Abstract
The isolation of mutants of Schizosaccharomyces pombe defective in the synthesis of phosphatidylcholine via the methylation of phosphatidylethanolamine is reported. These mutants are choline auxotrophs and fall into two unlinked complementation groups, cho1 and cho2. We also report the analysis of the cho1+ gene, the first structural gene encoding a phospholipid biosynthetic enzyme from S. pombe to be cloned and characterized. The cho1+ gene disruption mutant (cho1Delta) is viable if choline is supplied and resembles the cho1 mutants isolated after mutagenesis. Sequence analysis of the cho1+ gene indicates that it encodes a protein closely related to phospholipid methyltransferases from Saccharomyces cerevisiae and rat. Phospholipid methyltransferases encoded by a rat liver cDNA and the S. cerevisiae OPI3 gene are both able to complement the choline auxotrophy of the S. pombe cho1 mutants. These results suggest that both the structure and function of the phospholipid N-methyltransferases are broadly conserved among eukaryotic organisms.
Full Text
The Full Text of this article is available as a PDF (190.5 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
- Atkinson K. D., Jensen B., Kolat A. I., Storm E. M., Henry S. A., Fogel S. Yeast mutants auxotrophic for choline or ethanolamine. J Bacteriol. 1980 Feb;141(2):558–564. doi: 10.1128/jb.141.2.558-564.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Atkinson K., Fogel S., Henry S. A. Yeast mutant defective in phosphatidylserine synthesis. J Biol Chem. 1980 Jul 25;255(14):6653–6661. [PubMed] [Google Scholar]
- CROCKEN B. J., NYC J. F. PHOSPHOLIPID VARIATIONS IN MUTANT STRAINS OF NEUROSPORA CRASSA. J Biol Chem. 1964 Jun;239:1727–1730. [PubMed] [Google Scholar]
- Carman G. M., Zeimetz G. M. Regulation of phospholipid biosynthesis in the yeast Saccharomyces cerevisiae. J Biol Chem. 1996 Jun 7;271(23):13293–13296. doi: 10.1074/jbc.271.23.13293. [DOI] [PubMed] [Google Scholar]
- Cui Z., Houweling M., Vance D. E. Expression of phosphatidylethanolamine N-methyltransferase-2 in McArdle-RH7777 hepatoma cells inhibits the CDP-choline pathway for phosphatidylcholine biosynthesis via decreased gene expression of CTP:phosphocholine cytidylyltransferase. Biochem J. 1995 Dec 15;312(Pt 3):939–945. doi: 10.1042/bj3120939. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cui Z., Vance J. E., Chen M. H., Voelker D. R., Vance D. E. Cloning and expression of a novel phosphatidylethanolamine N-methyltransferase. A specific biochemical and cytological marker for a unique membrane fraction in rat liver. J Biol Chem. 1993 Aug 5;268(22):16655–16663. [PubMed] [Google Scholar]
- Fernandez S., Homann M. J., Henry S. A., Carman G. M. Metabolism of the phospholipid precursor inositol and its relationship to growth and viability in the natural auxotroph Schizosaccharomyces pombe. J Bacteriol. 1986 Jun;166(3):779–786. doi: 10.1128/jb.166.3.779-786.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Greenberg M. L., Klig L. S., Letts V. A., Loewy B. S., Henry S. A. Yeast mutant defective in phosphatidylcholine synthesis. J Bacteriol. 1983 Feb;153(2):791–799. doi: 10.1128/jb.153.2.791-799.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Griac P., Swede M. J., Henry S. A. The role of phosphatidylcholine biosynthesis in the regulation of the INO1 gene of yeast. J Biol Chem. 1996 Oct 11;271(41):25692–25698. doi: 10.1074/jbc.271.41.25692. [DOI] [PubMed] [Google Scholar]
- Henry S. A., Patton-Vogt J. L. Genetic regulation of phospholipid metabolism: yeast as a model eukaryote. Prog Nucleic Acid Res Mol Biol. 1998;61:133–179. doi: 10.1016/s0079-6603(08)60826-0. [DOI] [PubMed] [Google Scholar]
- Hubbard S. C., Brody S. Glycerophospholipid variation in choline and inositol auxotrophs of Neurospora crassa. Internal compensation among zwitterionic and anionic species. J Biol Chem. 1975 Sep 25;250(18):7173–7181. [PubMed] [Google Scholar]
- Ingrosso D., Fowler A. V., Bleibaum J., Clarke S. Sequence of the D-aspartyl/L-isoaspartyl protein methyltransferase from human erythrocytes. Common sequence motifs for protein, DNA, RNA, and small molecule S-adenosylmethionine-dependent methyltransferases. J Biol Chem. 1989 Nov 25;264(33):20131–20139. [PubMed] [Google Scholar]
- Johnson B. F. Growth of the fission yeast, Schizosaccharomyces pombe, with late, eccentric, lytic fission in an unbalanced medium. J Bacteriol. 1967 Jul;94(1):192–195. doi: 10.1128/jb.94.1.192-195.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
- KENNEDY E. P., WEISS S. B. The function of cytidine coenzymes in the biosynthesis of phospholipides. J Biol Chem. 1956 Sep;222(1):193–214. [PubMed] [Google Scholar]
- Kanipes M. I., Henry S. A. The phospholipid methyltransferases in yeast. Biochim Biophys Acta. 1997 Sep 4;1348(1-2):134–141. doi: 10.1016/s0005-2760(97)00121-5. [DOI] [PubMed] [Google Scholar]
- Kent C. Eukaryotic phospholipid biosynthesis. Annu Rev Biochem. 1995;64:315–343. doi: 10.1146/annurev.bi.64.070195.001531. [DOI] [PubMed] [Google Scholar]
- Kodaki T., Yamashita S. Characterization of the methyltransferases in the yeast phosphatidylethanolamine methylation pathway by selective gene disruption. Eur J Biochem. 1989 Nov 6;185(2):243–251. doi: 10.1111/j.1432-1033.1989.tb15109.x. [DOI] [PubMed] [Google Scholar]
- Kodaki T., Yamashita S. Yeast phosphatidylethanolamine methylation pathway. Cloning and characterization of two distinct methyltransferase genes. J Biol Chem. 1987 Nov 15;262(32):15428–15435. [PubMed] [Google Scholar]
- Kohli J., Hottinger H., Munz P., Strauss A., Thuriaux P. Genetic Mapping in SCHIZOSACCHAROMYCES POMBE by Mitotic and Meiotic Analysis and Induced Haploidization. Genetics. 1977 Nov;87(3):471–489. doi: 10.1093/genetics/87.3.471. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kovác L., Gbelská I., Poliachová V., Subík J., Kovácová V. Membrane mutants: a yeast mutant with a lesion in phosphatidylserine biosynthesis. Eur J Biochem. 1980 Oct;111(2):491–501. doi: 10.1111/j.1432-1033.1980.tb04965.x. [DOI] [PubMed] [Google Scholar]
- Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
- Loewy B. S., Henry S. A. The INO2 and INO4 loci of Saccharomyces cerevisiae are pleiotropic regulatory genes. Mol Cell Biol. 1984 Nov;4(11):2479–2485. doi: 10.1128/mcb.4.11.2479. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Majumder A. L., Johnson M. D., Henry S. A. 1L-myo-inositol-1-phosphate synthase. Biochim Biophys Acta. 1997 Sep 4;1348(1-2):245–256. doi: 10.1016/s0005-2760(97)00122-7. [DOI] [PubMed] [Google Scholar]
- Markham P., Bainbridge B. W. Characterization of a new choline locus in Aspergillus nidulans and its significance for choline metabolism. Genet Res. 1978 Nov;32(3):303–310. doi: 10.1017/s0016672300018802. [DOI] [PubMed] [Google Scholar]
- Maundrell K. Thiamine-repressible expression vectors pREP and pRIP for fission yeast. Gene. 1993 Jan 15;123(1):127–130. doi: 10.1016/0378-1119(93)90551-d. [DOI] [PubMed] [Google Scholar]
- McGraw P., Henry S. A. Mutations in the Saccharomyces cerevisiae opi3 gene: effects on phospholipid methylation, growth and cross-pathway regulation of inositol synthesis. Genetics. 1989 Jun;122(2):317–330. doi: 10.1093/genetics/122.2.317. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Patton-Vogt J. L., Griac P., Sreenivas A., Bruno V., Dowd S., Swede M. J., Henry S. A. Role of the yeast phosphatidylinositol/phosphatidylcholine transfer protein (Sec14p) in phosphatidylcholine turnover and INO1 regulation. J Biol Chem. 1997 Aug 15;272(33):20873–20883. doi: 10.1074/jbc.272.33.20873. [DOI] [PubMed] [Google Scholar]
- Perkins D. D. Biochemical Mutants in the Smut Fungus Ustilago Maydis. Genetics. 1949 Sep;34(5):607–626. doi: 10.1093/genetics/34.5.607. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ridgway N. D., Vance D. E. Purification of phosphatidylethanolamine N-methyltransferase from rat liver. J Biol Chem. 1987 Dec 15;262(35):17231–17239. [PubMed] [Google Scholar]
- Rothstein R. J. One-step gene disruption in yeast. Methods Enzymol. 1983;101:202–211. doi: 10.1016/0076-6879(83)01015-0. [DOI] [PubMed] [Google Scholar]
- Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Scarborough G. A., Nyc J. F. Methylation of ethanolamine phosphatides by microsomes from normal and mutant strains of Neurospora crassa. J Biol Chem. 1967 Jan 25;242(2):238–242. [PubMed] [Google Scholar]
- Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Steiner M. R., Lester R. L. In vitro studies of phospholipid biosynthesis in Saccharomyces cerevisiae. Biochim Biophys Acta. 1972 Feb 21;260(2):222–243. doi: 10.1016/0005-2760(72)90035-5. [DOI] [PubMed] [Google Scholar]
- Summers E. F., Letts V. A., McGraw P., Henry S. A. Saccharomyces cerevisiae cho2 mutants are deficient in phospholipid methylation and cross-pathway regulation of inositol synthesis. Genetics. 1988 Dec;120(4):909–922. doi: 10.1093/genetics/120.4.909. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tessitore L., Cui Z., Vance D. E. Transient inactivation of phosphatidylethanolamine N-methyltransferase-2 and activation of cytidine triphosphate: phosphocholine cytidylyltransferase during non-neoplastic liver growth. Biochem J. 1997 Feb 15;322(Pt 1):151–154. doi: 10.1042/bj3220151. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vance D. E., Walkey C. J., Cui Z. Phosphatidylethanolamine N-methyltransferase from liver. Biochim Biophys Acta. 1997 Sep 4;1348(1-2):142–150. doi: 10.1016/s0005-2760(97)00108-2. [DOI] [PubMed] [Google Scholar]
- Waechter C. J., Lester R. L. Regulation of phosphatidylcholine biosynthesis in Saccharomyces cerevisiae. J Bacteriol. 1971 Mar;105(3):837–843. doi: 10.1128/jb.105.3.837-843.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wright A., Maundrell K., Heyer W. D., Beach D., Nurse P. Vectors for the construction of gene banks and the integration of cloned genes in Schizosaccharomyces pombe and Saccharomyces cerevisiae. Plasmid. 1986 Mar;15(2):156–158. doi: 10.1016/0147-619x(86)90051-x. [DOI] [PubMed] [Google Scholar]