Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1989 May 11;17(9):3435–3446. doi: 10.1093/nar/17.9.3435

Expression of pGKL killer 28K subunit in Saccharomyces cerevisiae: identification of 28K subunit as a killer protein.

M Tokunaga 1, A Kawamura 1, F Hishinuma 1
PMCID: PMC317786  PMID: 2657659

Abstract

Saccharomyces cerevisiae and other yeast cells harboring the linear double stranded (ds) DNA plasmids pGKL1 and pGKL2 secrete a killer toxin consisting of 97K, 31K and 28K subunits into the culture medium (EMBO J. 5, 1995-2002 (1986), Nucleic Acids Res., 15, 1031-1046 (1987]. The 28K subunit of the killer toxin was successfully expressed in S. cerevisiae when it was cloned on a circular plasmid with its putative promoter region replaced with that of S. cerevisiae chromosomal genes. The expression of the 28K subunit of the killer toxin in killer-sensitive cells resulted in the death of the host cells. This killing activity by the 28K subunit was prevented by the expression of the killer immunity, indicating that the killing activity of the killer toxin complex was carried out by the 28K subunit. Although the 28K subunit was synthesized as a intact precursor protein with its own signal sequence, it was not secreted into the culture medium but remained in the host cells. This indicated that 28K subunit killed host cells from inside of the cells rather than from outside. We further suggested that 28K killer subunit without 97K and 31K subunits did not kill the killer-sensitive cells from outside.

Full text

PDF
3435

Images in this article

3441Image
on p.3441
3443Image
on p.3443

Selected References

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

  1. Guarente L., Yocum R. R., Gifford P. A GAL10-CYC1 hybrid yeast promoter identifies the GAL4 regulatory region as an upstream site. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7410–7414. doi: 10.1073/pnas.79.23.7410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Gunge N., Murata K., Sakaguchi K. Transformation of Saccharomyces cerevisiae with linear DNA killer plasmids from Kluyveromyces lactis. J Bacteriol. 1982 Jul;151(1):462–464. doi: 10.1128/jb.151.1.462-464.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Gunge N., Sakaguchi K. Intergeneric transfer of deoxyribonucleic acid killer plasmids, pGKl1 and pGKl2, from Kluyveromyces lactis into Saccharomyces cerevisiae by cell fusion. J Bacteriol. 1981 Jul;147(1):155–160. doi: 10.1128/jb.147.1.155-160.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Gunge N., Tamaru A., Ozawa F., Sakaguchi K. Isolation and characterization of linear deoxyribonucleic acid plasmids from Kluyveromyces lactis and the plasmid-associated killer character. J Bacteriol. 1981 Jan;145(1):382–390. doi: 10.1128/jb.145.1.382-390.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Hishinuma F., Nakamura K., Hirai K., Nishizawa R., Gunge N., Maeda T. Cloning and nucleotide sequences of the linear DNA killer plasmids from yeast. Nucleic Acids Res. 1984 Oct 11;12(19):7581–7597. doi: 10.1093/nar/12.19.7581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Inokuchi K., Nakayama A., Hishinuma F. Identification of sequence elements that confer cell-type-specific control of MF alpha 1 expression in Saccharomyces cerevisiae. Mol Cell Biol. 1987 Sep;7(9):3185–3193. doi: 10.1128/mcb.7.9.3185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Nilsson B., Abrahmsén L., Uhlén M. Immobilization and purification of enzymes with staphylococcal protein A gene fusion vectors. EMBO J. 1985 Apr;4(4):1075–1080. doi: 10.1002/j.1460-2075.1985.tb03741.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Niwa O., Sakaguchi K., Gunge N. Curing of the killer deoxyribonucleic acid plasmids of Kluyveromyces lactis. J Bacteriol. 1981 Dec;148(3):988–990. doi: 10.1128/jb.148.3.988-990.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Romanos M. A., Boyd A. A transcriptional barrier to expression of cloned toxin genes of the linear plasmid k1 of Kluyveromyces lactis: evidence that native k1 has novel promoters. Nucleic Acids Res. 1988 Aug 11;16(15):7333–7350. doi: 10.1093/nar/16.15.7333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Stam J. C., Kwakman J., Meijer M., Stuitje A. R. Efficient isolation of the linear DNA killer plasmid of Kluyveromyces lactis: evidence for location and expression in the cytoplasm and characterization of their terminally bound proteins. Nucleic Acids Res. 1986 Sep 11;14(17):6871–6884. doi: 10.1093/nar/14.17.6871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Stark M. J., Boyd A. The killer toxin of Kluyveromyces lactis: characterization of the toxin subunits and identification of the genes which encode them. EMBO J. 1986 Aug;5(8):1995–2002. doi: 10.1002/j.1460-2075.1986.tb04455.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Stark M. J., Mileham A. J., Romanos M. A., Boyd A. Nucleotide sequence and transcription analysis of a linear DNA plasmid associated with the killer character of the yeast Kluyveromyces lactis. Nucleic Acids Res. 1984 Aug 10;12(15):6011–6030. doi: 10.1093/nar/12.15.6011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Sugisaki Y., Gunge N., Sakaguchi K., Yamasaki M., Tamura G. Characterization of a novel killer toxin encoded by a double-stranded linear DNA plasmid of Kluyveromyces lactis. Eur J Biochem. 1984 Jun 1;141(2):241–245. doi: 10.1111/j.1432-1033.1984.tb08183.x. [DOI] [PubMed] [Google Scholar]
  15. Tajima M., Nogi Y., Fukasawa T. Duplicate upstream activating sequences in the promoter region of the Saccharomyces cerevisiae GAL7 gene. Mol Cell Biol. 1986 Jan;6(1):246–256. doi: 10.1128/mcb.6.1.246. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Tokunaga M., Wada N., Hishinuma F. A novel yeast secretion signal isolated from 28K killer precursor protein encoded on the linear DNA plasmid pGKL1. Nucleic Acids Res. 1988 Aug 11;16(15):7499–7511. doi: 10.1093/nar/16.15.7499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Tokunaga M., Wada N., Hishinuma F. A novel yeast secretion vector utilizing secretion signal of killer toxin encoded on the yeast linear DNA plasmid pGKL1. Biochem Biophys Res Commun. 1987 Apr 29;144(2):613–619. doi: 10.1016/s0006-291x(87)80010-4. [DOI] [PubMed] [Google Scholar]
  18. Tokunaga M., Wada N., Hishinuma F. Expression and identification of immunity determinants on linear DNA killer plasmids pGKL1 and pGKL2 in Kluyveromyces lactis. Nucleic Acids Res. 1987 Feb 11;15(3):1031–1046. doi: 10.1093/nar/15.3.1031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Wilson D. W., Meacock P. A. Extranuclear gene expression in yeast: evidence for a plasmid-encoded RNA polymerase of unique structure. Nucleic Acids Res. 1988 Aug 25;16(16):8097–8112. [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES