Abstract
The GGP1/GAS1 gene codes for a glycosylphosphatidylinositol-anchored plasma membrane glycoprotein of Saccharomyces cerevisiae. The ggp1delta mutant shows morphogenetic defects which suggest changes in the cell wall matrix. In this work, we have investigated cell wall glucan levels and the increase of chitin in ggp1delta mutant cells. In these cells, the level of alkali-insoluble 1,6-beta-D-glucan was found to be 50% of that of wild-type cells and was responsible for the observed decrease in the total alkali-insoluble glucan. Moreover, the ratio of alkali-soluble to alkali-insoluble glucan almost doubled, suggesting a change in glucan solubility. The increase of chitin in ggp1delta cells was found to be essential since the chs3delta ggp1delta mutations determined a severe reduction in the growth rate and in cell viability. Electron microscopy analysis showed the loss of the typical structure of yeast cell walls. Furthermore, in the chs3delta ggp1delta cells, the level of alkali-insoluble glucan was 57% of that of wild-type cells and the alkali-soluble/alkali-insoluble glucan ratio was doubled. We tested the effect of inhibition of chitin synthesis also by a different approach. The ggp1delta cells were treated with nikkomycin Z, a well-known inhibitor of chitin synthesis, and showed a hypersensitivity to this drug. In addition, studies of genetic interactions with genes related to the construction of the cell wall indicate a synthetic lethal effect of the ggp1delta kre6delta and the ggp1delta pkc1delta combined mutations. Our data point to an involvement of the GGP1 gene product in the cross-links between cell wall glucans (1,3-beta-D-glucans with 1,6-beta-D-glucans and with chitin). Chitin is essential to compensate for the defects due to the lack of Ggp1p. Moreover, the activities of Ggp1p and Chs3p are essential to the formation of the organized structure of the cell wall in vegetative cells.
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- BADIN J., JACKSON C., SCHUBERT M. Improved method for determination of plasma polysaccharides with tryptophan. Proc Soc Exp Biol Med. 1953 Nov;84(2):289–291. [PubMed] [Google Scholar]
- 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]
- 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]
- Choi W. J., Santos B., Durán A., Cabib E. Are yeast chitin synthases regulated at the transcriptional or the posttranslational level? Mol Cell Biol. 1994 Dec;14(12):7685–7694. doi: 10.1128/mcb.14.12.7685. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cid V. J., Durán A., del Rey F., Snyder M. P., Nombela C., Sánchez M. Molecular basis of cell integrity and morphogenesis in Saccharomyces cerevisiae. Microbiol Rev. 1995 Sep;59(3):345–386. doi: 10.1128/mr.59.3.345-386.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Fleet G. H., Manners D. J. The enzymic degradation of an alkali-soluble glucan from the cell walls of Saccharomyces cerevisiae. J Gen Microbiol. 1977 Feb;98(2):315–327. doi: 10.1099/00221287-98-2-315. [DOI] [PubMed] [Google Scholar]
- Gaughran J. P., Lai M. H., Kirsch D. R., Silverman S. J. Nikkomycin Z is a specific inhibitor of Saccharomyces cerevisiae chitin synthase isozyme Chs3 in vitro and in vivo. J Bacteriol. 1994 Sep;176(18):5857–5860. doi: 10.1128/jb.176.18.5857-5860.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hartland R. P., Vermeulen C. A., Klis F. M., Sietsma J. H., Wessels J. G. The linkage of (1-3)-beta-glucan to chitin during cell wall assembly in Saccharomyces cerevisiae. Yeast. 1994 Dec;10(12):1591–1599. doi: 10.1002/yea.320101208. [DOI] [PubMed] [Google Scholar]
- Hill J., Donald K. A., Griffiths D. E., Donald G. DMSO-enhanced whole cell yeast transformation. Nucleic Acids Res. 1991 Oct 25;19(20):5791–5791. doi: 10.1093/nar/19.20.5791. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hong Z., Mann P., Brown N. H., Tran L. E., Shaw K. J., Hare R. S., DiDomenico B. Cloning and characterization of KNR4, a yeast gene involved in (1,3)-beta-glucan synthesis. Mol Cell Biol. 1994 Feb;14(2):1017–1025. doi: 10.1128/mcb.14.2.1017. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Häusler A., Robbins P. W. Glycosylation in Saccharomyces cerevisiae: cloning and characterization of an alpha-1,2-mannosyltransferase structural gene. Glycobiology. 1992 Feb;2(1):77–84. doi: 10.1093/glycob/2.1.77. [DOI] [PubMed] [Google Scholar]
- 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]
- Kapteyn J. C., Montijn R. C., Vink E., de la Cruz J., Llobell A., Douwes J. E., Shimoi H., Lipke P. N., Klis F. M. Retention of Saccharomyces cerevisiae cell wall proteins through a phosphodiester-linked beta-1,3-/beta-1,6-glucan heteropolymer. Glycobiology. 1996 Apr;6(3):337–345. doi: 10.1093/glycob/6.3.337. [DOI] [PubMed] [Google Scholar]
- 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]
- Klis F. M. Review: cell wall assembly in yeast. Yeast. 1994 Jul;10(7):851–869. doi: 10.1002/yea.320100702. [DOI] [PubMed] [Google Scholar]
- 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]
- Kuranda M. J., Robbins P. W. Chitinase is required for cell separation during growth of Saccharomyces cerevisiae. J Biol Chem. 1991 Oct 15;266(29):19758–19767. [PubMed] [Google Scholar]
- Lu C. F., Montijn R. C., Brown J. L., Klis F., Kurjan J., Bussey H., Lipke P. N. Glycosyl phosphatidylinositol-dependent cross-linking of alpha-agglutinin and beta 1,6-glucan in the Saccharomyces cerevisiae cell wall. J Cell Biol. 1995 Feb;128(3):333–340. doi: 10.1083/jcb.128.3.333. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nuoffer C., Jenö P., Conzelmann A., Riezman H. Determinants for glycophospholipid anchoring of the Saccharomyces cerevisiae GAS1 protein to the plasma membrane. Mol Cell Biol. 1991 Jan;11(1):27–37. doi: 10.1128/mcb.11.1.27. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Popolo L., Vai M., Gatti E., Porello S., Bonfante P., Balestrini R., Alberghina L. Physiological analysis of mutants indicates involvement of the Saccharomyces cerevisiae GPI-anchored protein gp115 in morphogenesis and cell separation. J Bacteriol. 1993 Apr;175(7):1879–1885. doi: 10.1128/jb.175.7.1879-1885.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- Roemer T., Paravicini G., Payton M. A., Bussey H. Characterization of the yeast (1-->6)-beta-glucan biosynthetic components, Kre6p and Skn1p, and genetic interactions between the PKC1 pathway and extracellular matrix assembly. J Cell Biol. 1994 Oct;127(2):567–579. doi: 10.1083/jcb.127.2.567. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Roncero C., Valdivieso M. H., Ribas J. C., Durán A. Isolation and characterization of Saccharomyces cerevisiae mutants resistant to Calcofluor white. J Bacteriol. 1988 Apr;170(4):1950–1954. doi: 10.1128/jb.170.4.1950-1954.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Saporito-Irwin S. M., Birse C. E., Sypherd P. S., Fonzi W. A. PHR1, a pH-regulated gene of Candida albicans, is required for morphogenesis. Mol Cell Biol. 1995 Feb;15(2):601–613. doi: 10.1128/mcb.15.2.601. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Vai M., Gatti E., Lacanà E., Popolo L., Alberghina L. Isolation and deduced amino acid sequence of the gene encoding gp115, a yeast glycophospholipid-anchored protein containing a serine-rich region. J Biol Chem. 1991 Jul 5;266(19):12242–12248. [PubMed] [Google Scholar]
- Vai M., Orlandi I., Cavadini P., Alberghina L., Popolo L. Candida albicans homologue of GGP1/GAS1 gene is functional in Saccharomyces cerevisiae and contains the determinants for glycosylphosphatidylinositol attachment. Yeast. 1996 Mar 30;12(4):361–368. doi: 10.1002/(SICI)1097-0061(19960330)12:4%3C361::AID-YEA920%3E3.0.CO;2-T. [DOI] [PubMed] [Google Scholar]
- Valdivieso M. H., Mol P. C., Shaw J. A., Cabib E., Durán A. CAL1, a gene required for activity of chitin synthase 3 in Saccharomyces cerevisiae. J Cell Biol. 1991 Jul;114(1):101–109. doi: 10.1083/jcb.114.1.101. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vanoni M., Vai M., Popolo L., Alberghina L. Structural heterogeneity in populations of the budding yeast Saccharomyces cerevisiae. J Bacteriol. 1983 Dec;156(3):1282–1291. doi: 10.1128/jb.156.3.1282-1291.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- de Nobel H., Lipke P. N. Is there a role for GPIs in yeast cell-wall assembly? Trends Cell Biol. 1994 Feb;4(2):42–45. doi: 10.1016/0962-8924(94)90003-5. [DOI] [PubMed] [Google Scholar]
- 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]