In eukaryotic cells, phosphatidylserine (PtdSer)
is synthesized by two distinct synthases on the endoplasmic reticulum
by a base-exchange reaction in which the polar head-group of an
existing phospholipid is replaced with serine. We report the cloning
and expression of a cDNA for mouse liver PtdSer synthase-1. The
deduced protein sequence is >90% identical to that of PtdSer
synthase-1 from Chinese hamster ovary cells and a sequence from a human
myeloblast cell line. PtdSer synthase-1 cDNA was stably expressed
in M.9.1.1 cells which are mutant Chinese hamster ovary cells defective
in PtdSer synthase-1 activity, are ethanolamine auxotrophs, and have a
reduced content of PtdSer and phosphatidylethanolamine (PtdEtn). The
growth defect of M.9.1.1 cells was eliminated, and a normal phospholipid composition was restored in the absence of exogenous ethanolamine, implying that the cloned cDNA encoded PtdSer
synthase. Mouse liver PtdSer synthase-1 was also expressed in McArdle
7777 rat hepatoma cells. In addition to a 3-fold higher in
vitro serine-exchange activity, these cells also exhibited
enhanced choline- and ethanolamine-exchange activities and incorporated
more [3H]serine into PtdSer than did control cells.
However, the levels of PtdSer and PtdEtn in cells overexpressing PtdSer
synthase-1 activity were not increased. Excess PtdSer produced by the
transfected cells was rapidly decarboxylated to PtdEtn and the
degradation of PtdSer, and/or PtdEtn derived from PtdSer, was
increased. Moreover, the CDP-ethanolamine pathway for PtdEtn
biosynthesis was inhibited. These data suggest that (i) cellular levels
of PtdSer and PtdEtn are tightly controlled, and (ii) the metabolism of
PtdSer and PtdEtn is coordinately regulated to maintain phospholipid
homeostasis.
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INTRODUCTION |
Phosphatidylserine
(PtdSer)1 is an amino
phospholipid component of all animal cell membranes, accounting for
~5-10% of membrane phospholipids. In addition to a presumed
structural role in membranes, PtdSer is required for activation of
protein kinase C (1) and for progression of the blood coagulation
cascade (2, 3). PtdSer exposure on the cell surface also serves as a
signal for recognition and removal of apoptotic cells by macrophages
(4, 5). In mammalian cells, PtdSer is synthesized on ER membranes in a
calcium-dependent base-exchange reaction catalyzed by
PtdSer synthases (6). In this reaction, the polar head-group of an existing phospholipid, such as PtdCho or PtdEtn, is replaced by L-serine (Fig. 1). The
pathway by which PtdSer is synthesized in eukaryotes is different from
that in bacteria (7) and Saccharomyces cerevisiae (8, 9), in
both of which PtdSer is synthesized from L-serine, with
CDP-diacylglycerol as the donor of the phosphatidyl group.

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Fig. 1.
Biosynthetic pathways for PtdSer and PtdEtn
in mammalian cells. The abbreviations used are: PSS,
PtdSer synthase; PSD, PtdSer decarboxylase; Etn,
ethanolamine; DAG, diacylglycerol; EK,
ethanolamine kinase; ET, CTP:phosphoethanolamine
cytidylyltransferase; EPT,
CDP-ethanolamine:1,2-diacylglycerol ethanolamine-
phosphotransferase.
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A cDNA encoding PtdSer synthase-1 was cloned from CHO-K1 cells by
complementation of PSA-3 mutant cells that are defective in PtdSer
synthase-1 activity (10). Expression of the cloned cDNA in PSA-3
cells showed that the enzyme catalyzed a base-exchange reaction with
serine, choline, and ethanolamine. Immunoblot analysis (11) revealed
that PtdSer synthase-1 is present in both microsomes and
mitochondria-associated membranes (12). More recently, a cDNA that
encodes PtdSer synthase-2 was also isolated from CHO-K1 cells
(GenBankTM data bank accession number AB004109) (13).
Although the cDNAs for PtdSer synthase-1 and -2 share little
similarity, the corresponding amino acid sequences are 32% identical.
Expression of PtdSer synthase-2 cDNA showed that, as predicted,
this enzyme uses PtdEtn, but not PtdCho, as phosphatidyl group donor.
The PtdSer synthase-2 cDNA also transformed PSA-3 cells, which
lacked PtdSer synthase-1 activity, into PtdSer prototrophs (13).
M.9.1.1 cells are phenotypically similar to PSA-3 cells. In M.9.1.1
cells, the PtdSer synthase activity is ~50% that of parental CHO-K1
cells (14). Consequently, in M.9.1.1 cells that are deprived of
ethanolamine, and therefore are unable to make PtdEtn via the CDP-ethanolamine pathway (Fig. 1), PtdSer and PtdEtn levels are lower
than in CHO-K1 cells. The growth defect of M.9.1.1 cells is overcome
when ethanolamine, PtdSer, lyso-PtdEtn, or PtdEtn are included in the
culture medium. M.9.1.1 cells also do not utilize PtdCho as a substrate
for PtdSer biosynthesis, although PtdEtn is converted into PtdSer
almost as efficiently as in wild-type CHO-K1 cells. Therefore, the
primary defect in M.9.1.1 cells was deduced to be in the
PtdCho-dependent synthesis of PtdSer (14).
PtdSer synthesized on the ER is transported to mitochondria and
decarboxylated to PtdEtn by the enzyme PtdSer decarboxylase (Fig. 1)
(15). Nearly all mitochondrial PtdEtn in CHO cells is synthesized
in situ in mitochondria by this reaction (16). PtdEtn is
also synthesized on the ER by the CDP-ethanolamine pathway (Fig. 1)
(17). In this pathway, ethanolamine is first phosphorylated by
ethanolamine kinase to phosphoethanolamine, which is converted to
CDP-ethanolamine by CTP:ethanolamine-phosphate cytidylyltransferase (ET). In the final step of the pathway,
CDP-ethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase
transfers phosphoethanolamine from CDP-ethanolamine to diacylglycerol
to produce PtdEtn. A third, and quantitatively minor, pathway for the
biosynthesis of PtdEtn is the base-exchange reaction (6). The relative
importance of the two major pathways for PtdEtn biosynthesis appears to
depend on the cell type. In some studies, for example in rat liver (18, 19) and hamster heart (20), PtdEtn has been reported to be synthesized
primarily via the CDP-ethanolamine pathway, although in another study
Yeung and Kuksis (21) found that the decarboxylation of PtdSer was the
major route of PtdEtn biosynthesis in rat liver. Other cells, such as
CHO and BHK-21 cells, synthesize at least 80% of their PtdEtn from
PtdSer decarboxylation, even in the presence of ethanolamine (22, 23).
In all these studies difficulties in measurement of pool sizes and
possible non-homogeneous radiolabeling of lipid precursor pools
complicate interpretation of the data. Several cell types, for example
rat mammary carcinoma cells (24), hybridoma cells (25), and human
keratinocytes (26), have an absolute requirement for ethanolamine for
growth and/or proliferation (27). One cannot, however, conclude that
the ethanolamine requirement of these cells is necessarily for PtdEtn
synthesis, since ethanolamine is also used for the
glycosylphosphatidylinositol anchors of proteins (28).
Little information is available on how PtdSer biosynthesis is regulated
and whether or not the two pathways for PtdEtn synthesis are
coordinately regulated. We have therefore cloned and expressed a
cDNA that encodes mouse liver PtdSer synthase-1. First, we
demonstrate that this cDNA complements the ethanolamine auxotrophy
of M.9.1.1 mutant CHO-K1 cells. We also show that when PtdSer synthesis
is increased severalfold in hepatoma cells overexpressing mouse liver PtdSer synthase-1 cDNA, PtdSer and PtdEtn levels remain the same as
in control cells. Apparently, as compensation for overexpression of
PtdSer synthase-1 activity, the catabolism of PtdSer and/or PtdSer-derived PtdEtn is increased, and the CDP-ethanolamine pathway for PtdEtn biosynthesis is inhibited.
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EXPERIMENTAL PROCEDURES |
Materials--
CHO-K1 cells and McArdle 7777 cells were obtained
from the American Type Tissue Culture Collection. The radiochemicals
[3-3H]serine, [1-3H]ethanolamine,
[methyl14C]choline, and
[32P]ATP were from Amersham Pharmacia Biotech (Oakville,
Ontario, Canada).
Cytidine-5'-diphospho-[1,2-14C]ethanolamine was from ICN
Radiochemicals (Montreal, Quebec, Canada). Thin layer chromatography
plates were purchased from BDH Chemicals. Fetal bovine serum, horse
serum, tissue culture media, and DNA modifying enzymes were purchased
from Life Technologies, Inc. Authentic phospholipid standards were from
Avanti Polar Lipids (Birmingham, AL). Sodium orthovanadate was
purchased from Calbiochem. The phosphatase 1/2A inhibitor, microcystin,
was a generous gift from Dr. C. Holmes, University of Alberta. All
other chemicals were from Sigma or Fisher.
Oligonucleotides were synthesized as primers for PCR. Modified
gt11
forward (FP) and reverse (RP) sequencing primers, and two gene-specific
primers based on the CHO-K1 PtdSer synthase-1 cDNA sequence (10),
were synthesized. The oligonucleotide 520S was complementary to the
sense strand of PtdSer synthase-1, and 913AS was complementary
to the antisense strand (FP, GCGACGACTCCTGGAGCCCG; RP,
TGACACCAGACCAACTGGTAATG; 520S, GGCCATGAAGGCCTTGTTGATCCGTAGT; 913AS,
TATGAATGTCCTTGAAGCTTGCCCA).
PCR and Cloning--
The cDNA from a
gt11 mouse liver
expression library (CLONTECH, Palo Alto, CA) was
isolated by modification of the method described by Sambrook et
al. (29). Luria-Bertani medium (500 ml) was inoculated with
Y1090R
strain Escherichia coli infected with
phage from the mouse liver cDNA library. Cells were grown at
37 °C until lysis was apparent, after which addition of 10 ml of
chloroform and further incubation at 37 °C for 10 min completed
lysis. DNase I and RNase A were added to the lysed cultures at final
concentrations of 1 and 5 µg/ml, respectively, and incubated at
37 °C for 30 min. Polyethylene glycol and NaCl were added at final
concentrations of 10% and 1 M, respectively. Cultures were
maintained at 4 °C for 1 h to precipitate the phage, which were
isolated by centrifugation for 10 min at 10,000 × g.
The phage-containing pellet was resuspended in 10 mM
Tris-HCl buffer (pH 8.0) containing 10 mM EDTA and 0.5% SDS, followed by incubation at 68 °C for 20 min to release the
phage cDNA. The DNA solution was extracted three times with an
equal volume of phenol:chloroform (1:1), and the DNA was precipitated by addition of NaCl (to a final concentration of 0.25 M)
and 2 volumes of 95% ethanol. The DNA was pelleted by centrifugation for 10 min at 10,000 × g. The pellet was washed with
80% ethanol, and the DNA was resuspended at a final concentration of 1 µg/µl in 10 mM Tris-HCl buffer (pH 8.0) containing 1 mM EDTA and used as a template for PCR reactions. The PCR
reaction mixture (50 µl) contained 25 ng of
phage cDNA, 5 µl of the supplied 10-fold concentrated reaction buffer (Panvera,
Madison, WI), 2.5 mM MgCl, 0.25 mM of each
nucleotide triphosphate, 10 pmol of
gt11 sequencing primer (FP or
RP), and 10 pmol of PtdSer synthase-1-specific primers (520S or 913AS),
as well as 2.5 units of TaKaRa Ex Taq DNA polymerase (Panvera, Madison, WI). The PCR reaction was performed for 30 cycles at
94 °C for 1 min, 65 °C for 2 min, 72 °C for 1.5 min, and a
final 10-min extension at 72 °C. The products of the PCR reaction
were separated by agarose gel electrophoresis. The DNA fragments were
eluted using a gel extraction kit (Qiagen, Missisauga, Ontario, Canada)
and cloned into the pCRII vector (Invitrogen, San Diego, CA). Inserts
were sequenced using the dideoxy termination/polyacrylamide gel method
(30) by the DNA Core facility, at the University of Alberta. The
full-length clone was sequenced in both directions.
Construction of a Full-length PtdSer Synthase-1 cDNA and
Expression of Mouse Liver PtdSer Synthase-1 cDNA in Mammalian
Cells--
The 950-bp 5' end generated by 913AS/FP primers was ligated
into the KpnI/HindIII sites of pBluescript (SK+)
(Stratagene). The 1840-bp 3' end generated by 520S/RP primers was
inserted into the ApaI/SpeI sites of pBluescript
(SK+). The 3' end was then digested with
HindIII/NotI and joined in-frame to the 5'
fragment. A 2-kilobase pair fragment containing the entire coding
region of PtdSer synthase-1 was inserted into the eukaryotic expression vector pRc/CMV (Invitrogen).
M.9.1.1 cells (14) (a gift from Dr. D. R. Voelker, National Jewish
Research Center, Denver, CO) and McArdle 7777 rat hepatoma cells were
grown in a 5% CO2 incubator at 37 °C. M.9.1.1
cells were routinely maintained in Ham's F-12 medium containing 10% delipidated fetal bovine serum (31) and 20 µM
ethanolamine. McArdle 7777 cells were maintained in Dulbecco's
modified Eagle's medium containing 10% fetal bovine serum and 10%
horse serum. Both types of cells were transfected by the calcium
phosphate precipitation method (32) using 10 µg of DNA. For selection of stable transfectants, cells were cultured in medium containing 400 µg/ml G418. Individual colonies were isolated, and once cell lines
were established the concentration of G418 was reduced to 200 µg/ml.
The control cells for the experiments with M.9.1.1 and McArdle 7777 were transfected with vector alone.
Enzyme Activities--
PtdSer synthase activity was measured as
described previously (33). Briefly, cells were scraped from dishes and
disrupted by sonication with a probe sonicator, 2 × 10 s, in
10 mM HEPES buffer (pH 7.5) containing 0.25 M
sucrose. Lysates were centrifuged for 2 min at 600 × g
to pellet cellular debris, and PtdSer synthase-1 activity was measured
in the supernatant using [3-3H]serine,
[1-3H]ethanolamine, or
[methyl-14C]choline. The activities of
the three enzymes of the CDP-ethanolamine pathway were determined as
described previously (33-35). The products of the ethanolamine kinase
and ET assays were separated by thin layer chromatography on silica gel
G-60 thin layer plates in the solvent system methanol, 0.6% NaCl,
NH4OH (10:10:1, v/v) and identified by comparison to
authentic standards. In some experiments, as indicated, ET was assayed
in lysates that had been prepared in the presence of the phosphoprotein
phosphatase inhibitors microcystin (5 µM) (36), sodium
orthovanadate (1 mM), and sodium fluoride (10 mM).
Radioabeling of Cells--
PtdSer and PtdEtn were radiolabeled
by incubation of cultured cells with [3-3H]serine (5 µCi/ml). For experiments in which cells were labeled with
[1-3H]ethanolamine, radioactivity in PtdEtn was
determined. In addition, water-soluble intermediates of the
CDP-ethanolamine pathway were separated by thin layer chromatography on
silica gel G-60 thin layer plates in a combination of two different
solvent systems. Ethanolamine and phosphoethanolamine were separated in
the solvent system methanol, 0.6% NaCl, NH4OH (10:10:1,
v/v), whereas CDP-ethanolamine and glycerophosphoethanolamine were
separated in the solvent system ethanol, 0.9% NaCl, NH4OH
(80:10:26, v/v). Ethanolamine, phosphoethanolamine, CDP-ethanolamine,
and glycerophosphoethanolamine standards were added as carriers. The
bands corresponding to these intermediates were scraped, and
radioactivity was measured.
Other Analyses--
Phospholipids were extracted from cells by
the method of Bligh and Dyer (37) and separated by thin layer
chromatography on silica gel G-60 thin layer chromatography plates in
the solvent chloroform:methanol:acetic acid:formic acid:water
(70:30:12:4:2, v/v). Phospholipids were visualized by exposure to
iodine vapors and identified by comparison with authentic standards.
The phosphorus content of each phospholipid was determined as described
previously (38). For measurement of the cellular content of
diacylglycerols, lipids were separated by thin layer chromatography in
the solvent system heptane:isopropyl ether:acetic acid (60:40:4, v/v).
The thin layer plate was immersed in a solution of cupric
acetate/phosphoric acid and then heated at 180 °C for 5 min to
visualize the lipids (39). The amount of diacylglycerol was
determined by densitometric scanning of the bands and comparison with
known amounts of standard diacylglycerol. The triacylglycerol content
of the cells was determined in a total lipid extract using the
Triglyceride E Kit (Wako Chemicals, Richmond, VA). Protein
concentrations were determined by the BCA method (Pierce).
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RESULTS |
Isolation of the cDNA for Mouse Liver PtdSer
Synthase-1--
PCR cloning was used to isolate the cDNA for
PtdSer synthase-1 from mouse liver. cDNA from a
gt11 cDNA
library containing 2 × 106 clones was isolated and
used as a template for PCR reactions. The 5' and 3' ends of PtdSer
synthase-1 were amplified in two separate PCR reactions. One fragment,
520S/RP, contained the 3' end, and the other fragment, 913AS/FP,
contained the 5' end. These two fragments possessed a 235-bp
overlapping region and were ligated at a common HindIII
restriction site. The assembled PtdSer synthase-1 clone was 2362 bp in
length with the longest open reading frame encoding a 473-amino acid
protein with a predicted molecular weight of 55,617 (Fig.
2). The 5'-untranslated region contained
an in-frame stop codon suggesting that the clone was full length.

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Fig. 2.
Nucleotide and predicted amino acid sequences
of mouse liver PtdSer synthase-1 cDNA. The translational start
codon is numbered +1. The first ATG and the stop codon of
the largest open reading frame are underlined. An in-frame
stop codon (TAG) in the 5'-untranslated region is in
boldface. Five putative transmembrane regions are indicated
by broken underlines.
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The PtdSer synthase-1 sequence lacks a typical N-terminal signal
sequence for targeting the protein to the ER but contains five putative
membrane spanning regions according to hydropathy plot analysis (40).
In addition, the C terminus contains the Lys-Lys motif that has been
proposed to be an ER membrane retention signal (41). These findings, as
well as the observation that immunoreactive PtdSer synthase-1 protein
is present in the ER of CHO cells (11), are consistent with the
enzymatic activity of PtdSer synthase-1 being primarily localized to
the ER (33). A human myeloblast cDNA sequence encoding a putative
PtdSer synthase-1 was identified by sequence analysis from
GenBankTM (accession number D14694). Comparison of the
PtdSer synthase-1 sequences of mouse liver, CHO-K1
(GenBankTM accession number D90468), and human myeloblast
cells revealed a very high degree of conservation across species (Fig.
3). However, the mouse liver PtdSer
synthase-1 cDNA was not homologous to the PtdSer synthase cDNAs
isolated from E. coli, S. cerevisiae, or Bacillus subtilis (7, 8, 42, 43). This lack of homology was
not unexpected since the yeast and bacterial enzymes synthesize PtdSer
by a reaction different from that in mammalian cells and use
CDP-diacylglycerol as a substrate instead of catalyzing a base-exchange
reaction. At the level of their cDNAs, the three mammalian clones
are ~80% identical, whereas their amino acid sequences are >90%
identical (Fig. 3). In contrast, only 30% of the amino acids in the
mouse liver PtdSer synthase-1 are identical to those deduced from the
PtdSer synthase-2 cDNA which has recently been cloned from CHO-K1
cells (13).

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Fig. 3.
Comparison of the predicted amino acid
sequences of PtdSer synthases-1 from CHO-K1 cells, human myeloblastic
cells, and mouse liver. Sequences of PtdSer synthase-1 from CHO-K1
(10) and human myeloblastic cells (GenBankTM accession
number D14694) were aligned with the deduced amino acid sequence of
mouse liver PtdSer synthase-1. Identical amino acids are enclosed
in boxes.
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Complementation of the CHO-K1 Mutant Cells, M.9.1.1--
To
confirm that the isolated mouse liver cDNA encoded PtdSer
synthase-1, we first determined whether or not the cDNA
complemented the growth defect of M.9.1.1 cells and restored the
phospholipid composition of these cells. Voelker and Frazier (14) have
reported that PtdSer synthase activity in M.9.1.1 cells is 50% lower
than in wild-type CHO-K1 cells, an observation that our data confirm (Fig. 4). Stable expression of PtdSer
synthase-1 cDNA in M.9.1.1 cells resulted in an approximately
5-fold increase in the incorporation of [3-3H]serine into
PtdSer in a cell-free assay (Fig. 4). In addition, PtdSer synthase-1
utilized both ethanolamine and choline as substrates for base-exchange
reactions. Compared with control M.9.1.1 cells that were transfected
with vector alone, a 4-fold increase in base exchange activity for the
incorporation of [1-3H]ethanolamine into PtdEtn and a
6-fold increase in the in vitro incorporation of
[methyl-14C]choline into PtdCho were
observed in M.9.1.1 cells expressing mouse liver PtdSer synthase-1
(Fig. 4). Since the cloned mouse liver PtdSer synthase-1 increased the
choline exchange activity, the isolated cDNA does not encode PtdSer
synthase-2 which lacks this activity (13).

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Fig. 4.
Base exchange enzyme activities of
wild-type CHO-K1 cells, M.9.1.1 cells, and control cells expressing
mouse liver PtdSer synthase-1 (M.9.1.1/MLPSS1). Base exchange
activities were measured using [3-3H]serine
(Ser), [1-3H]ethanolamine (Etn),
and [methyl-14C]choline (Cho)
in cellular lysates from CHO-K1 cells (solid bars), control
M.9.1.1 cells transfected with vector alone (open
bars), and M.9.1.1/MLPSS1 cells (stippled bars).
Data are averages ± S.D. of triplicate analyses from one
experiment that was representative of three similar experiments.
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M.9.1.1 cells are defective in the synthesis of PtdSer and,
consequently, in the production of PtdEtn from PtdSer decarboxylation. In these cells ethanolamine (20 µM) is required in the
culture medium to support sufficient PtdEtn synthesis via the
CDP-ethanolamine pathway for normal growth (14). We therefore examined
whether or not M.9.1.1 cells could be transformed into ethanolamine
prototrophs by expression of mouse liver PtdSer synthase-1 cDNA. As
shown in Fig. 5, and as previously
reported (14), M.9.1.1 cells grew in the absence of ethanolamine for
approximately three generations, then died. Addition of 20 µM ethanolamine restored normal growth. However, M.9.1.1
cells expressing PtdSer synthase-1 grew at the same rate regardless of
the presence of ethanolamine (Fig. 5). Thus, expression of PtdSer
synthase-1 cDNA complemented the ethanolamine auxotrophy of M.9.1.1
cells.

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Fig. 5.
Growth of M.9.1.1 cells and M.9.1.1 cells
expressing mouse liver PtdSer synthase-1 (M.9.1.1/MLPSS1 cells).
Control M.9.1.1 cells transfected with vector alone
(squares) and M.9.1.1/MLPSS1 cells (circles) were
plated at a density of 5 × 104 cells/60-mm dish and
cultured in Ham's F-12 medium supplemented with 10% delipidated fetal
bovine serum with (open symbols) or without (solid
symbols) 20 µM ethanolamine. Cells were harvested at
24-h intervals by trypsinization and counted. Data are averages ± S.D. of triplicate analyses from one representative experiment of three
similar experiments. Some error bars are hidden by the symbols.
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Analysis of the phospholipid composition of M.9.1.1 cells revealed that
PtdSer and PtdEtn levels were significantly (57 and 51%, respectively
(p < 0.05)) lower when the cells were cultured in the
absence of ethanolamine than in the presence of ethanolamine (Fig.
6), in agreement with previous data (14).
However, the content of PtdSer and PtdEtn in M.9.1.1 cells expressing
mouse liver PtdSer synthase-1 cDNA, cultured in either the presence or absence of exogenous ethanolamine, was very similar to that of
M.91.1 cells grown in the presence of 20 µM ethanolamine
(Fig. 6). These results are consistent with those from studies by Kuge et al. (10) in which expression of PtdSer synthase-1
cDNA from CHO-K1 cells corrected the growth defect and normalized
the phospholipid composition of PSA-3 cells.

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Fig. 6.
PtdSer and PtdEtn content of control M.9.1.1
cells and M.9.1.1 cells expressing mouse liver PtdSer synthase-1.
Cells were grown in medium supplemented with 20 µM
ethanolamine for 48 h. Medium with (+) or without ( ) 20 µM ethanolamine (Eth) was then added for an
additional 48 h after which cells were harvested, and the content
of PtdSer and PtdEtn was determined. PSS (+) denotes M.9.1.1 cells
transfected with PtdSer synthase-1 cDNA; PSS ( ) denotes control
M.9.1.1 cells transfected with vector alone. Data are averages ± S.D. of triplicate analyses from three independent experiments. *,
statistical significance (p < 0.05) of differences between cells cultured in the presence and absence of 20 µM ethanolamine was evaluated by the Student's
t test.
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Expression of Mouse Liver PtdSer Synthase-1 in McArdle 7777 Cells--
We next expressed PtdSer synthase-1 cDNA in McArdle
7777 rat hepatoma cells that possess endogenous PtdSer synthase-1
activity. Several stably transfected McArdle 7777 cell lines expressing mouse liver PtdSer synthase-1 were generated. We selected one cell line
(designated Mc/PSS1) that exhibited a 2-3-fold increase in serine,
ethanolamine, and choline exchange activities when the cell lysate was
assayed in vitro (Fig. 7).
Although we screened about 50 transfectants, a 3-fold increase in
PtdSer synthase-1 activity was the maximum achieved. One explanation
for this observation is that a higher expression of PtdSer synthase-1
might be lethal. Mc/PSS1 cells and control cells (i.e.
McArdle 7777 cells transfected with vector alone) were labeled with
[3-3H]serine for up to 1 h, and incorporation of
radiolabel into PtdSer and PtdEtn was determined. The total uptake of
[3H]serine was the same in the two cell types. In Mc/PSS1
cells the rate of incorporation of [3H]serine into PtdSer
was 3-fold higher than in control cells, and the rate of incorporation
into PtdEtn was double that of control cells (Fig.
8). These data indicate that the
synthesis of both PtdSer and PtdEtn was increased in Mc/PSS1 cells
compared with control cells. Fig. 9 shows
that the amounts of the major phospholipids were not significantly
different in Mc/PSS1 cells and control cells.

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Fig. 7.
Base exchange activities of control McArdle
7777 cells and McArdle 7777 cells expressing mouse liver PtdSer
synthase-1 (Mc/PSS1). Base exchange activities were measured in
cellular lysates using [3-3H]serine (Ser),
[methyl-14C]choline (Cho), and
[1-3H]ethanolamine (Etn). Solid
symbols, control McArdle 7777 cells transfected with vector alone;
open symbols, Mc/PSS1 cells. Data are averages ± S.D.
of triplicate analyses from three independent experiments.
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Fig. 8.
Incorporation of [3H]serine
into PtdSer and PtdEtn of control cells and Mc/PSS1 cells. Cells
were incubated in medium containing [3-3H]serine (20 µCi/dish). At the indicated times, cells were harvested, and PtdSer
and PtdEtn were extracted and separated by thin layer chromatography.
Open symbols, control McArdle cells transfected with vector
alone; solid symbols, Mc/PSS1 cells. Data are averages ± S.D. of triplicate analyses from one experiment which was repeated twice with similar results. Some error bars are obscured by
the symbols.
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Fig. 9.
Phospholipid composition of control cells and
Mc/PSS1 cells. Cells were grown to 80% confluence and then were
harvested, and the phospholipids were extracted and separated by thin
layer chromatography. The phosphorous content of PtdSer, PtdEtn,
PtdCho, sphingomyelin (SM), and phosphatidylinositol
(PtdIns) was determined. Solid symbols, control
McArdle 7777 cells transfected with vector alone; open
symbols, Mc/PSS1 cells. Data are averages ± S.D. of triplicate analyses from three independent experiments.
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These observations suggested that the levels of PtdSer and PtdEtn might
be tightly controlled and that synthesis and/or degradation of these
lipids might be regulated. We therefore examined further the metabolism
of PtdSer and PtdEtn in control cells and Mc/PSS1 cells. The cells were
pulse-labeled for 1 h with [3-3H]serine, then the
radiolabel was chased for up to 12 h (Fig. 10). As expected from the data shown in
Fig. 8, incorporation of [3H]serine into PtdSer in
Mc/PSS1 cells at the end of the 1-h pulse was approximately 2.5-fold
higher than in control cells (Fig. 10, upper panel).
However, radioactivity in PtdSer declined more rapidly in Mc/PSS1 cells
than in control cells, indicating an increased rate of degradation of
PtdSer in the cells overexpressing PtdSer synthase-1. Fig. 10
(lower panel) also shows that the incorporation of
[3H]serine into PtdEtn was consistently higher in Mc/PSS1
cells than in control cells, suggesting that at least a portion of the increased radiolabel lost from PtdSer in Mc/PSS1 cells was due to an
increased conversion of PtdSer to PtdEtn. However, in neither Mc/PSS1
nor control cells was the radioactivity lost from PtdSer quantitatively
recovered in PtdEtn. For example, in Mc/PSS1 cells during the first
6 h of the chase period, 107 × 103 dpm/mg
protein were lost from PtdSer, whereas the 3H content of
PtdEtn increased by only 25 × 103 dpm/mg protein.
Similarly, during the same period, 35 × 103 dpm/mg
protein were lost from PtdSer of control cells, and only 15 × 103 dpm/mg protein accumulated in PtdEtn (Fig. 10). These
data show the following: (i) in both types of cells some PtdSer was
converted into PtdEtn; (ii) the conversion of PtdSer to PtdEtn was
increased in Mc/PSS1 cells compared with control cells; (iii) some
radiolabeled PtdSer and/or PtdEtn was degraded in both Mc/PSS1 and
control cells; and (iv) in Mc/PSS1 cells, ~four times as much
radiolabeled PtdSer and/or PtdEtn was degraded as in control cells.
Presumably, these metabolic events are coordinated to ensure constant
cellular levels of PtdSer and PtdEtn.

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Fig. 10.
Metabolism of
[3H]serine-derived PtdSer and PtdEtn in control cells and
Mc/PSS1 cells. Cells were incubated for 1 h in medium
containing [3H]serine (20 µCi/dish). The medium was
then removed, and fresh medium containing 1 mM unlabeled
serine was added. At the indicated times cells were harvested, and
PtdSer (upper panel) and PtdEtn (lower panel)
were extracted and separated by thin layer chromatography. Open
symbols, control McArdle 7777 cells transfected with vector alone; solid symbols, Mc/PSS1 cells. Data are averages ± S.D. from triplicate analyses from one experiment which was repeated at twice with similar results. Some error bars are too small
to be visible.
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Overexpression of PtdSer Synthase-1 Inhibits PtdEtn Synthesis via
the CDP-ethanolamine Pathway--
Figs. 8 and 10 show that Mc/PSS1
cells produced more PtdEtn by decarboxylation of PtdSer than did
control cells. However, the total cellular mass of PtdEtn did not
increase (Fig. 9). We therefore examined whether or not the increased
expression of mouse liver PtdSer synthase-1 in McArdle 7777 cells
inhibited the synthesis of PtdEtn via the other major route for PtdEtn
biosynthesis, the CDP-ethanolamine pathway. Mc/PSS1 cells were
incubated with [1-3H]ethanolamine for up to 1 h, and
incorporation of radioactivity was measured in PtdEtn and intermediates
of the CDP-ethanolamine pathway (Fig.
11). The total amount of
[3H]ethanolamine taken up by control and Mc/PSS1 cells
was the same. In Mc/PSS1 cells, more radiolabeled ethanolamine,
phosphoethanolamine, and CDP-ethanolamine was present than in control
cells. In contrast, the incorporation of radiolabel into PtdEtn was
greatly reduced in Mc/PSS1 cells compared with control cells. The
relative pool sizes of radiolabeled ethanolamine metabolites in Mc/PSS1
cells and control cells were also compared after labeling with
[3H]ethanolamine for 24 h. As expected from the data
shown in Fig. 11, more radiolabeled ethanolamine and
phosphoethanolamine (2.5- and 13-fold, respectively) were present in
Mc/PSS1 cells than in control cells. In control cells, the dpm/mg
protein in ethanolamine were (3.30 ± 0.50) × 103,
and in Mc/PSS1 cells the dpm/mg protein were (8.20 ± 2.50) × 103. In phosphoethanolamine the dpm/mg protein were
(6.60 ± 2.50) × 103 in control cells and (84.7 ± 20.5) × 103 in Mc/PSS1 cells. For CDP-ethanolamine,
(2.10 ± 0.20) × 103 dpm/mg protein were recovered
from control cells and (3.40 ± 1.40) × 103 dpm/mg
protein from Mc/PSS1 cells.

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Fig. 11.
Incorporation of
[3H]ethanolamine into PtdEtn and ethanolamine-derived
metabolites in control McArdle 7777 cells and Mc/PSS1 cells. Cells
were incubated in medium containing [3H]ethanolamine (1.5 µCi/dish) for the indicated times and then harvested, and PtdEtn and
water-soluble intermediates of the CDP-ethanolamine pathway
(Etn, ethanolamine; Etn-P, phosphoethanolamine;
CDP-Etn, CDP-ethanolamine) were extracted and separated by
thin layer chromatography. Open symbols, control McArdle
7777 cells transfected with vector alone; solid symbols,
Mc/PSS1 cells. Data are averages ± S.D. of triplicate analyses
from one experiment which was repeated twice with similar results. Some
error bars are obscured by symbols.
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Two likely explanations for the decreased labeling of PtdEtn from
[3H]ethanolamine when PtdSer synthase-1 was overexpressed
are as follows: (i) PtdEtn synthesis via the CDP-ethanolamine pathway was inhibited, or (ii) the rate of degradation of ethanolamine-derived PtdEtn was increased. These two possibilities were distinguished by
incubation of Mc/PSS1 cells with [3H]ethanolamine for
24 h. The radioactivity was chased with 2 mM unlabeled
ethanolamine, and the amounts of [3H]PtdEtn and
ethanolamine-derived intermediates were measured. Fig.
12 shows that the rate of loss of
radiolabel from PtdEtn was the same in Mc/PSS1 cells as in control
cells. In addition, the amounts of labeled degradation products
of PtdEtn (i.e.
[3H]phosphoethanolamine, [3H]ethanolamine,
and [3H]glycerophosphoethanolamine) were the same in
Mc/PSS1 and control cells (data not shown). The combined data,
therefore, indicate that the increased content of
[3H]ethanolamine-labeled intermediates in Mc/PSS1 cells,
as shown in Fig. 11, was due to inhibition of PtdEtn synthesis via the
CDP-ethanolamine pathway rather than increased degradation of
ethanolamine-derived PtdEtn.

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Fig. 12.
Degradation of
[3H]ethanolamine-derived PtdEtn. Control McArdle
7777 cells transfected with vector alone (closed symbols) and Mc/PSS1 cells (open symbols) were labeled with
[3H]ethanolamine (1.5 µCi/dish) for 24 h. The
radiolabeled medium was removed and replaced with medium containing (2 mM) unlabeled ethanolamine. Cells were harvested at the
indicated times, and incorporation of radiolabel into PtdEtn was
determined. Data are averages ± S.D. of triplicate analyses from
one experiment which was repeated twice with similar results.
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Since in Mc/PSS1 cells less PtdEtn was synthesized by the
CDP-ethanolamine pathway than in control cells, we considered the possibility that the activity of one of the enzymes of this pathway, most likely ET, was decreased in Mc/PSS1 cells. ET has been suggested to be the rate-limiting enzyme of the CDP-ethanolamine pathway under
most metabolic conditions (44-46). However, Table
I shows that the activities of
ethanolamine kinase, ET, and ethanolaminephosphotransferase in Mc/PSS1
cells were essentially the same as in control cells. Since many enzymes
are regulated by phosphorylation-dephosphorylation events, ET was also
assayed in cell lysates that had been prepared in the presence of the
phosphoprotein phosphatase inhibitors microcystin (36), vanadate, and
fluoride, and the same results were obtained.
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Table I
Activities of enzymes of the CDP-ethanolamine pathway in control
cells and Mc/PSS1 cells
Total cellular membranes and cytosol were prepared by centrifugation of
the cell lysate at 400,000 × g for 30 min. Cytosol was
assayed for ethanolamine kinase and ET activity. Membranes were
assayed for CDP-ethanolamine:1,2-diacylglycerol
ethanolaminephosphotransferase activity. The data for each enzyme assay
are from triplicate analyses of one experiment which was repeated twice
with similar results. ET activity was assayed in the presence (+) or
absence ( ) of 5 µM microcystin, 1 mM sodium
orthovanadate, and 10 mM NaF.
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The level of diacylglycerol has also been reported to regulate the
CDP-ethanolamine pathway (47, 48) since this lipid is a substrate for
ethanolaminephosphotransferase. We therefore measured the
diacylglycerol content of control and Mc/PSS1 cells and found that the
amount of this lipid was approximately the same in control cells
(4.4 ± 0.5 nmol/mg protein) and (4.0 ± 0.5 nmol/mg protein)
Mc/PSS1 cells. Since diacylglycerol is an intermediate in the
biosynthesis of several important glycerolipids and has also been
implicated as a lipid second messenger, the cellular content of this
lipid probably fluctuates only transiently and locally. Consequently,
changes in the supply of diacylglycerols for
ethanolaminephosphotransferase might not be detected by measurement of
the total cellular diacylglycerol content. We therefore measured the
cellular content of triacylglycerols as a potential "reserve" precursor pool of diacylglycerols. The content of triacylglycerols was
40% less in Mc/PSS1 cells overexpressing PtdSer synthase-1 (14.2 ± 1.2 nmol/mg protein) than in control cells (23.7 ± 2.1 nmol/mg
protein).
 |
DISCUSSION |
We report the cloning and expression of a cDNA that encodes
PtdSer synthase-1 from mouse liver and show that the PtdSer synthesis and/or decarboxylation pathways can coordinately regulate the production of PtdEtn via the CDP-ethanolamine pathway.
As evidence that the cloned cDNA encoded PtdSer synthase-1, the
cDNA for mouse liver PtdSer synthase-1 was heterologously expressed
in M.9.1.1 cells that are mutant CHO cells deficient in PtdSer
synthase-1 activity (14). When PtdSer synthase-1 cDNA was
expressed, the ethanolamine auxotrophy of the M.9.1.1 cells was
eliminated, the base exchange activities with serine, choline, and
ethanolamine were increased, and the levels of PtdSer and PtdEtn were
restored to normal. In addition, expression of mouse liver PtdSer
synthase-1 in McArdle 7777 rat hepatoma cells resulted in increased
in vitro base exchange activity and an increased rate of
incorporation of [3H]serine into PtdSer and PtdEtn in
intact cells. These observations are consistent with previous data
showing that a PtdSer synthase-1 cDNA from CHO-K1 cells
complemented the ethanolamine auxotrophy of a mutant CHO cell line,
PSA-3 (10).
Our studies in McArdle hepatoma cells imply that the metabolism of
PtdSer and PtdEtn is coordinately regulated so that cellular levels of
these lipids remain constant. When PtdSer synthase-1 activity was
overexpressed, homeostasis of PtdSer and PtdEtn was maintained by the
following: (i) increased conversion of PtdSer to PtdEtn, (ii) increased
degradation of PtdSer and/or PtdEtn derived from PtdSer, and (iii)
inhibition of the CDP-ethanolamine pathway for PtdEtn synthesis. These
findings are reminiscent of those from a study in which
CTP:phosphocholine cytidylyltransferase, the rate-limiting enzyme of
PtdCho biosynthesis via the CDP-choline pathway, was overexpressed in
COS cells (49). Despite a greatly increased rate of PtdCho synthesis in
these transfected cells, the mass of PtdCho was barely increased.
However, the rate of PtdCho degradation was increased, presumably as a
mechanism for maintaining a constant level of PtdCho. Similarly,
overexpression of E. coli PtdSer synthase in E. coli did not alter the phospholipid composition of these cells
(50).
The increased conversion of PtdSer to PtdEtn in cells overexpressing
PtdSer synthase is most likely the direct result of increased PtdSer
synthesis. Production of PtdSer-derived PtdEtn appears not to be
limited by the activity of the decarboxylase but rather by availability
of the substrate, PtdSer (51). PtdSer supply is, in turn, regulated by
either the rate of PtdSer synthesis or the rate of translocation of
PtdSer from its site of synthesis on the ER to the site of the
decarboxylase on the outer aspect of inner mitochondrial membranes
(15). Hence, an increased supply of PtdSer to mitochondria would be
expected to be translated into an increased production of PtdEtn by the
decarboxylase.
A second consequence of overexpression of PtdSer synthase-1 was that
degradation of PtdSer and/or PtdSer-derived PtdEtn was increased,
although the degradation of PtdEtn derived from the CDP-ethanolamine
pathway was not increased. Most likely the enhanced degradation was of
PtdSer rather than of PtdEtn since in primary hepatocytes newly made
PtdSer is rapidly degraded, whereas PtdSer-derived PtdEtn is not
significantly degraded (52).
A third consequence of overexpression of PtdSer synthase-1 in McArdle
7777 cells was that the synthesis of PtdEtn via the CDP-ethanolamine
pathway was inhibited, as was demonstrated by the greatly reduced
incorporation of [3H]ethanolamine into PtdEtn. This
reduction in incorporation of radioactivity could not be accounted for
by an increased degradation of ethanolamine-derived PtdEtn. Moreover,
the amounts of [3H]ethanolamine-labeled precursors of
PtdEtn were increased indicating that the CDP-ethanolamine pathway for
PtdEtn synthesis was inhibited. We therefore investigated which step of
the CDP-ethanolamine pathway was modified when PtdSer synthase-1 was
overexpressed. Under most metabolic conditions the rate-limiting enzyme
of this pathway is thought to be ET (44-46). However, the total
in vitro activity of ET was unaffected by overexpression of
PtdSer synthase-1. We cannot, however, exclude the possibility that the
distribution of ET between a small active pool and a large inactive
pool had been altered since such a change would not have been detected in our experiments. Unlike the cytidylyltransferase of the CDP-choline biosynthetic pathway, there is no evidence that the activity of ET is
regulated by reversible translocation to and from membranes (44). ET is
presumed to be a cytosolic protein that neither requires lipids for
activity nor is tightly associated with membranes (47, 53-55).
However, some association of ET with ER membranes has been detected in
immunogold electron microscopy studies (53). The cDNA sequences of
ET (56) and CTP:phosphocholine cytidylyltransferase (57) share some
similarities, especially at the N termini of the corresponding
proteins. CTP:phosphocholine cytidylyltransferase contains an
amphipathic
-helical C-terminal domain that has been proposed to
mediate membrane association (58). From analysis of the cDNA
sequence of ET (56) it appears that ET might also have an
amphipathic
-helical domain close to the C terminus that could
potentially form a loose association with membranes.
The supply of diacylglycerols has also been implicated in the
regulation of the CDP-ethanolamine pathway (47, 48) by virtue of this
lipid being a substrate for ethanolaminephosphotransferase. Similarly,
under some metabolic conditions, diacylglycerols can regulate the
CDP-choline pathway for PtdCho synthesis at the level of the
cholinephosphotransferase (59-61). We detected no difference in the
diacylglycerol content of control cells and Mc/PSS1 cells although our
experiments would not have detected a change in a small localized pool
of this lipid. However, in cells overexpressing PtdSer synthase-1, the
cellular content of triacylglycerols was 40% less than in control
cells. For each molecule of PtdSer synthesized, one molecule of
phospholipid, and hence one molecule of diacylglycerol, is consumed. If
the "reservoir" of stored triacylglycerols in the cell served as a
precursor pool of diacylglycerols, a reduction in triacylglycerol mass
might result from the increased demand for diacylglycerols in the cells
overexpressing PtdSer synthase-1. Therefore, overexpression of PtdSer
synthase-1 might deplete a specific pool of diacylglycerols that is
used for PtdEtn synthesis via the ethanolaminephosphotransferase
reaction.
In conclusion, an increased synthesis of PtdSer, induced by
overexpression of mouse liver PtdSer synthase-1 cDNA, increases the
production of PtdEtn from the PtdSer decarboxylation pathway and
inhibits the synthesis of PtdEtn via the CDP-ethanolamine pathway.
These observations suggest that the synthesis and decarboxylation of
PtdSer, as well as the synthesis of PtdEtn from the CDP-ethanolamine pathway and the degradation of PtdSer and/or PtdEtn, are coordinately regulated so that constant cellular levels of these phospholipids are
maintained. Experiments are presently underway in our laboratory to
determine whether or not increasing in the synthesis of PtdEtn via the
CDP-ethanolamine pathway, by overexpression of ET, reciprocally inhibits the synthesis and/or decarboxylation of PtdSer.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF042731.