Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1991 Mar;173(6):2134–2136. doi: 10.1128/jb.173.6.2134-2136.1991

Expression in Escherichia coli of the Saccharomyces cerevisiae CCT gene encoding cholinephosphate cytidylyltransferase.

Y Tsukagoshi 1, J Nikawa 1, K Hosaka 1, S Yamashita 1
PMCID: PMC207753  PMID: 1848222

Abstract

The coding region of the CCT gene from the yeast Saccharomyces cerevisiae was cloned into the pUC18 expression vector. The plasmid directed the synthesis of an active cholinephosphate cytidylyltransferase in Escherichia coli, confirming that CCT is the structural gene for this enzyme. The enzyme produced in E. coli efficiently utilized cholinephosphate and N,N-dimethylethanolaminephosphate, but N-methylethanolamine-phosphate and ethanolaminephosphate were poor substrates. Consistently, disruption of the CCT locus in the wild-type yeast cells resulted in a drastic decrease in activities with respect to the former two substrates. When activity was expressed in E. coli, over 90% was recovered in the cytosol, whereas most of the activity of yeast cells was associated with membranes, suggesting that yeast cells possess a mechanism that promotes membrane association of cytidylyltransferase.

Full text

PDF

Images in this article

2135Image
on p.2135

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Feldman D. A., Weinhold P. A. CTP:phosphorylcholine cytidylyltransferase from rat liver. Isolation and characterization of the catalytic subunit. J Biol Chem. 1987 Jul 5;262(19):9075–9081. [PubMed] [Google Scholar]
  2. Hosaka K., Kodaki T., Yamashita S. Cloning and characterization of the yeast CKI gene encoding choline kinase and its expression in Escherichia coli. J Biol Chem. 1989 Feb 5;264(4):2053–2059. [PubMed] [Google Scholar]
  3. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Nikawa J., Kodaki T., Yamashita S. Expression of the Saccharomyces cerevisiae PIS gene and synthesis of phosphatidylinositol in Escherichia coli. J Bacteriol. 1988 Oct;170(10):4727–4731. doi: 10.1128/jb.170.10.4727-4731.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Nikawa J., Yonemura K., Yamashita S. Yeast mutant with thermolabile CDP-choline synthesis. Isolation and characterization of a cholinephosphate cytidyltransferase mutant. Eur J Biochem. 1983 Mar 1;131(1):223–229. doi: 10.1111/j.1432-1033.1983.tb07253.x. [DOI] [PubMed] [Google Scholar]
  7. Pelech S. L., Paddon H. B., Vance D. E. Phorbol esters stimulate phosphatidylcholine biosynthesis by translocation of CTP:phosphocholine cytidylyltransferase from cytosol to microsomes. Biochim Biophys Acta. 1984 Oct 4;795(3):447–451. doi: 10.1016/0005-2760(84)90171-1. [DOI] [PubMed] [Google Scholar]
  8. Pelech S. L., Pritchard P. H., Brindley D. N., Vance D. E. Fatty acids promote translocation of CTP:phosphocholine cytidylyltransferase to the endoplasmic reticulum and stimulate rat hepatic phosphatidylcholine synthesis. J Biol Chem. 1983 Jun 10;258(11):6782–6788. [PubMed] [Google Scholar]
  9. Pelech S. L., Vance D. E. Regulation of phosphatidylcholine biosynthesis. Biochim Biophys Acta. 1984 Jun 25;779(2):217–251. doi: 10.1016/0304-4157(84)90010-8. [DOI] [PubMed] [Google Scholar]
  10. Rose M., Grisafi P., Botstein D. Structure and function of the yeast URA3 gene: expression in Escherichia coli. Gene. 1984 Jul-Aug;29(1-2):113–124. doi: 10.1016/0378-1119(84)90172-0. [DOI] [PubMed] [Google Scholar]
  11. Sanghera J. S., Vance D. E. CTP:phosphocholine cytidylyltransferase is a substrate for cAMP-dependent protein kinase in vitro. J Biol Chem. 1989 Jan 15;264(2):1215–1223. [PubMed] [Google Scholar]
  12. Sleight R., Kent C. Regulation of phosphatidylcholine biosynthesis in cultured chick embryonic muscle treated with phospholipase C. J Biol Chem. 1980 Nov 25;255(22):10644–10650. [PubMed] [Google Scholar]
  13. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Sundler R. Ethanolaminephosphate cytidylyltransferase. Purification and characterization of the enzyme from rat liver. J Biol Chem. 1975 Nov 25;250(22):8585–8590. [PubMed] [Google Scholar]
  16. Tsukagoshi Y., Nikawa J., Yamashita S. Molecular cloning and characterization of the gene encoding cholinephosphate cytidylyltransferase in Saccharomyces cerevisiae. Eur J Biochem. 1987 Dec 15;169(3):477–486. doi: 10.1111/j.1432-1033.1987.tb13635.x. [DOI] [PubMed] [Google Scholar]
  17. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  18. Yao Z. M., Jamil H., Vance D. E. Choline deficiency causes translocation of CTP:phosphocholine cytidylyltransferase from cytosol to endoplasmic reticulum in rat liver. J Biol Chem. 1990 Mar 15;265(8):4326–4331. [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES