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. 1997 Jul;179(13):4096–4105. doi: 10.1128/jb.179.13.4096-4105.1997

Cloning of the Candida albicans homolog of Saccharomyces cerevisiae GSC1/FKS1 and its involvement in beta-1,3-glucan synthesis.

T Mio 1, M Adachi-Shimizu 1, Y Tachibana 1, H Tabuchi 1, S B Inoue 1, T Yabe 1, T Yamada-Okabe 1, M Arisawa 1, T Watanabe 1, H Yamada-Okabe 1
PMCID: PMC179227  PMID: 9209021

Abstract

Saccharomyces cerevisiae GSC1 (also called FKS1) and GSC2 (also called FKS2) have been identified as the genes for putative catalytic subunits of beta-1,3-glucan synthase. We have cloned three Candida albicans genes, GSC1, GSL1, and GSL2, that have significant sequence homologies with S. cerevisiae GSC1/FKS1, GSC2/FKS2, and the recently identified FKSA of Aspergillus nidulans at both nucleotide and amino acid levels. Like S. cerevisiae Gsc/Fks proteins, none of the predicted products of C. albicans GSC1, GSL1, or GSL2 displayed obvious signal sequences at their N-terminal ends, but each product possessed 10 to 16 potential transmembrane helices with a relatively long cytoplasmic domain in the middle of the protein. Northern blotting demonstrated that C. albicans GSC1 and GSL1 but not GSL2 mRNAs were expressed in the growing yeast-phase cells. Three copies of GSC1 were found in the diploid genome of C. albicans CAI4. Although we could not establish the null mutation of C. albicans GSC1, disruption of two of the three GSC1 alleles decreased both GSC1 mRNA and cell wall beta-glucan levels by about 50%. The purified C. albicans beta-1,3-glucan synthase was a 210-kDa protein as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and all sequences determined with peptides obtained by lysyl endopeptidase digestion of the 210-kDa protein were found in the deduced amino acid sequence of C. albicans Gsc1p. Furthermore, the monoclonal antibody raised against the purified beta-1,3-glucan synthase specifically reacted with the 210-kDa protein and could immunoprecipitate beta-1,3-glucan synthase activity. These results demonstrate that C. albicans GSC1 is the gene for a subunit of beta-1,3-glucan synthase.

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Selected References

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  1. Alani E., Cao L., Kleckner N. A method for gene disruption that allows repeated use of URA3 selection in the construction of multiply disrupted yeast strains. Genetics. 1987 Aug;116(4):541–545. doi: 10.1534/genetics.112.541.test. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Au-Young J., Robbins P. W. Isolation of a chitin synthase gene (CHS1) from Candida albicans by expression in Saccharomyces cerevisiae. Mol Microbiol. 1990 Feb;4(2):197–207. doi: 10.1111/j.1365-2958.1990.tb00587.x. [DOI] [PubMed] [Google Scholar]
  3. Botstein D., Falco S. C., Stewart S. E., Brennan M., Scherer S., Stinchcomb D. T., Struhl K., Davis R. W. Sterile host yeasts (SHY): a eukaryotic system of biological containment for recombinant DNA experiments. Gene. 1979 Dec;8(1):17–24. doi: 10.1016/0378-1119(79)90004-0. [DOI] [PubMed] [Google Scholar]
  4. Castro C., Ribas J. C., Valdivieso M. H., Varona R., del Rey F., Duran A. Papulacandin B resistance in budding and fission yeasts: isolation and characterization of a gene involved in (1,3)beta-D-glucan synthesis in Saccharomyces cerevisiae. J Bacteriol. 1995 Oct;177(20):5732–5739. doi: 10.1128/jb.177.20.5732-5739.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chen-Wu J. L., Zwicker J., Bowen A. R., Robbins P. W. Expression of chitin synthase genes during yeast and hyphal growth phases of Candida albicans. Mol Microbiol. 1992 Feb;6(4):497–502. doi: 10.1111/j.1365-2958.1992.tb01494.x. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Drgonová J., Drgon T., Tanaka K., Kollár R., Chen G. C., Ford R. A., Chan C. S., Takai Y., Cabib E. Rho1p, a yeast protein at the interface between cell polarization and morphogenesis. Science. 1996 Apr 12;272(5259):277–279. doi: 10.1126/science.272.5259.277. [DOI] [PubMed] [Google Scholar]
  9. Eng W. K., Faucette L., McLaughlin M. M., Cafferkey R., Koltin Y., Morris R. A., Young P. R., Johnson R. K., Livi G. P. The yeast FKS1 gene encodes a novel membrane protein, mutations in which confer FK506 and cyclosporin A hypersensitivity and calcineurin-dependent growth. Gene. 1994 Dec 30;151(1-2):61–71. doi: 10.1016/0378-1119(94)90633-5. [DOI] [PubMed] [Google Scholar]
  10. Garrett-Engele P., Moilanen B., Cyert M. S. Calcineurin, the Ca2+/calmodulin-dependent protein phosphatase, is essential in yeast mutants with cell integrity defects and in mutants that lack a functional vacuolar H(+)-ATPase. Mol Cell Biol. 1995 Aug;15(8):4103–4114. doi: 10.1128/mcb.15.8.4103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Inoue S. B., Takewaki N., Takasuka T., Mio T., Adachi M., Fujii Y., Miyamoto C., Arisawa M., Furuichi Y., Watanabe T. Characterization and gene cloning of 1,3-beta-D-glucan synthase from Saccharomyces cerevisiae. Eur J Biochem. 1995 Aug 1;231(3):845–854. doi: 10.1111/j.1432-1033.1995.tb20770.x. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. Kasahara S., Ben Inoue S., Mio T., Yamada T., Nakajima T., Ichishima E., Furuichi Y., Yamada H. Involvement of cell wall beta-glucan in the action of HM-1 killer toxin. FEBS Lett. 1994 Jul 4;348(1):27–32. doi: 10.1016/0014-5793(94)00575-3. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Kelly R., Register E., Hsu M. J., Kurtz M., Nielsen J. Isolation of a gene involved in 1,3-beta-glucan synthesis in Aspergillus nidulans and purification of the corresponding protein. J Bacteriol. 1996 Aug;178(15):4381–4391. doi: 10.1128/jb.178.15.4381-4391.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kollár R., Petráková E., Ashwell G., Robbins P. W., Cabib E. Architecture of the yeast cell wall. The linkage between chitin and beta(1-->3)-glucan. J Biol Chem. 1995 Jan 20;270(3):1170–1178. doi: 10.1074/jbc.270.3.1170. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Mazur P., Morin N., Baginsky W., el-Sherbeini M., Clemas J. A., Nielsen J. B., Foor F. Differential expression and function of two homologous subunits of yeast 1,3-beta-D-glucan synthase. Mol Cell Biol. 1995 Oct;15(10):5671–5681. doi: 10.1128/mcb.15.10.5671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mio T., Yabe T., Sudoh M., Satoh Y., Nakajima T., Arisawa M., Yamada-Okabe H. Role of three chitin synthase genes in the growth of Candida albicans. J Bacteriol. 1996 Apr;178(8):2416–2419. doi: 10.1128/jb.178.8.2416-2419.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mio T., Yamada-Okabe T., Yabe T., Nakajima T., Arisawa M., Yamada-Okabe H. Isolation of the Candida albicans homologs of Saccharomyces cerevisiae KRE6 and SKN1: expression and physiological function. J Bacteriol. 1997 Apr;179(7):2363–2372. doi: 10.1128/jb.179.7.2363-2372.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mol P. C., Park H. M., Mullins J. T., Cabib E. A GTP-binding protein regulates the activity of (1-->3)-beta-glucan synthase, an enzyme directly involved in yeast cell wall morphogenesis. J Biol Chem. 1994 Dec 9;269(49):31267–31274. [PubMed] [Google Scholar]
  23. Odds F. C. Candida infections: an overview. Crit Rev Microbiol. 1987;15(1):1–5. doi: 10.3109/10408418709104444. [DOI] [PubMed] [Google Scholar]
  24. Orlean P. A. (1,3)-beta-D-Glucan synthase from budding and filamentous cultures of the dimorphic fungus Candida albicans. Eur J Biochem. 1982 Oct;127(2):397–403. doi: 10.1111/j.1432-1033.1982.tb06885.x. [DOI] [PubMed] [Google Scholar]
  25. Qadota H., Python C. P., Inoue S. B., Arisawa M., Anraku Y., Zheng Y., Watanabe T., Levin D. E., Ohya Y. Identification of yeast Rho1p GTPase as a regulatory subunit of 1,3-beta-glucan synthase. Science. 1996 Apr 12;272(5259):279–281. doi: 10.1126/science.272.5259.279. [DOI] [PubMed] [Google Scholar]
  26. Ram A. F., Brekelmans S. S., Oehlen L. J., Klis F. M. Identification of two cell cycle regulated genes affecting the beta 1,3-glucan content of cell walls in Saccharomyces cerevisiae. FEBS Lett. 1995 Jan 23;358(2):165–170. doi: 10.1016/0014-5793(94)01418-z. [DOI] [PubMed] [Google Scholar]
  27. Ram A. F., Wolters A., Ten Hoopen R., Klis F. M. A new approach for isolating cell wall mutants in Saccharomyces cerevisiae by screening for hypersensitivity to calcofluor white. Yeast. 1994 Aug;10(8):1019–1030. doi: 10.1002/yea.320100804. [DOI] [PubMed] [Google Scholar]
  28. Santos M. A., Tuite M. F. The CUG codon is decoded in vivo as serine and not leucine in Candida albicans. Nucleic Acids Res. 1995 May 11;23(9):1481–1486. doi: 10.1093/nar/23.9.1481. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sawistowska-Schröder E. T., Kerridge D., Perry H. Echinocandin inhibition of 1,3-beta-D-glucan synthase from Candida albicans. FEBS Lett. 1984 Jul 23;173(1):134–138. doi: 10.1016/0014-5793(84)81032-7. [DOI] [PubMed] [Google Scholar]
  30. Shaw J. A., Mol P. C., Bowers B., Silverman S. J., Valdivieso M. H., Durán A., Cabib E. The function of chitin synthases 2 and 3 in the Saccharomyces cerevisiae cell cycle. J Cell Biol. 1991 Jul;114(1):111–123. doi: 10.1083/jcb.114.1.111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Shematek E. M., Braatz J. A., Cabib E. Biosynthesis of the yeast cell wall. I. Preparation and properties of beta-(1 leads to 3)glucan synthetase. J Biol Chem. 1980 Feb 10;255(3):888–894. [PubMed] [Google Scholar]
  32. Short J. M., Fernandez J. M., Sorge J. A., Huse W. D. Lambda ZAP: a bacteriophage lambda expression vector with in vivo excision properties. Nucleic Acids Res. 1988 Aug 11;16(15):7583–7600. doi: 10.1093/nar/16.15.7583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sudoh M., Nagahashi S., Doi M., Ohta A., Takagi M., Arisawa M. Cloning of the chitin synthase 3 gene from Candida albicans and its expression during yeast-hyphal transition. Mol Gen Genet. 1993 Nov;241(3-4):351–358. doi: 10.1007/BF00284688. [DOI] [PubMed] [Google Scholar]
  34. 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]

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