From the
Phosphatidylcholine is a product of the CDP-choline pathway and
the pathway that methylates phosphatidylethanolamine. We have asked the
question: are the two pathways functionally interchangeable? We
addressed this question by investigating the expression of
phosphatidylethanolamine N-methyltransferase-2 (PEMT2) of rat
liver in mutant Chinese hamster ovary cells (MT-58) (Esko, J. D.,
Wermuth, M. M., and Raetz, C. R. H.(1981) J. Biol. Chem. 256,
7388-7393) defective in the CDP-choline pathway for
phosphatidylcholine biosynthesis. Cell lines stably expressing
different amounts of PEMT2 activity (up to 700 pmol/min
Phosphatidylcholine (PC)
Liver is unique among
animal tissues in that it can synthesize substantial amounts of PC from
phosphatidylethanolamine (PE) in a reaction catalyzed by PE N-methyltransferase (PEMT) (EC. 2.1.1.17) (Vance and Ridgway,
1988). Most PEMT activity is localized to the cytosolic surface of the
endoplasmic reticulum, but its activity is also found in a
mitochondria-associated membrane fraction (Cui et al., 1993),
which appears to be distinct from the endoplasmic reticulum and
mitochondria (Vance, 1990b). The cDNA for PEMT of the
mitochondria-associated membrane has been recently cloned and named
PEMT2 (Cui et al., 1993).
It is not obvious why PEMT
activity has survived in evolution largely as a liver-specific enzyme
when all nucleated cells have the capacity to make PC via the
CDP-choline pathway. If PEMT-catalyzed methylation of PE were simply a
backup for the biosynthesis of PC via the CDP-choline pathway, why is
the PEMT activity so low in non-hepatic tissues and cells?
Quantitatively, methylation of the ethanolamine moiety of PE by PEMT in
liver may be significant for the generation of choline that is required
for growth of all animal cells (Eagle, 1955). Alternatively, PEMT may
have an unknown function that is required by the liver, such as
involvement in the regulation of cell division (Cui et al.,
1994). To obtain more insight into the role of PEMT and to see if the
PC generated by this enzyme is interchangeable with PC derived from the
CDP-choline pathway, we expressed the cDNA for PEMT2 in a mutant CHO
cell line that was defective in the CDP-choline pathway.
This CHO
mutant (MT-58) was first described by Esko et al. (1981). The
mutant had diminished ability to incorporate
[methyl-
shows the activity of
soluble and membrane-bound CT in the WT-K1 and MT-58 cells and three
mutant cell lines that express different amounts of CT activity as
determined in cells grown at 33 °C. These data confirmed that CT
activity in the mutant is approximately 12% of that found in WT-K1
cells (Esko et al., 1981). further shows that
cell lines were isolated that stably expressed CT activity in the range
of twice the activity of MT-58 cells to 3 times the activity found in
WT-K1 cells. The elevated levels of CT activity in these cells resulted
from an increase in the level of CT protein as determined by
immunoblotting with a specific antibody (Jamil et al.(1992)
and data not shown).
Next, the incorporation of
[
We next examined the ability of these different
cell lines to grow at the restrictive temperature (40 °C). The data
in demonstrate that the rate of
[
The growth of
the mutants stably expressing CT was examined at 40 °C. Although
all three cell lines (MT+CT-A, -B, and -C) were rescued by
transfection with CT, only MT+CT-A had a growth rate similar to
that of the wild type cells (Fig. 1). MT+CT-B grew
significantly slower than wild type cells, and MT+CT-C grew only
slightly better than the MT-58 cells.
Next, the
WT-K1 and MT-58 cells and all the cell lines stably expressing PEMT2
activity were shifted from 33 to 40 °C. The WT-K1 cells (control)
grew normally at the restrictive temperature, whereas the MT-58 cells
divided only once and then stopped growing. As published before (Esko et al., 1982), the addition of lyso-PC (30 µM) to
MT-58 cells suppressed the phenotype. To our surprise, all cell lines
stably expressing PEMT2 divided only once and then stopped growing,
like the MT-58 cells (Fig. 3). The observations that the
MT+PEMT-D cell line could be rescued by the addition of lyso-PC
and that parental cells expressing PEMT2 (WT-K1+PEMT; 440
pmol/min
The major conclusion from this study is that expression of
PEMT2 in mutant CHO MT-58 cells, in contrast to the expression of CT in
these cells, does not suppress the temperature-sensitive phenotype. The
data suggest that PC made via the methylation of PE is not simply an
alternative source for PC made via the CDP-choline pathway.
There is
one other example in the literature (Cleves et al., 1991) that
has suggested a functional difference between the CDP-choline pathway
and the methylation of PE. Cleves et al. (1991) have studied a
yeast mutant (sec14) that has no functional
phosphatidylinositol/PC transfer protein. The mutant is defective in
growth and in secretion. Bypass mutants of sec14 have been
isolated that are defective in the CDP-choline pathway. Thus, by
attenuation of the synthesis of PC via the CDP-choline pathway, the sec14 mutants were rescued. Subsequent studies by McGee et
al.(1994) and Skinner et al.(1995) suggest that SEC14p,
when PC is bound to the protein, decreases the activity of CT in the
CDP-choline pathway. Their data suggest that in sec14 mutants
this regulatory capacity was lost, and, consequently, too much PC was
made in the Golgi. Apparently a defect in the CDP-choline pathway
compensated for the over-production of PC, and, therefore, the
phenotype was suppressed. In contrast, mutants in the PE methylation
pathway failed to rescue the sec14 phenotype (Cleves et
al., 1991). Hence, their results point to a functional difference
in yeast between the CDP-choline and PE methylation pathways. Unlike
mammalian cells, yeast cells can grow normally in the absence of
choline as long as they have a functional methylation pathway (Carman
and Henry, 1989).
The failure of PEMT2 expression in MT-58 cells to
rescue growth at 40 °C was unexpected. Earlier studies (Esko et
al., 1982; Esko and Matsuoka, 1983) demonstrated that the addition
of exogenous PC emulsions or lyso-PC to the cells suppressed the
phenotype. Moreover, supplementation of MT-58 cells with
phosphatidyldimethylethanolamine, an intermediate in the conversion of
PE to PC, rescued the MT-58 cells at 40 °C. There was wide
flexibilty in the fatty acid compositions of the exogenous PC or
lyso-PC that allowed the MT-58 cells to grow. On the other hand, alkyl-
or alkenyl-PC or lipoproteins that contained PC were ineffective.
There are several possible explanations for why PEMT2 expression in
MT-58 cells failed to permit growth of these cells at 40 °C, even
though the amount of PC was restored to control values. 1) Perhaps
PEMT2 has some specialized function in the cell, and expression of
PEMT1 (the putative endoplasmic reticulum isoenzyme) would rescue the
mutant. This possibility cannot be discounted until PEMT1 has been
cloned and expressed in MT-58 cells. It would also be interesting to
know if the bacterial PEMT (Arondel et al., 1993) or the yeast
PEMT (Kodaki and Yamashita, 1987) could rescue MT-58 cells. 2) Another
possible explanation that cannot be eliminated at this point is that PC
made via the methylation route cannot rescue the MT-58 phenotype,
because this pathway might not produce the molecular species of PC
important for the signal transduction cascade leading to cell
proliferation. 3) We have not eliminated the possibility that the MT-58
cells may have a second mutation that can also be compensated by
over-expression of CT but not PEMT2. There is some support for this
idea. From immunoblot analyses, CT protein appeared to be completely
absent from the mutant at 33 and 40 °C (data not shown and Wang et al., 1993), yet the cells survived at 33 °C but not at
40 °C. Even though the CT protein appeared to be absent, CT
activity was 12% of the wild type level as shown in and by
Esko et al.(1981). Moreover, the mRNA for CT in MT-58 cells
was 90% of that found in wild type cells (Sweitzer and Kent, 1994).
Support for a second mutation in addition to the CT mutation was also
suggested, because complete rescue of the mutant (i.e. similar
growth rate as wild type) required 3-fold higher expression of CT
activity than that observed in the wild type (). In
addition, two transfected cell lines (MT+CT-B and MT+CT-C)
showed a 75% decrease in choline incorporation into PC at 40 °C
compared with 33 °C. In contrast, wild type and MT+CT-A cells
showed a near doubling of incorporation into PC when shifted to the
restrictive temperature.
Whatever the reason for the failure of
PEMT2 to rescue MT-58 cells, the data support the idea that the
CDP-choline pathway is vital for cell growth. At this point we
speculate that PC made via the CDP-choline route or a metabolite
(CDP-choline) is required for normal progression through the cell cycle
and that PC made via the methylation of PE is not suitable for this
purpose. This speculation agrees with recent publications (Jackowski,
1994; Tercé et al., 1994) that suggest that PC derived
from CDP-choline is required for the progression of cells from the
G
Cells were grown at 33 °C on 100-mm culture dishes to near
confluency. Soluble and membrane fractions were prepared and assayed
for CT activity as described under ``Experimental
Procedures.'' The rate of PC synthesis was estimated after
incubation for 22 h either at 33 °C or at 40 °C, by adding 5
µCi of [
Cells were grown to near confluency on
100-mm culture dishes at 33 °C and harvested, and PEMT activity was
assayed in a 600
We are very grateful to Sandra Ungarian for excellent
technical assistance and Dr. Jean Vance for helpful comments. We thank
Dr. Marek Michalek for kindly providing the protein disulfide isomerase
antibody and Dr. Mark Lee for the WT-K1+PEMT cell line.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
mg
protein) were isolated. A positive correlation between the amount of
PEMT2 activity expressed and the incorporation of
[
H]methionine into phosphatidylcholine at both
the permissive and restrictive temperatures showed that PEMT2 was
functional in the Chinese hamster ovary MT-58 cells. In contrast to
mutant cell lines stably expressing transfected CTP:phosphocholine
cytidylyltransferase, the cell lines stably expressing PEMT2 did not
survive at the restrictive temperature. Determination of the
phosphatidylcholine mass in wild type cells, mutant MT-58 cells, and
cells with the highest level of PEMT2 expression showed that PEMT2 was
functional and synthesized the same amount of phosphatidylcholine as
did wild type cells at the restrictive temperature. Indirect
immunofluorescence studiesshowed that localization of the
over-expressed cytidylyltransferase in MT-58 cells was largely nuclear,
whereas PEMT2 was predominantly located outside the nucleus. Our data
show that methylation of phosphatidylethanolamine to
phosphatidylcholine cannot substitute for the CDP-choline pathway.
(
)is the most
abundant phospholipid in cellular membranes of mammalian tissues. In
addition to its structural role in membranes and lipoproteins, PC has
recently been identified as a major source of intracellular signaling
molecules (reviewed in Billah and Anthes(1990) and Exton(1990)). PC is
synthesized de novo primarily via the CDP-choline pathway
(Kennedy, 1986), which is often regulated by the activity of
CTP:phosphocholine cytidylyltransferase (CT) (EC 2.7.7.15) (Tijburg et al., 1990; Vance, 1990a; Kent, 1991; Tronchère et al., 1994). CT is present in both the soluble and membrane
fractions of all nucleated animal cells.
C]choline into PC at the
restrictive temperature of 40 °C. The defect was due to a lack of
CT activity. The parental cell line (WT-K1) and MT-58 cells grow
normally at the permissive temperature of 33 °C, whereas only the
WT-K1 cell line and not MT-58 cells survived when grown at the
restrictive temperature. The MT-58 mutant, therefore, provided a
suitable model to determine if, by the introduction of PEMT2 activity
into the MT-58 cells, PC made via the methylation of PE could
compensate for the defective CDP-choline pathway.
Materials
[methyl-H]Choline
chloride (15 Ci/mmol),
[methyl-
H]methionine (70 Ci/mmol), S-adenosyl-L-[methyl-
H]methionine
(15 Ci/mmol), and the ECL
kit were purchased from Amersham
International. The substrate for CT assays,
phospho[methyl-
H]choline, was
synthesized enzymatically from
[methyl-
H]choline and ATP with choline
kinase as described (Vance et al., 1982). Cell culture media,
Ham's F-12 Nutrient Mixture, and fetal bovine serum were obtained
from Life Technologies, Inc. Culture dishes and flasks were from Becton
Dickinson, and Silica Gel G60 thin-layer chromatography plates were
purchased from Merck (Darmstadt, Germany). All other chemicals (unless
specified) were from Sigma or Fisher.
Cell Culture
CHO WT-K1 and MT-58 cells (kindly
donated by Drs. C. Kent and C. Raetz) were cultured in Ham's F-12
medium supplemented with 10% fetal bovine serum. Cells were maintained
in 100-mm culture dishes at either 33 or 40 °C, 5% CO,
and 90% relative humidity.
Introduction of CT or PEMT2 Expression Plasmids into CHO
MT-58 Cells
The construction of PEMT2 (Cui et al.,
1993) and CT (kindly donated by Dr. R. Cornell) (Walkey et
al., 1994) expression plasmids has been described. 10 µg of
the expression plasmids for CT or PEMT2 were co-transfected with 0.3
µg of pSV-neo (Southern and Berg, 1982) into CHO MT-58 cells by
calcium phosphate precipitation (Chen and Okayama, 1987). Individual
neomycin-resistant colonies were selected with 0.6 µg/ml G418 in
Ham's F-12 medium supplemented with 10% fetal bovine serum. The
colonies were picked and grown in the presence of 0.3 µg/ml G418.
Each cell line was assayed for either PEMT or CT activity to confirm
expression.
Incorporation of Radioactive Precursors into
PC
Cells were grown in 100-mm culture dishes to 60-80%
confluency and labeled with either [H]choline or
[
H]methionine for 2 h. Cells were washed three
times with ice-cold phosphate-buffered saline and harvested in 2 ml of
buffer A (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1.0
mM phenylmethylsulfonyl fluoride, 1.0 mM EDTA, 2.0
mM dithiothreitol, and 0.025% sodium azide). Lipids were
extracted from the cells as described by Sundler et al.(1974),
and phospholipids were separated by thin-layer chromatography on Silica
Gel G60 plates with chloroform/methanol/acetic acid/formic
acid/H
O (70:30:12:4:1) as developing agents. The PC band
was visualized by iodine vapor and scraped, and the radiolabel
incorporated into PC was quantified by liquid scintillation counting.
Determination of Phospholipid Mass, Enzyme Activities,
and Amount of Protein
Cells were cultured as described above and
collected in 2 ml of buffer A. Cells were homogenized using a
glass-Teflon homogenizer, and aliquots were taken for phospholipid
determination, enzyme assays, and immunoblotting. Phospholipids were
extracted and separated as described above, and the phospholipid mass
was determined (Rouser et al., 1966). Aliquots of the
homogenate were centrifuged for 5 min at 600 g to
remove unbroken cells. The resulting supernatant was directly used for
PEMT activity and protein measurement, whereas, for the determination
of CT activity and protein, the resulting supernatant was further
centrifuged at 350,000
g for 15 min to prepare soluble
and membrane fractions. The activity of PEMT was assayed as described
(Ridgway and Vance, 1992), and the activity of CT in both the soluble
and the membrane fractions was assayed in the presence of PC:oleate
vesicles essentially as described (Weinhold et al., 1986) with
modifications (Yao et al., 1990). The amounts of PEMT and CT
protein were determined using immunoblot analysis. Briefly, proteins
(50 µg/lane) were boiled for 5 min in Laemmli(1970) buffer and
separated on a 12.5% polyacrylamide gel in the presence of 0.1% SDS.
Proteins were transferred to Immobilon-P membranes by electrophoretic
blotting (Towbin et al., 1979). The PEMT2-specific antibody
was described previously (Cui et al., 1993), as was the
antibody to CT (Jamil et al., 1992). The membranes probed with
specific antibodies were visualized by the ECL
system
(Amersham) according to the manufacturer's instructions.
Localization of the Expressed CT and PEMT2 in CHO MT-58
Cells by Indirect Immunofluorescence
Cells were grown on 12-mm
diameter poly-L-lysine-coated glass coverslips in Ham's
F-12 medium supplemented with 10% fetal bovine serum at 33 °C.
Cells were fixed and permeabilized by treatment for 10 min in methanol
at -20 °C and incubated with a primary antibody against
either CT or PEMT2 for 16-18 h at 4 °C. After extensive
washing, the cells were incubated with a secondary antibody,
anti-rabbit IgG conjugated to fluorescein (1000-fold dilution in
phosphate-buffered saline), for 1 h. The cells were washed extensively
and subsequently examined with a Zeiss fluorescence microscope.
Expression of CT cDNA in CHO MT-58 Cells
Esko et al.(1981) proposed that a single mutation in the structural
gene of CT was most likely responsible for the temperature sensitivity
of growth and the conditional decrease in CT activity and PC
biosynthesis in CHO MT-58 cells. Therefore, our first goal was to
determine whether or not expression of the cDNA for CT would rescue the
MT-58 cells when grown at the restrictive temperature of 40 °C.
These experiments were important as a control for subsequent expression
studies with PEMT2. CHO MT-58 cells were co-transfected with the rat
liver CT cDNA expression plasmid and a G418-resistant gene vector.
Transfected mutants resistant to G418 and expressing CT activity were
selected for further studies.
H]choline into PC via the CDP-choline pathway in
the different cell lines at 33 °C was measured (). At
33 °C, a positive correlation existed between the amount of
membrane-bound CT activity present in the cells and the labeling of PC.
Hence, the enzyme was functionally expressed at the permissive
temperature in the transfected cells. The MT+CT-A cell line
exhibited a lower rate of [
H]choline
incorporation into PC than would be predicted from the amount of
membrane-bound CT activity present in these cells. In agreement with
this result, Walkey et al.(1994) have reported that
over-expression of CT in COS cells resulted in a 20-100-fold
increase in the specific activity of CT in the membrane fraction but
only a 3-5-fold increase in the incorporation of
[
H]choline into PC. The apparent discrepancy may
be due to enhanced degradation of the newly labeled PC (Walkey et
al., 1994).
H]choline incorporation into PC at 40 °C was
approximately twice as high as that at 33 °C for the WT-K1 and
MT+CT-A cell lines. Surprisingly, even though the expressed CT is
a wild type protein and presumably temperature-insensitive,
[
H]choline incorporation into PC in MT+CT-B
cells at 40 °C decreased to 25-30% of that found at 33
°C. We have no explanation for this observation.
Figure 1:
Growth curve at 40 °C of different
cell lines stably expressing CT activity. Cells cultured at 33 °C
were harvested with trypsin and added to 60-mm diameter culture dishes
containing 3 ml of Ham's F-12 medium supplemented with 10% fetal
bovine serum (approximately 5 10
cells/dish). The
cells were incubated at 40 °C. At the indicated times the cells
were harvested with trypsin, and viable cells that excluded trypan blue
were counted. The experiment was repeated twice with similar results.
MT+CT-A, -B, and -C refer to cells lines
transfected with a pCMV vector that contained a cDNA that encoded
CT.
The conclusion from these
studies is that over-expression of CT activity corrects the mutant
phenotype in choline incorporation into PC, CT activity, and growth at
40 °C. This result further supports the statement that the major
defect is likely to be in the structural gene for CT (Esko et
al., 1981). While this manuscript was in preparation, Sweitzer and
Kent(1994) reported that transfection of MT-58 cells with the cDNA for
CT rescued the temperature-sensitive phenotype and that the temperature
sensitivity of CT is due to a single base change in the CT cDNA from
strain MT-58, which results in conversion of Arg to His.
Expression of the PEMT2 cDNA in CHO MT-58
Cells
Because the lethal phenotype of MT-58 cells was rescued by
expression of CT, these cells seemed to be a suitable host to test
whether PEMT2 could compensate for a defective CDP-choline pathway. The
cDNA for PEMT2, which was placed behind the cytomegalovirus promotor,
was introduced into MT-58 cells using the calcium-phosphate
precipitation method. Individual colonies resistant to G418 were
isolated and screened for PEMT activity (). As expected
from a non-hepatic cell line, wild type and mutant CHO cell lines did
not have significant endogenous PEMT activity. Four transfected cell
lines were isolated that expressed PEMT2 activities ranging from 95 to
700 pmol/minmg protein (). The PEMT activity of the
highest PEMT2 expresser (MT+PEMT-D) was similar to that found in
homogenates of rat hepatocytes (652 pmol/min
mg protein). Because
the PEMT activity in hepatocytes is a combination of PEMT1 (the
putative endoplasmic reticulum form of the enzyme) and PEMT2 (the
mitochondria-associated membrane form of the enzyme), the level of the
expressed PEMT2 in MT-58 cells was much higher than the amount of PEMT2
normally present in rat hepatocytes. Fig. 2shows that the
increase in PEMT activity in the different cell lines was accompanied
by an elevated level of PEMT2 protein, as determined by immunoblotting
with a specific antibody against PEMT2 (Cui et al., 1993).
Figure 2:
Expression of PEMT2 protein in
different cell lines. Cells were grown to near confluency on 100-mm
dishes in Ham's F-12 medium that contained 10% fetal bovine
serum. The plates were washed 3 times with ice-cold phosphate-buffered
saline, and the cells were harvested and subsequently homogenized.
Unbroken cells were removed by centrifugation of the homogenates for 5
min at 600 g. Aliquots were taken for immunoblot
analysis of PEMT2 protein. The experiment was repeated twice with
similar results. Molecular mass markers (in kDa) are indicated on the left of the figure. The film was overexposed to show the
complete lack of PEMT2 in WT-K1 and MT-58 cells. Control Plasmid is the pCMV vector without the cDNA insert. MT+PEMT-A, -B,
-C, and -D are cells lines that have been transfected with pCMV
carrying a cDNA insert for PEMT2 (Cui et al.,
1993).
We determined whether or not the expressed PEMT2 was active in the
conversion of PE to PC via the methylation pathway in these cells.
Radioactive methionine, the precursor of S-adenosylmethionine,
was used to label PC. As the level of PEMT2 expression increases, one
would expect a corresponding increase in the incorporation of
radioactivity into PC from [H]methionine. A
positive correlation existed between the amount of label incorporated
into PC via the methylation route and the corresponding PEMT activity
in these cells grown at 33 °C (). The rate of
[
H]methionine incorporation into PC was also
measured at 40 °C. As observed for PC synthesis via the CDP-choline
pathway (), the rate of incorporation of label into PC via
the methylation of PE at 40 °C was approximately 1.5 times higher
than at 33 °C (). Thus, the expressed rat liver PEMT2
enzyme was fully active at the restrictive temperature.
mg protein) grew normally at the restrictive temperature
(data not shown) demonstrated that there was no unrelated physiological
complication due to the over-expression of PEMT2 that interferes with
cell growth.
Figure 3:
Growth
curve at 40 °C of different cell lines stably expressing PEMT2
activity. The experiment was performed exactly as described in the
legend of Fig. 1. Control Plasmid refers to MT-58 cells
transfected with pCMV vector alone. MT+PEMT-D refers to a
cell line derived from MT-58 cells transfected with pCMV vector that
contained a cDNA that encoded PEMT2. LPC indicates that 30
µM lyso-PC was added to the cell cultures at the start of
the incubation at 40 °C.
Determination of the Phospholipid Composition of Cell
Lines Stably Expressing CT or PEMT2
In MT-58 cells, the impaired
CT activity resulted in a reduction in total cellular PC mass at 40
°C to approximately half the value found in wild type cells. The
observation that expressed PEMT2 did not rescue the MT-58 cells when
grown at 40 °C raised the question of whether the amount of PEMT2
activity present in these cells could restore PC mass to wild type
levels at this temperature. Increased PC synthesis does not necessarily
lead to increased mass of PC because the expression of CT in COS cells
stimulated PC degradation (Walkey et al., 1994). Therefore,
the phospholipid composition of the different cell lines was determined
at both the permissive temperature and 24 h after being incubated at 40
°C (Fig. 4). As published before (Esko et al.,
1981), the percentage of total phospholipid mass that was PC in MT-58
cells dropped dramatically upon a temperature shift from 46% at 33
°C to 26% at 40 °C. In contrast, in the wild type cell line,
the percentage of PC was approximately 53% at both temperatures (Fig. 4). As a control, the PC mass in the MT+CT-A cell line
was measured. This cell line, which stably over-expressed CT activity,
grew normally at 40 °C and had the same PC content as the wild type
cells at both temperatures. Determination of the amount of PC in the
cell lines that expressed PEMT2 revealed that, as the expression of
PEMT2 increased, the percentage of PC approached that of the wild type
CHO cell line (Fig. 4). Similar results were obtained if the
amount of PC/cell was determined 72 h after cells were shifted to 40
°C (WT-K1, 30.9 nmol/10 cells; MT-58, 8.8 nmol/10
cells; control plasmid, 9.0 nmol/10
cells;
MT+PEMT-D, 24.8 nmol/10
cells; and MT+CT-A, 23.2
nmol/10
cells). These data lead to the conclusion that the
amount of enzyme activity present in the highest PEMT2 expresser
(MT+PEMT-D) can generate the amount of PC required for cell
proliferation at the restrictive temperature.
Figure 4:
Effect of a temperature shift from 33 to
40 °C on the amount of PC in different lines of CHO cells. Cells
were grown on 100-mm dishes in Ham's F-12 medium, containing 10%
fetal bovine serum at 33 °C. When the cells were approximately 50%
confluent, some cells were kept at this temperature (hatched
bars), and others were incubated at 40 °C (solid
bars). 24 h later, cells were washed 3 times with ice-cold
phosphate-buffered saline, harvested, and homogenized by sonication. An
aliquot of the homogenate was taken to measure the protein content, and
the remainder was used for phospholipid determination as described
under ``Experimental Procedures.'' Data are the means
± S.D. of three separate experiments. The amount of
phosphatidylcholine is expressed as a percentage of total phospholipid.
Explanations of the various cell lines is found in the legends to Figs.
1 and 2.
Localization of the Expressed CT and PEMT2 in CHO MT-58
Cells
CT is exclusively localized in the nucleus of wild type
CHO WT-K1 cells (Watkins and Kent, 1992; Wang et al., 1993).
PEMT2, on the other hand, is localized exclusively to the
mitochondria-associated membrane in rat hepatocytes (Cui et
al., 1993). Indirect immunofluorescence studies were performed to
determine the localization of the expressed CT and PEMT2 in mutant CHO
cells. Fig. 5demonstrates that the expressed CT was largely
nuclear (Fig. 5D), as shown for the WT-K1 cells (Fig. 5C), whereas the expressed PEMT2 was predominantly
localized to regions outside the nucleus (Fig. 5E) but
was different from protein disulfide isomerase, a protein found in the
endoplasmic reticulum (Fig. 5F). No fluorescence signal
was detectable when WT-K1 and MT-58 cells were stained with anti-PEMT2
(results not shown). This agrees very well with the observation that
both cell lines had very low levels of PEMT activity ().
Therefore, the cells that expressed PEMT2 showed an immunofluorescence
pattern distinct from that of both CT and protein disulfide isomerase.
At this point it is difficult to exclude the possibility that some of
the expressed PEMT2 is localized to the nucleoplasm.
Figure 5:
Localization of the expressed CT and PEMT2
by indirect immunofluorescence microscopy. Cells plated on glass
coverslips were fixed and permeabilized by methanol (-20 °C)
treatment for 10 min. The cells in B (MT-58), C (WT-K1), and D (MT+CT-A) were incubated with a
primary antibody specific for CT. The cells expressing PEMT2
(MT+PEMT-D) were incubated with an affinity-purified antibody
specific for PEMT2 (E) or an antibody specific for rat protein
disulfide isomerase (F). A is a control sample
incubated in an identical fashion but without primary antibody. All
cells were incubated with secondary antibody (anti-rabbit IgG
conjugated to fluorescein). G and H show phase
contrast pictures from WT-K1 and MT-58 cells,
respectively.
to the S phase of the cell cycle.
Table: Expression of rat liver CT in CHO MT-58 cells
H]choline to each dish. After 2 h the
incubation was stopped, and the incorporation of
[
H]choline into PC was determined. Data are the
means ± S.D. of three separate experiments. MT+CT-A, -B,
and -C refer to MT-58 cells transfected with a pCMV vector that
contains a cDNA that encodes CT.
Table: Expression of rat liver
PEMT2 in CHO MT-58 cells
g supernatant. The rate of PC
synthesis was estimated after incubation for 22 h either at 33 °C
or at 40 °C, by the addition of 10 µCi of
[
H]methionine to each dish. After a 2-h
incubation the reaction was stopped, and the radiolabel that was
incorporated into PC was determined. Data are the means ± S.D.
of three separate experiments. Control plasmid refers to MT-58 cells
transfected with the pCMV vector alone. MT+PEMT-A, -B, -C, and -D
refer to cells lines derived from MT-58 cells transfected with pCMV
that contained a cDNA that encodes for PEMT2.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.