline pathway, either genetically (44) or by using enzyme inhibitors (45), causes apoptosis in several mammalian cell types. Elevated levels of GroPC are found in Alzheimer's disease brain compared with normal controls (46, 47). Oncogenic transformation results in alterations in the activity of enzymes involved in PC biosynthesis and turnover, thus affecting the cellular levels of Cho-P and GroPC (14, 48, 49). The existence of regulatory mechanisms that coordinate PC biosynthesis with PC hydrolysis has been suggested by a number of studies (13-17). In particular, overexpression of CTP:phosphocholine cytidylyltransferase (a key regulatory enzyme of the CDP-choline pathway) in COS cells (17), Chinese hamster ovary cells (16), or HeLa cells (15) results in increased PC biosynthesis and increased PC degradation to GroPC, thereby maintaining PC homeostasis. There is also evidence to suggest that GroPC produced through PC deacylation may inhibit lysophospholipid hydrolysis, providing a feedback mechanism for reducing the rate of phospholipid degradation (50).
Studies in S. cerevisiae indicate not only that phospholipid
synthesis and turnover are highly integrated but that PC metabolism, in
particular, plays a central role in the regulation of this metabolic
network. Many of the phospholipid biosynthetic genes are
transcriptionally repressed by the addition of inositol to the culture
medium (38). Additionally, regulation in response to inositol
availability requires ongoing PC biosynthesis (38), suggesting that a
signal emanating from the metabolism itself is responsible for
controlling the transcription of the coordinately regulated
phospholipid biosynthetic genes. By using mutants blocked at various
steps of PC biosynthesis and mutants exhibiting alterations in PC
turnover, it has been shown that conditions that result in elevated
PLD-mediated turnover of PC, and therefore, elevated levels of PA or a
closely related metabolite cause constitutive expression of the
phospholipid biosynthetic genes (10, 11). These results and others
indicate that delineation of the molecular mechanisms controlling
phospholipid metabolism will require a detailed knowledge of the
factors affecting all pathways for PC synthesis and degradation. In the
present study we continue the analysis of PC metabolism, focusing on
the deacylation of PC to GroPC. In particular, we have employed
S. cerevisiae strains bearing mutations in the genes
encoding enzymes of the CDP-choline and the methylation pathways for PC
biosynthesis to examine how the route and extent of PC biosynthesis are
coupled to the turnover of PC to GroPC.
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EXPERIMENTAL PROCEDURES |
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Culture Conditions and Strains--
Strains were maintained on
YEPD medium (1% yeast extract, 2% Bactopeptone, 2% glucose, Difco
YPD Broth). All experiments were performed in chemically defined
synthetic media (18) without inositol and containing 20 µM, 1 mM, or 10 mM choline, as
detailed below. Where indicated, the media also contained
[14C]choline or [14C]methylmethionine
(American Radiolabeled Chemicals, Inc.). The cki1 strain
(MATa, ura3, his3, cki1::HIS3) and its congenic
wild type, WT-A (MATa, ura3, his3, trp1), were constructed
as described (11). The PCT1 gene of WT-A (nucleotides
41-2274) was replaced with the dominant resistance marker
KanMX (19) by a one-step gene replacement to produce
pct1
(MATa, ura3, his3, trp1,
pct1::KanMX). Correct placement of the
KanMX marker at the PCT1 locus was verified by
polymerase chain reaction. The cpt1
strain (MATa
leu2 ura3 trp1 his3 cpt1::LEU2) and its congenic wild
type, WT-B (MATa leu2 ura3 trp1 his3), were isolated by
crossing HJ100 (MATa his3 leu2 ura3 trp1
cpt1::LEU2 ept1::URA3) (18) × JAGWT
(MAT
his3 leu2 trp1 ura3),
sporulating the resulting diploid, and performing tetrad analysis. The
cho2
opi3
strain (MAT
his3 ade2 leu2 ura3 cho2::LEU2
opi3::URA3) and its congenic wild type, WT-C
(MAT
leu2 ade2 ura3), were isolated by
crossing SH414 (MATa opi3::URA3 ade2 can1 his3 leu2
trp1 ura3) × SH458 (MAT
ura3 leu2 his3
cho2::LEU2), sporulating the resulting diploid, and
performing tetrad analysis. The following strains and their isogenic
wild types were generously provided by Fritz Paltauf, Technical
University, Graz: plb1
(MAT
his3 trp1
leu2 ade2 ura3 plb1::URA3), plb2
(MATa his3 trp1 leu2 ade2 ura3 plb2::KanMX),
plb3
(MAT
his3 trp1 leu2 ade2 ura3
plb3::KanMX), plb1
plb2
plb3
(MATa his3 trp1 leu2 ura3 plb1::URA3
plb2::KanMX plb3::KanMX).
Analysis of [14C]Choline-labeled PC
Turnover--
Cells were grown to uniform labeling in medium
containing 1 µCi/ml [14C]choline (20 µM),
harvested in logarithmic phase, washed twice in fresh nonradioactive
medium, and inoculated at A600 = 0.1 in medium
containing 10 mM nonradioactive choline. At various times, 1-ml aliquots of the cultures were removed, and the cells were pelleted
by centrifugation. The supernatant was saved as the "medium" fraction. The cell pellet was suspended in 0.5 ml of 5%
trichloroacetic acid and incubated on ice for 20 min with occasional
vortexing. Following centrifugation, the supernatant containing the
acid extract was saved as the "intracellular water-soluble
fraction" of the cell (20). The cell pellet was suspended in 0.5 ml
of 1 M Tris buffer, pH 8, centrifuged, and the resulting
supernatant was added to the intracellular water-soluble
fraction, effectively neutralizing the acidic extract. The cell
pellet was saved as the "membrane" fraction. The recovery of total
counts (medium, membrane, and intracellular water-soluble fraction) at
each time point was 90%.
Phosphatidylcholine Analysis-- Phospholipids were extracted from the membrane fraction as described previously (21). Radiolabeled phosphatidylcholine was resolved by SG81 (Whatman) paper chromatography in chloroform:methanol:NH4OH (66:17:3) and the spot excised, and concentrations were determined by liquid scintillation counting. Alternatively, PC content was determined by subjecting the chromatogram to PhosphorImager (Molecular Dynamics) analysis.
Analysis of Choline-containing Water-soluble Metabolites-- The choline-containing compounds (i.e. choline, choline-phosphate (Cho-P), and GroPC) in the medium and the intracellular water-soluble fractions were separated by either TLC (22) or ion-exchange chromatography (23), and concentrations were determined by liquid scintillation counting.
Determination of [14C]Choline and [14C]Methionine Incorporation into PC-- Cells were grown to mid-logarithmic phase in synthetic medium containing 1 mM choline. The cultures were split into two flasks; the A600 of each was adjusted to 0.5, one flask incubated at 30 °C and the other flask at 37 °C. After 15 min of incubation for temperature adjustment, 1 µCi/ml of either [14C]choline or [14C]methionine was added to the cultures. At 20-, 40-, and 60-min intervals, 900-µl aliquots of the cultures were removed to tubes containing 100 µl of 50% trichloroacetic acid to kill the cells, and the tubes were incubated on ice for 30 min. Radiolabeled PC was extracted and quantified as described above.
Measurement of Water-soluble Metabolites by NMR--
For
cultures grown at 30 °C in plus and minus 1.0 mM choline
media, 1-liter cultures were grown overnight to mid-logarithmic phase
(A600 0.5-0.7). For cultures grown under
temperature shift conditions, strains grown overnight at 30 °C in 10 µM choline were diluted into 500 ml of media
(A600 0.2) containing 10 mM choline
at the requisite temperature of either 30 or 37 °C and shaken for
6 h. To harvest the cells, the cultures were rapidly cooled in an
ice bath, the cells collected by centrifugation, washed with water, and
the wet paste placed at 70 °C. The frozen cell paste was treated
with 2 volumes of 1 N perchloric acid at 5 °C, vortexed,
and freeze-thawed 2 times (24). After centrifugation, the supernatant
was neutralized with 2.5 N KOH, centrifuged to remove the
precipitated KClO4, and the solution lyophilized. The dried
samples were dissolved in 0.8 ml of 50 mM Tris, 10 mM EDTA, pH 8.0, plus 10% D2O as an internal
lock source. 31P NMR analysis was performed on 0.6 ml of
the sample plus 20 µl of triethylphosphate (TEP) as an internal
standard for both chemical shift and calibration (final concentration,
0.16 mM). The 31P NMR spectra were acquired
using a 300-MHz Bruker DXR spectrometer with a multinuclear probe set
to 121.5 MHz for phosphorus. The spectra were accumulated for 1 h
to ensure sufficient signal and processed with integration using Bruker
software. Metabolites were identified by comparison to known compounds
and published chemical shifts (25).
Mass Spectrometry Analysis of PC--
Strains grown to an
A600 of 0.5-0.7 in 25-50 ml of the indicated
media were harvested, washed with water, and suspended in 5%
trichloroacetic acid for 30 min on ice. The cells were harvested and
washed two times with water, and 50 µg of
dimyristoylphosphatidylcholine was added to the cell pellet as a
standard. Phospholipids were extracted as described (21), and the dried
lipid extract was dissolved to a concentration of ~10 pmol of lipid
species/µl in CH3OH/CHCl3 (2:1) containing 5 mM LiOH (26). Electrospray ionization-mass spectrometry (ESI-MS) (Finnigan LCQ) was used to determine the molecular species of the cellular PCs.
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RESULTS |
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Previous reports have shown that PC in S. cerevisiae is hydrolyzed by PLD to produce PA and choline (10, 11) and by PLB to produce GroPC and fatty acids (3, 4, 27). However, the factors controlling these routes of PC degradation are largely unstudied. Thus, we undertook a thorough analysis of the regulation of PC turnover in response to choline availability, temperature, and genetic perturbations of the biosynthetic routes for PC production.
PC Turnover in Wild-type Cells Grown at 30 °C--
Wild-type
cells prelabeled with [14C]choline, washed free of
radioactivity, and chased with excess choline at 30 °C (Fig.
2A), exhibited a pattern of
label movement indicative of PLD-mediated turnover. Radiolabel
associated with PC slowly diminished, and a concomitant amount of label
appeared in the medium, primarily as free choline (Table I, part B).
The appearance of labeled choline in the medium suggested that the
choline produced through PLD-mediated turnover exited the cell, as is
known to occur in mammalian cells (28). Since S. cerevisiae
rapidly incorporates free choline into PC via the CDP-choline pathway,
the nonradioactive choline chase was necessary to dilute out the
[14C]choline produced through PC turnover and, thereby,
obtain an accurate assessment of the turnover.
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PC Turnover in Wild-Type Cells Grown at 37 °C-- In contrast to the PC turnover observed at 30 °C, a wild-type strain uniformly labeled with [14C]choline at 30 °C and then shifted from 30 to 37 °C in the presence of a cold choline chase exhibited massive PC degradation (Fig. 2B). The label lost from PC appeared in the intracellular fraction, primarily as GroPC (Table I, part A). Since there is no evidence for synthesis of GroPC de novo in any eukaryotic cell (29, 30), the shift from 30 to 37 °C is most likely to have induced the deacylation of PC to GroPC. These patterns of PC turnover occurred in all three wild-type strains that were tested (data not shown).
Effect of CDP-choline Pathway Mutations on PC Turnover at
37 °C--
A mutant lacking choline kinase (cki1),
the first enzyme of the CDP-choline pathway for PC biosynthesis,
incorporates [14C]choline into PC at 10% of the
wild-type level (10). Even though the cki1
mutant lacks
choline kinase, the action of an ethanolamine kinase that has some
specificity for choline (31) can explain the minor degree of
incorporation found. This mutant, which was used to monitor the effect
of decreased production of CDP-choline pathway-derived PC, exhibited a
pattern of radiolabel distribution different than that of the wild
type. Taken as a percentage of total choline-associated radiolabel, the
cki1
mutant contained less intracellular GroPC than wild
type at 30 °C, and neither PC degradation nor GroPC levels increased
at 37 °C when 10 mM choline was present (Table II). One
interpretation of this result is that Cki1p itself is required for
cells to induce GroPC formation in response to temperature elevation.
Alternatively, the CDP-choline pathway, in general, may be necessary
for the response. To distinguish between these possibilities, a
cpt1
deletion mutant, which lacks the enzyme responsible
for the final step of the CDP-choline pathway for PC biosynthesis (Fig.
1), but which can incorporate [14C]choline label into PC
due to the action of an ethanolamine phosphotransferase (31), was also
analyzed. The cpt1
mutant, like the cki1
mutant, was unable to induce PC deacylation when shifted from 30 to
37 °C (data not shown), suggesting that it is a functional
CDP-choline pathway that is required to induce PC deacylation upon
temperature elevation. The pct1
mutant could not be
analyzed in this manner, as it incorporated no
[14C]choline label into PC.
Effect of Choline Supplementation and Temperature Elevation on
GroPC Levels in a Wild-type Strain as Analyzed by 31P NMR
Spectroscopy--
To explore further the role of the CDP-choline
pathway for PC biosynthesis in PC deacylation, 31P NMR
spectroscopy was employed, as it overcomes the drawbacks associated
with radiolabeling cells with [14C]choline.
[14C]Choline labeling monitors only the PC derived from
the CDP-choline pathway and not the PC derived from the methylation of
PE. Furthermore, the labeling of CDP-choline pathway mutants with
[14C]choline is necessarily limited, as the incorporation
of choline into PC depends upon the CDP-choline pathway. Finally,
following [14C]choline-labeled PC turnover requires a
cold choline chase to prevent the labeled metabolites from being
rapidly recycled into PC. 31P NMR spectroscopy provides a
quantitative method of measuring the intracellular levels of all
phosphate-containing metabolites produced through PC turnover and does
not require the presence of exogenous choline. A typical
31P NMR spectrum is shown in Fig.
3.
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By using 31P NMR spectroscopy, the effects of both choline
supplementation and temperature elevation on the intracellular
concentration of GroPC were analyzed. As shown in Fig.
4, a wild-type strain grown in the
presence of exogenous choline exhibited ~7-fold more intracellular
GroPC than did the same strain grown in the absence of choline
supplementation. In addition, a wild type strain shifted from 30 to
37 °C in choline-containing medium exhibited a further 4-fold
increase in intracellular GroPC (Fig. 5),
in general agreement with the results obtained through radiolabeling
(Fig. 2 and Tables I and II).
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Effect of Choline Supplementation and Temperature Elevation on
GroPC Levels in CDP-choline Pathway Mutants as Analyzed by
31P NMR Spectroscopy--
The mutants (cki1,
pct1
, or cpt1
) bearing deletions in genes
encoding the CDP-choline pathway enzymes exhibited no increase in GroPC
levels in response to choline supplementation when examined by
31P NMR spectroscopy (Fig. 4). The pct1
and
cpt1
strains did, however, display elevated levels of
Cho-P, as might be expected for strains blocked in the reaction steps
subsequent to the formation of Cho-P by choline kinase. Cho-P levels
were highest in the presence of choline, where increased substrate for
the CDP-choline pathway could lead to the build up of an intermediate,
such as Cho-P, if Pct1p or Cpt1p were absent. In addition to choline
supplementation, the CDP-choline pathway mutants were also largely
unresponsive to temperature elevation. The cki1
and
pct1
strains exhibited no increase in GroPC levels when
shifted from 30 to 37 °C in the presence of choline (Fig. 5).
Although the cpt1
strain did exhibit an increase in GroPC
concentration upon shift to 37 °C, the total amount produced under
these circumstances was ~8-fold less than that seen in the wild-type
strain (Fig. 5). The pct1
and cpt1
strains
also exhibited increased levels of Cho-P at both 30 and 37 °C,
consistent with their being blocked in the reaction steps subsequent to
the formation of Cho-P by choline kinase.
Effect of Temperature Elevation on GroPC Levels in a Methylation
Pathway Mutant Grown in Choline-containing Medium as Analyzed by
31P NMR Spectroscopy--
Because disruption of the
CDP-choline pathway resulted in the inability of the cell to induce PC
deacylation in response to choline supplementation or temperature
elevation, we next investigated the effect of blocking the alternate
route for PC biosynthesis, the stepwise methylation of PE. A
cho2 opi3
mutant is blocked in all three
methylation steps converting PE to PC, making it a choline auxotroph.
Interestingly, a cho2
opi3
mutant contained more intracellular GroPC than the congenic wild type when both were
grown in choline-containing medium at 30 °C (Fig.
6A). The amount of GroPC in
the cho2
opi3
mutant increased ~2-fold
when the temperature was shifted to 37 °C (Fig. 6B).
Although the wild-type strain congenic to the cho2
opi3
mutant (Fig. 6A) had roughly half as much
GroPC at 30 °C as did the wild-type strain shown in Fig. 4, the
pattern of PC deacylation in response to temperature shift was the
same, with both strains exhibiting a 3-4-fold increase in GroPC when
the temperature was raised to 37 °C. Since wild-type strains with
different genetic backgrounds can exhibit differences in lipid
composition (32), it is not surprising that the levels of phospholipid
metabolites, such as GroPC, can also vary.
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The cho2 opi3
mutant exhibited roughly
10-fold higher levels of GroPE than any other strain used in these
studies both at 30 and 37 °C (Fig. 6). As expected for a strain that
cannot carry out the methylation steps subsequent to PE formation (Fig.
1), a cho2opi3 mutant was shown previously to contain
elevated levels of PE compared with wild type (33). Thus, the build up
of GroPE seen in the cho2
opi3
mutant may
be a reflection of increased levels of its catabolic precursor, PE.
Increased PC Turnover May be Coupled to an Increase in PC Synthesis
upon Temperature Elevation--
The increased PC turnover observed in
a wild-type strain upon shifting from 30 to 37 °C may be a
reflection of an increase in PC biosynthesis. To test this hypothesis,
the incorporation of [14C]choline and
[14C]methionine into the PC of wild-type cells was
measured at 30 and 37 °C (Fig. 7).
Approximately 2-fold more [14C]choline was found to be
associated with PC at 37 than at 30 °C following 20, 40, and 60 min
of labeling. In contrast, the incorporation of
[14C]methionine into PC was relatively unaffected by
temperature. This result suggests that the two pathways for PC
biosynthesis are differentially affected by temperature elevation.
Although Fig. 7 shows the data obtained for WT-A (the wild type
congenic to cki1, cpt1
, and
pct1
), WT-C (the wild type congenic to cho2
opi3
) was also analyzed with similar results (data not
shown).
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Analysis of PC Molecular Species by Mass Spectroscopy--
Since
the turnover and synthesis of CDP-choline-derived PC and methylation
pathway-derived PC appeared to be differentially regulated, we
hypothesized that their fatty acid contents might also differ. To test
this possibility, the molecular species of PC were analyzed by mass
spectrometry in pct1 (which makes all of its PC via
methylation of PE), cho2
opi3
(which makes
all of its PC via the CDP-choline pathway), and their
congenic wild-type strains (Table III). Although no drastic alteration
in fatty acid composition among the strains was observed, the
pct1
strain appeared to have a somewhat higher percentage
of C32:2 PC species (32%) at the expense of C32:1 PC species (26%) as
compared with the wild-type strain (C32:2, 25%; C32:1, 35%).
Effect of PLB1, PLB2, and PLB3 Deletion Mutations on GroPC
Production--
The production of GroPC presumably occurs through the
action of a PLB or a phospholipase A1 or A2 in
conjunction with a lysophospholipase. Based on homology to a PLB from
Penicillium notatum (34), three S. cerevisiae
PLB-encoding genes have been characterized, PLB1 (3),
PLB2 (4), and PLB3 (4). The production of intracellular GroPC in a plb1 mutant, a plb2
mutant, a
plb3
mutant, and in a plb1
plb2
plb3
triple mutant was
indistinguishable from wild type when 14C-labeled cells
were chased with excess choline at both 30 and 37 °C (data not
shown). Thus, the PLB homologs identified to date (3, 4) do not appear
to be involved in the regulated PC deacylation reported here. Clearly
other deacylating phospholipases must exist in yeast, although their
corresponding genes have yet to be characterized. An example of
potential phospholipase-encoding genes can be found using the Yeast
Proteome Data Base (35), which identifies open reading frames of
YDR444w, YJR107c, and YOR084w as having lipase/esterase motifs
and ORF YNL040w as having a PLA2 active site
signature (36, 37).
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DISCUSSION |
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PC turnover in S. cerevisiae varies as a function of
the environmental conditions in which the cells are grown and as a
function of the route by which the PC is synthesized. We demonstrate
that wild-type S. cerevisiae cells grown in the presence of
exogenous choline exhibit severalfold greater concentration of GroPC
than cells grown in the absence of exogenous choline (Fig. 4).
Furthermore, we demonstrate that a strain bearing a deletion mutation
in any gene encoding an enzyme of the CDP-choline pathway for PC
biosynthesis (cki1, pct1
, or
cpt1
) is unable to induce PC deacylation and generate
GroPC in response to choline availability (Fig. 4). In contrast, a
disruption in the alternate route for PC biosynthesis, the stepwise
methylation of PE, has the opposite effect. A cho2
opi3
mutant grown in the presence of choline exhibits
roughly 4-fold more GroPC than does its congenic wild type (Fig. 6).
Temperature also affects PC turnover, as wild-type cells shifted from
30 to 37 °C in choline-containing medium exhibit rapid PC
deacylation, resulting in intracellular GroPC levels roughly 4-fold
greater than those observed at 30 °C (Figs. 2 and 5 and Table I).
However, cki1
and pct1
strains are unable
to induce PC deacylation in response to temperature elevation (Table
II and Fig. 5), and the cpt1
strain exhibits only a modest increase. Disruption
of the methylation pathway, again, leads to a different result, as
shown by a cho2
opi3
mutant that exhibits
roughly 2-fold more GroPC when shifted from 30 to 37 °C (Fig. 6). An
analysis of PC biosynthesis in response to temperature elevation
indicates that although the CDP-choline pathway for PC biosynthesis is
induced at 37 °C, the methylation pathway is not (Fig. 7). Finally,
mass spectrometry analysis (Table III)
indicates that there are no major differences in the fatty acyl
composition of PC generated via the CDP-choline and methylation
pathways. These findings suggest two major conclusions as follows. (i)
The catabolism of PC to GroPC is dependent upon an active CDP-choline
pathway for PC biosynthesis but not an active methylation pathway. In
fact, disruption of the methylation pathway for PC synthesis seems to
enhance the deacylation of both PC and PE in cells grown at 30 °C.
(ii) The stress of temperature elevation accelerates the synthesis and
turnover of CDP-choline derived PC but not methylation-derived PC.
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One explanation for the observed increase in GroPC that occurs upon choline supplementation in S. cerevisiae (as well as in cultured rat hepatocytes (13)) is that having a functional CDP-choline pathway in the presence of exogenous choline results in excess biosynthesis of PC that must be counterbalanced by increased PC degradation. Since exogenous choline would only increase PC synthesis in strains having a functional CDP-choline pathway, the finding that choline supplementation does not increase PC deacylation in CDP-choline pathway mutants is consistent with this hypothesis (Fig. 4). Also in agreement with this interpretation is the finding that overexpression of CTP:phosphocholine cytidylyltransferase (the rate-limiting step of the CDP-choline pathway) in a number of mammalian cell types (15-17) results in increased PC biosynthesis and increased PC degradation to GroPC. In extending these studies to yeast, we demonstrate that the presence of exogenous choline only results in increased PC deacylation if it is successfully converted to PC. Mutants blocked at any step of the CDP-choline pathway (giving rise to the accumulation of pathway intermediates) do not exhibit increased PC catabolism when choline is added.
Temperature elevation from 30 to 37 °C also causes wild-type yeast cells to undergo increased PC deacylation and concomitant GroPC production. Although strains bearing mutations in the CDP-choline pathway for PC biosynthesis do not produce increased GroPC upon temperature elevation, a strain blocked in the methylation pathway does. Furthermore, temperature elevation causes an increase in PC biosynthesis via the CDP-choline pathway but not through the methylation pathway. These results are also in agreement with the hypothesis that conditions that cause an increase in PC biosynthesis result in an increase in PC deacylation. According to this interpretation of the data, the CDP-choline pathway mutants do not experience an increase in PC deacylation upon shifting to 37 °C, because there is no increase in PC biosynthesis upon shifting to 37 °C. This interpretation also implies that the cell has a means of monitoring its PC levels and using that information to induce PC turnover when appropriate.
Changes in phospholipid metabolism associated with temperature elevation have been well documented (24, 33, 39, 40). It has long been speculated that such changes are due to a need to alter the cellular phospholipid composition to one more favorable to higher temperature. If that hypothesis is correct, it is indeed surprising that only the CDP-choline pathway is affected by temperature, since the cell would presumably also need to alter its phospholipid composition when PC is being made primarily through the methylation pathway. Furthermore, if temperature elevation leads to the activation of a phospholipase(s) that catalyzes PC deacylation, our data would suggest that methylation pathway-derived PC is not a substrate for those phospholipases.
The insensitivity of methylation pathway-derived PC to deacylation upon
temperature shift could be explained if the PC generated by these two
pathways differs in some regard, perhaps in their subcellular location
or in the composition of their fatty acyl chains. Since neither the
distribution of CDP-choline derived versus methylation
pathway-derived PC is known nor the location of the phospholipase(s)
involved, the importance of subcellular location is currently
impossible to address. With regard to fatty acid composition, we report
(Table III) that a small shift toward C32:2 PC species at the expense
of C32:1 PC species occurs in the cpt1 mutant (which
makes its PC solely through the methylation pathway). It is unclear
whether this rather minor change contributes to the drastic difference
in deacylation sensitivity observed between PC generated by the two
pathways. Waite and Vance (41) recently reported finding only
negligible differences in the fatty acid composition of PC produced
through the methylation and CDP-choline pathways in a Chinese hamster
ovary cell line. In contrast, DeLong et al. (42) report that
in rat primary hepatocytes, CDP-choline-derived PC is composed mainly
of medium chain saturated species, whereas methylation pathway-derived
PC is more diverse and contains more polyunsaturated species.
Previous studies have suggested that the pathways for PC biosynthesis
in S. cerevisiae are not functionally identical. Mutations in the genes (CHO2 or OPI3) encoding the
phospholipid methyltransferases that convert PE to PC, but not
mutations in genes encoding enzymes of the CDP-choline pathway for PC
biosynthesis, result in an overproduction of inositol (Opi)
phenotype. An Opi
phenotype indicates overexpression of
INO1, which encodes inositol-1-phosphate synthase, the
rate-limiting step of inositol biosynthesis (38). Next, mutations in
the CDP-choline pathway, but not the methylation pathway, suppress the
temperature-sensitive phenotype of a sec14ts
mutation (43).
PC is the major phospholipid found in eukaryotic cells and the
precursor of various signaling molecules such as PA and diacylglycerol. The regulation of PC metabolism is crucial to cell function, as evidenced by the existence of physiological and pathological events (apoptosis (44, 45) and oncogenic transformation (14, 48, 49), for
example) associated with alterations in PC metabolism. Future studies
will focus on the identification of the phospholipase(s) involved in PC
deacylation and on the determination of how the cell senses the amount
of its PC.
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ACKNOWLEDGEMENTS |
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We thank Susan A. Henry for her generous financial support. We thank Chien Ho for use of the Bruker WH300 spectrometer and Virgil Simplaceanu for providing technical assistance with the NMR analysis. We thank the Center for Molecular Analysis at Carnegie Mellon University (supported by National Science Foundation Grants CHE-9808188 and DBI-9729351) for the use of the mass spectrometer. We thank Susan A. Henry, Toon DeKroon, and Christopher R. McMaster for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants GM-19629 (to Susan A. Henry) and GM59817 (to J. L. P-V).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 412-268-5122; Fax: 412-268-7129; E-mail: jp5s@andrew.cmu.edu.
Published, JBC Papers in Press, November 14, 2000, DOI 10.1074/jbc.M003694200
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ABBREVIATIONS |
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The abbreviations used are: PC, phosphatidylcholine; CDP-choline, cytidine diphosphate-choline; Cho-P, choline-phosphate; GroPC, glycerophosphocholine; PA, phosphatidic acid; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PLB, phospholipase B; PLD, phospholipase D; TEP, triethylphosphate; WT, wild type.
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