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. 1991 Jan;11(1):175–181. doi: 10.1128/mcb.11.1.175

Mutational analysis of the functional domains of yeast K1 killer toxin.

H Zhu 1, H Bussey 1
PMCID: PMC359607  PMID: 1702512

Abstract

To determine the functional domains of K1 killer toxin, we analyzed the phenotypes of a set of mutations throughout regions encoding the alpha- and beta-toxin subunits that allow secretion of mutant toxins. A range of techniques have been used to examine the ability of mutant toxins to bind to beta-glucan cell wall receptor and to form lethal ion channels. Our results indicate that both the alpha and beta subunits are involved in beta-glucan receptor binding. Defects in ion channel formation and toxin immunity are confined to the hydrophobic alpha subunit of the toxin.

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Selected References

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  1. Al-Aidroos K., Bussey H. Chromosomal mutants of Saccharomyces cerevisiae affecting the cell wall binding site for killer factor. Can J Microbiol. 1978 Mar;24(3):228–237. doi: 10.1139/m78-041. [DOI] [PubMed] [Google Scholar]
  2. Boone C., Bussey H., Greene D., Thomas D. Y., Vernet T. Yeast killer toxin: site-directed mutations implicate the precursor protein as the immunity component. Cell. 1986 Jul 4;46(1):105–113. doi: 10.1016/0092-8674(86)90864-0. [DOI] [PubMed] [Google Scholar]
  3. Boone C., Sommer S. S., Hensel A., Bussey H. Yeast KRE genes provide evidence for a pathway of cell wall beta-glucan assembly. J Cell Biol. 1990 May;110(5):1833–1843. doi: 10.1083/jcb.110.5.1833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bostian K. A., Elliott Q., Bussey H., Burn V., Smith A., Tipper D. J. Sequence of the preprotoxin dsRNA gene of type I killer yeast: multiple processing events produce a two-component toxin. Cell. 1984 Mar;36(3):741–751. doi: 10.1016/0092-8674(84)90354-4. [DOI] [PubMed] [Google Scholar]
  5. Bostian K. A., Jayachandran S., Tipper D. J. A glycosylated protoxin in killer yeast: models for its structure and maturation. Cell. 1983 Jan;32(1):169–180. doi: 10.1016/0092-8674(83)90507-x. [DOI] [PubMed] [Google Scholar]
  6. Bussey H., Boone C., Zhu H., Vernet T., Whiteway M., Thomas D. Y. Genetic and molecular approaches to synthesis and action of the yeast killer toxin. Experientia. 1990 Feb 15;46(2):193–200. doi: 10.1007/BF02027313. [DOI] [PubMed] [Google Scholar]
  7. Bussey H., Saville D., Greene D., Tipper D. J., Bostian K. A. Secretion of Saccharomyces cerevisiae killer toxin: processing of the glycosylated precursor. Mol Cell Biol. 1983 Aug;3(8):1362–1370. doi: 10.1128/mcb.3.8.1362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bussey H., Saville D., Hutchins K., Palfree R. G. Binding of yeast killer toxin to a cell wall receptor on sensitive Saccharomyces cerevisiae. J Bacteriol. 1979 Dec;140(3):888–892. doi: 10.1128/jb.140.3.888-892.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bussey H., Vernet T., Sdicu A. M. Mutual antagonism among killer yeasts: competition between K1 and K2 killers and a novel cDNA-based K1-K2 killer strain of Saccharomyces cerevisiae. Can J Microbiol. 1988 Jan;34(1):38–44. doi: 10.1139/m88-007. [DOI] [PubMed] [Google Scholar]
  10. Casadaban M. J., Cohen S. N. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol. 1980 Apr;138(2):179–207. doi: 10.1016/0022-2836(80)90283-1. [DOI] [PubMed] [Google Scholar]
  11. Davis R. W., Thomas M., Cameron J., St John T. P., Scherer S., Padgett R. A. Rapid DNA isolations for enzymatic and hybridization analysis. Methods Enzymol. 1980;65(1):404–411. doi: 10.1016/s0076-6879(80)65051-4. [DOI] [PubMed] [Google Scholar]
  12. Dmochowska A., Dignard D., Henning D., Thomas D. Y., Bussey H. Yeast KEX1 gene encodes a putative protease with a carboxypeptidase B-like function involved in killer toxin and alpha-factor precursor processing. Cell. 1987 Aug 14;50(4):573–584. doi: 10.1016/0092-8674(87)90030-4. [DOI] [PubMed] [Google Scholar]
  13. Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
  14. Hutchins K., Bussey H. Cell wall receptor for yeast killer toxin: involvement of (1 leads to 6)-beta-D-glucan. J Bacteriol. 1983 Apr;154(1):161–169. doi: 10.1128/jb.154.1.161-169.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Kagan B. L. Mode of action of yeast killer toxins: channel formation in lipid bilayer membranes. Nature. 1983 Apr 21;302(5910):709–711. doi: 10.1038/302709a0. [DOI] [PubMed] [Google Scholar]
  17. Kalderon D., Richardson W. D., Markham A. F., Smith A. E. Sequence requirements for nuclear location of simian virus 40 large-T antigen. Nature. 1984 Sep 6;311(5981):33–38. doi: 10.1038/311033a0. [DOI] [PubMed] [Google Scholar]
  18. Konisky J. Colicins and other bacteriocins with established modes of action. Annu Rev Microbiol. 1982;36:125–144. doi: 10.1146/annurev.mi.36.100182.001013. [DOI] [PubMed] [Google Scholar]
  19. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lolle S. J., Bussey H. In vivo evidence for posttranslational translocation and signal cleavage of the killer preprotoxin of Saccharomyces cerevisiae. Mol Cell Biol. 1986 Dec;6(12):4274–4280. doi: 10.1128/mcb.6.12.4274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lolle S., Skipper N., Bussey H., Thomas D. Y. The expression of cDNA clones of yeast M1 double-stranded RNA in yeast confers both killer and immunity phenotypes. EMBO J. 1984 Jun;3(6):1383–1387. doi: 10.1002/j.1460-2075.1984.tb01981.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Martinac B., Zhu H., Kubalski A., Zhou X. L., Culbertson M., Bussey H., Kung C. Yeast K1 killer toxin forms ion channels in sensitive yeast spheroplasts and in artificial liposomes. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6228–6232. doi: 10.1073/pnas.87.16.6228. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Meaden P., Hill K., Wagner J., Slipetz D., Sommer S. S., Bussey H. The yeast KRE5 gene encodes a probable endoplasmic reticulum protein required for (1----6)-beta-D-glucan synthesis and normal cell growth. Mol Cell Biol. 1990 Jun;10(6):3013–3019. doi: 10.1128/mcb.10.6.3013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Middelbeek E. J., Stumm C., Vogels G. D. Effects of Pichia kluyveri killer toxin on sensitive cells. Antonie Van Leeuwenhoek. 1980;46(2):205–220. doi: 10.1007/BF00444075. [DOI] [PubMed] [Google Scholar]
  25. Neville D. M., Jr, Hudson T. H. Transmembrane transport of diphtheria toxin, related toxins, and colicins. Annu Rev Biochem. 1986;55:195–224. doi: 10.1146/annurev.bi.55.070186.001211. [DOI] [PubMed] [Google Scholar]
  26. Olsnes S., Pihl A. Isolation and properties of abrin: a toxic protein inhibiting protein synthesis. Evidence for different biological functions of its two constituent-peptide chains. Eur J Biochem. 1973 May;35(1):179–185. doi: 10.1111/j.1432-1033.1973.tb02823.x. [DOI] [PubMed] [Google Scholar]
  27. Pattus F., Massotte D., Wilmsen H. U., Lakey J., Tsernoglou D., Tucker A., Parker M. W. Colicins: prokaryotic killer-pores. Experientia. 1990 Feb 15;46(2):180–192. [PubMed] [Google Scholar]
  28. 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]
  29. Schauer I., Emr S., Gross C., Schekman R. Invertase signal and mature sequence substitutions that delay intercompartmental transport of active enzyme. J Cell Biol. 1985 May;100(5):1664–1675. doi: 10.1083/jcb.100.5.1664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Sturley S. L., Elliot Q., LeVitre J., Tipper D. J., Bostian K. A. Mapping of functional domains within the Saccharomyces cerevisiae type 1 killer preprotoxin. EMBO J. 1986 Dec 1;5(12):3381–3389. doi: 10.1002/j.1460-2075.1986.tb04654.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Tao J., Ginsberg I., Banerjee N., Held W., Koltin Y., Bruenn J. A. Ustilago maydis KP6 killer toxin: structure, expression in Saccharomyces cerevisiae, and relationship to other cellular toxins. Mol Cell Biol. 1990 Apr;10(4):1373–1381. doi: 10.1128/mcb.10.4.1373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Tipper D. J., Bostian K. A. Double-stranded ribonucleic acid killer systems in yeasts. Microbiol Rev. 1984 Jun;48(2):125–156. doi: 10.1128/mr.48.2.125-156.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Van Ness B. G., Howard J. B., Bodley J. W. ADP-ribosylation of elongation factor 2 by diphtheria toxin. NMR spectra and proposed structures of ribosyl-diphthamide and its hydrolysis products. J Biol Chem. 1980 Nov 25;255(22):10710–10716. [PubMed] [Google Scholar]
  35. Vernet T., Dignard D., Thomas D. Y. A family of yeast expression vectors containing the phage f1 intergenic region. Gene. 1987;52(2-3):225–233. doi: 10.1016/0378-1119(87)90049-7. [DOI] [PubMed] [Google Scholar]
  36. Wickner R. B. Double-stranded RNA replication in yeast: the killer system. Annu Rev Biochem. 1986;55:373–395. doi: 10.1146/annurev.bi.55.070186.002105. [DOI] [PubMed] [Google Scholar]
  37. Wickner R. B., Leibowitz M. J. Two chromosomal genes required for killing expression in killer strains of Saccharomyces cerevisiae. Genetics. 1976 Mar 25;82(3):429–442. doi: 10.1093/genetics/82.3.429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Woods D. R., Bevan E. A. Studies on the nature of the killer factor produced by Saccharomyces cerevisiae. J Gen Microbiol. 1968 Apr;51(1):115–126. doi: 10.1099/00221287-51-1-115. [DOI] [PubMed] [Google Scholar]
  39. Wray W., Boulikas T., Wray V. P., Hancock R. Silver staining of proteins in polyacrylamide gels. Anal Biochem. 1981 Nov 15;118(1):197–203. doi: 10.1016/0003-2697(81)90179-2. [DOI] [PubMed] [Google Scholar]
  40. Young T. W., Yagiu M. A comparison of the killer character in different yeasts and its classification. Antonie Van Leeuwenhoek. 1978;44(1):59–77. doi: 10.1007/BF00400077. [DOI] [PubMed] [Google Scholar]
  41. Zhu H., Bussey H., Thomas D. Y., Gagnon J., Bell A. W. Determination of the carboxyl termini of the alpha and beta subunits of yeast K1 killer toxin. Requirement of a carboxypeptidase B-like activity for maturation. J Biol Chem. 1987 Aug 5;262(22):10728–10732. [PubMed] [Google Scholar]
  42. Zhu Hong, Bussey Howard. The K1 Toxin of Saccharomyces cerevisiae Kills Spheroplasts of Many Yeast Species. Appl Environ Microbiol. 1989 Aug;55(8):2105–2107. doi: 10.1128/aem.55.8.2105-2107.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA. Nucleic Acids Res. 1982 Oct 25;10(20):6487–6500. doi: 10.1093/nar/10.20.6487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. de la Peña P., Barros F., Gascón S., Lazo P. S., Ramos S. Effect of yeast killer toxin on sensitive cells of Saccharomyces cerevisiae. J Biol Chem. 1981 Oct 25;256(20):10420–10425. [PubMed] [Google Scholar]

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