Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1995 Jun;177(11):3227–3234. doi: 10.1128/jb.177.11.3227-3234.1995

Cloning and characterization of GNS1: a Saccharomyces cerevisiae gene involved in synthesis of 1,3-beta-glucan in vitro.

M el-Sherbeini 1, J A Clemas 1
PMCID: PMC177015  PMID: 7768822

Abstract

The GNS1 gene product is required for the synthesis of 1,3-beta-glucan in vitro, since mutations in this gene result in exhibit an 80 to 90% reduction in 1,3-beta-glucan synthase specific activity. gns1 mutant strains display a pleiotropic phenotype including resistance to a pneumocandin B0 analog (L-733,560), slow growth, and mating and sporulation defects. The gns1-1 mutation was genetically mapped to within 1.35 centimorgans from the MAT locus on chromosome III. The wild-type GNS1 gene was isolated by complementing the pneumocandin resistance phenotype of the gns1-1 mutation and by hybridization with a chromosome III-derived sequence being used as a probe. The nucleotide sequence of GNS1 was determined and compared with the homologous region of the chromosome. The genetic and nucleotide sequence analyzes revealed that GNS1 and the open reading frame, YCR34 [S. Oliver, Q. van der Aart, M. Agostoni-Carbone, and the Chromosome III Sequencing Group, Nature (London) 357:38-46, 1992], represent identical loci in the genome. Cells deleted for GNS1 are viable but exhibit slow growth as well as the pleiotropic phenotype of the gns1 mutants. The putative protein product is predicted to be an integral membrane protein with five transmembrane helices displaying an exoplasmic orientation for the N terminus and a cytoplasmic orientation for the C terminus. This protein may be a subunit of 1,3-beta-glucan synthase.

Full Text

The Full Text of this article is available as a PDF (366.8 KB).

Selected References

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

  1. Baguley B. C., Römmele G., Gruner J., Wehrli W. Papulacandin B: an inhibitor of glucan synthesis in yeast spheroplasts. Eur J Biochem. 1979 Jul;97(2):345–351. doi: 10.1111/j.1432-1033.1979.tb13120.x. [DOI] [PubMed] [Google Scholar]
  2. Bastide M., Jouvert S., Bastide J. M. A comparison of the effects of several antifungal imidazole derivatives and polyenes on Candida albicans: an ultrastructural study by scanning electron microscopy. Can J Microbiol. 1982 Oct;28(10):1119–1126. doi: 10.1139/m82-166. [DOI] [PubMed] [Google Scholar]
  3. Becker D. M., Guarente L. High-efficiency transformation of yeast by electroporation. Methods Enzymol. 1991;194:182–187. doi: 10.1016/0076-6879(91)94015-5. [DOI] [PubMed] [Google Scholar]
  4. Bouffard F. A., Zambias R. A., Dropinski J. F., Balkovec J. M., Hammond M. L., Abruzzo G. K., Bartizal K. F., Marrinan J. A., Kurtz M. B., McFadden D. C. Synthesis and antifungal activity of novel cationic pneumocandin B(o) derivatives. J Med Chem. 1994 Jan 21;37(2):222–225. doi: 10.1021/jm00028a003. [DOI] [PubMed] [Google Scholar]
  5. Cabib E. Differential inhibition of chitin synthetases 1 and 2 from Saccharomyces cerevisiae by polyoxin D and nikkomycins. Antimicrob Agents Chemother. 1991 Jan;35(1):170–173. doi: 10.1128/aac.35.1.170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cabib E., Kang M. S. Fungal 1,3-beta-glucan synthase. Methods Enzymol. 1987;138:637–642. doi: 10.1016/0076-6879(87)38057-7. [DOI] [PubMed] [Google Scholar]
  7. Cabib E., Roberts R., Bowers B. Synthesis of the yeast cell wall and its regulation. Annu Rev Biochem. 1982;51:763–793. doi: 10.1146/annurev.bi.51.070182.003555. [DOI] [PubMed] [Google Scholar]
  8. Decker H., Walz F., Bormann C., Zähner H., Fiedler H. P., Heitsch H., König W. A. Metabolic products of microorganisms. 255. Nikkomycins Wz and Wx, new chitin synthetase inhibitors from Streptomyces tendae. J Antibiot (Tokyo) 1990 Jan;43(1):43–48. doi: 10.7164/antibiotics.43.43. [DOI] [PubMed] [Google Scholar]
  9. Douglas C. M., Foor F., Marrinan J. A., Morin N., Nielsen J. B., Dahl A. M., Mazur P., Baginsky W., Li W., el-Sherbeini M. The Saccharomyces cerevisiae FKS1 (ETG1) gene encodes an integral membrane protein which is a subunit of 1,3-beta-D-glucan synthase. Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12907–12911. doi: 10.1073/pnas.91.26.12907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Douglas C. M., Marrinan J. A., Li W., Kurtz M. B. A Saccharomyces cerevisiae mutant with echinocandin-resistant 1,3-beta-D-glucan synthase. J Bacteriol. 1994 Sep;176(18):5686–5696. doi: 10.1128/jb.176.18.5686-5696.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Feinberg A. P., Vogelstein B. "A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity". Addendum. Anal Biochem. 1984 Feb;137(1):266–267. doi: 10.1016/0003-2697(84)90381-6. [DOI] [PubMed] [Google Scholar]
  12. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  13. Hoffman C. S., Winston F. A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene. 1987;57(2-3):267–272. doi: 10.1016/0378-1119(87)90131-4. [DOI] [PubMed] [Google Scholar]
  14. Kang M. S., Cabib E. Regulation of fungal cell wall growth: a guanine nucleotide-binding, proteinaceous component required for activity of (1----3)-beta-D-glucan synthase. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5808–5812. doi: 10.1073/pnas.83.16.5808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kang M. S., Elango N., Mattia E., Au-Young J., Robbins P. W., Cabib E. Isolation of chitin synthetase from Saccharomyces cerevisiae. Purification of an enzyme by entrapment in the reaction product. J Biol Chem. 1984 Dec 10;259(23):14966–14972. [PubMed] [Google Scholar]
  16. Kasahara S., Yamada H., Mio T., Shiratori Y., Miyamoto C., Yabe T., Nakajima T., Ichishima E., Furuichi Y. Cloning of the Saccharomyces cerevisiae gene whose overexpression overcomes the effects of HM-1 killer toxin, which inhibits beta-glucan synthesis. J Bacteriol. 1994 Mar;176(5):1488–1499. doi: 10.1128/jb.176.5.1488-1499.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kurtz M. B., Douglas C., Marrinan J., Nollstadt K., Onishi J., Dreikorn S., Milligan J., Mandala S., Thompson J., Balkovec J. M. Increased antifungal activity of L-733,560, a water-soluble, semisynthetic pneumocandin, is due to enhanced inhibition of cell wall synthesis. Antimicrob Agents Chemother. 1994 Dec;38(12):2750–2757. doi: 10.1128/aac.38.12.2750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lehle L., Tanner W. The specific site of tunicamycin inhibition in the formation of dolichol-bound N-acetylglucosamine derivatives. FEBS Lett. 1976 Nov 15;72(1):167–170. doi: 10.1016/0014-5793(76)80922-2. [DOI] [PubMed] [Google Scholar]
  19. Mizoguchi J., Saito T., Mizuno K., Hayano K. On the mode of action of a new antifungal antibiotic, aculeacin A: inhibition of cell wall synthesis in Saccharomyces cerevisiae. J Antibiot (Tokyo) 1977 Apr;30(4):308–313. doi: 10.7164/antibiotics.30.308. [DOI] [PubMed] [Google Scholar]
  20. Mullis K. B., Faloona F. A. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 1987;155:335–350. doi: 10.1016/0076-6879(87)55023-6. [DOI] [PubMed] [Google Scholar]
  21. O'Shea E. K., Rutkowski R., Kim P. S. Evidence that the leucine zipper is a coiled coil. Science. 1989 Jan 27;243(4890):538–542. doi: 10.1126/science.2911757. [DOI] [PubMed] [Google Scholar]
  22. Odds F. C., Cockayne A., Hayward J., Abbott A. B. Effects of imidazole- and triazole-derivative antifungal compounds on the growth and morphological development of Candida albicans hyphae. J Gen Microbiol. 1985 Oct;131(10):2581–2589. doi: 10.1099/00221287-131-10-2581. [DOI] [PubMed] [Google Scholar]
  23. Oliver S. G., van der Aart Q. J., Agostoni-Carbone M. L., Aigle M., Alberghina L., Alexandraki D., Antoine G., Anwar R., Ballesta J. P., Benit P. The complete DNA sequence of yeast chromosome III. Nature. 1992 May 7;357(6373):38–46. doi: 10.1038/357038a0. [DOI] [PubMed] [Google Scholar]
  24. Ribas J. C., Diaz M., Duran A., Perez P. Isolation and characterization of Schizosaccharomyces pombe mutants defective in cell wall (1-3)beta-D-glucan. J Bacteriol. 1991 Jun;173(11):3456–3462. doi: 10.1128/jb.173.11.3456-3462.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Roemer T., Bussey H. Yeast beta-glucan synthesis: KRE6 encodes a predicted type II membrane protein required for glucan synthesis in vivo and for glucan synthase activity in vitro. Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11295–11299. doi: 10.1073/pnas.88.24.11295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rose M. D., Novick P., Thomas J. H., Botstein D., Fink G. R. A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene. 1987;60(2-3):237–243. doi: 10.1016/0378-1119(87)90232-0. [DOI] [PubMed] [Google Scholar]
  27. 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]
  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. Schmatz D. M., Romancheck M. A., Pittarelli L. A., Schwartz R. E., Fromtling R. A., Nollstadt K. H., Vanmiddlesworth F. L., Wilson K. E., Turner M. J. Treatment of Pneumocystis carinii pneumonia with 1,3-beta-glucan synthesis inhibitors. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5950–5954. doi: 10.1073/pnas.87.15.5950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sipos L., von Heijne G. Predicting the topology of eukaryotic membrane proteins. Eur J Biochem. 1993 May 1;213(3):1333–1340. doi: 10.1111/j.1432-1033.1993.tb17885.x. [DOI] [PubMed] [Google Scholar]
  31. Taft C. S., Stark T., Selitrennikoff C. P. Cilofungin (LY121019) inhibits Candida albicans (1-3)-beta-D-glucan synthase activity. Antimicrob Agents Chemother. 1988 Dec;32(12):1901–1903. doi: 10.1128/aac.32.12.1901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. VanMiddlesworth F., Omstead M. N., Schmatz D., Bartizal K., Fromtling R., Bills G., Nollstadt K., Honeycutt S., Zweerink M., Garrity G. L-687,781, a new member of the papulacandin family of beta-1,3-D-glucan synthesis inhibitors. I. Fermentation, isolation, and biological activity. J Antibiot (Tokyo) 1991 Jan;44(1):45–51. doi: 10.7164/antibiotics.44.45. [DOI] [PubMed] [Google Scholar]
  33. Vanden Bossche H., Willemsens G., Marichal P. Anti-Candida drugs--the biochemical basis for their activity. Crit Rev Microbiol. 1987;15(1):57–72. doi: 10.3109/10408418709104448. [DOI] [PubMed] [Google Scholar]
  34. Yamaguchi H., Hiratani T., Iwata K., Yamamoto Y. Studies on the mechanism of antifungal action of aculeacin A. J Antibiot (Tokyo) 1982 Feb;35(2):210–219. doi: 10.7164/antibiotics.35.210. [DOI] [PubMed] [Google Scholar]
  35. Yamamoto T., Hiratani T., Hirata H., Imai M., Yamaguchi H. Killer toxin from Hansenula mrakii selectively inhibits cell wall synthesis in a sensitive yeast. FEBS Lett. 1986 Mar 3;197(1-2):50–54. doi: 10.1016/0014-5793(86)80296-4. [DOI] [PubMed] [Google Scholar]
  36. Zlotnik H., Fernandez M. P., Bowers B., Cabib E. Saccharomyces cerevisiae mannoproteins form an external cell wall layer that determines wall porosity. J Bacteriol. 1984 Sep;159(3):1018–1026. doi: 10.1128/jb.159.3.1018-1026.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. de Nobel J. G., Klis F. M., Priem J., Munnik T., van den Ende H. The glucanase-soluble mannoproteins limit cell wall porosity in Saccharomyces cerevisiae. Yeast. 1990 Nov-Dec;6(6):491–499. doi: 10.1002/yea.320060606. [DOI] [PubMed] [Google Scholar]
  38. el-Sherbeini M., Clemas J. A. Nikkomycin Z supersensitivity of an echinocandin-resistant mutant of Saccharomyces cerevisiae. Antimicrob Agents Chemother. 1995 Jan;39(1):200–207. doi: 10.1128/aac.39.1.200. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES