Expression of phosphatidylethanolamine N-methyltransferase-2 cannot compensate for an impaired CDP-choline pathway in mutant Chinese hamster ovary cells.
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 his 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.mg protein) were isolated. A positive correlation between the amount of PEMT2 activity expressed and the incorporation of [3H]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 studies showed 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.
Houweling
M
M
Lipid and Lipoprotein Research Group, University of Alberta, Edmonton, Canada.
Cui
Z
Z
Vance
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D000818:Animals; D016466:CHO Cells; D002451:Cell Compartmentation; D002455:Cell Division; D002460:Cell Line; D019916:Choline-Phosphate Cytidylyltransferase; D006224:Cricetinae; D003566:Cytidine Diphosphate Choline / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D008099:Liver / Q000201:enzymology; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism*; D009713:Nucleotidyltransferases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000096:biosynthesis*; D050918:Phosphatidylethanolamine N-Methyltransferase; D010714:Phosphatidylethanolamines / Q000378:metabolism*; D010743:Phospholipids / Q000032:analysis; D051381:Rats
D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010743:Phospholipids; D003566:Cytidine Diphosphate Choline; D008780:Methyltransferases; D050918:Phosphatidylethanolamine N-Methyltransferase; D009713:Nucleotidyltransferases; D019916:Choline-Phosphate Cytidylyltransferase