©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Expression of Phosphatidylethanolamine N-Methyltransferase-2 Cannot Compensate for an Impaired CDP-choline Pathway in Mutant Chinese Hamster Ovary Cells (*)

Martin Houweling (§) , Zheng Cui , Dennis E. Vance (¶)

From the (1)Lipid and Lipoprotein Research Group and the Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2S2, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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/minmg 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.


INTRODUCTION

Phosphatidylcholine (PC)()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.

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-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.


EXPERIMENTAL PROCEDURES

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/HO (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.


RESULTS

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.

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 [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).

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 [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.

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.


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/minmg 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.

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/minmg 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.




DISCUSSION

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 to the S phase of the cell cycle.

  
Table: Expression of rat liver CT in CHO MT-58 cells

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 [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

Cells were grown to near confluency on 100-mm culture dishes at 33 °C and harvested, and PEMT activity was assayed in a 600 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.



FOOTNOTES

*
This research was supported by a grant from the Medical Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Postdoctoral Fellow of the Alberta Heritage Foundation for Medical Research.

Medical Scientist of the Alberta Heritage Foundation of Medical Research. To whom correspondence should be addressed. Fax: 403-492-3383; E-mail: dvance@gpu.srv.ualberta.ca.

The abbreviations used are: PC, phosphatidylcholine; CHO, Chinese hamster ovary; CT, CTP:phosphocholine cytidylyltransferase; PE, phosphatidylethanolamine; PEMT, phosphatidylethanolamine N-methyltransferase.


ACKNOWLEDGEMENTS

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.


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