Skip to main content
SpringerLink
Log in

Regulation of cell wallβ-glucan assembly:PTC1 Negatively affectsPBS2 Action in a pathway that includes modulation ofEXG1 transcription

  • Original Paper
  • Published:
Molecular and General Genetics MGG Aims and scope Submit manuscript

Abstract

Analysis of genes involved in yeast cell wallβ-glucan assembly has led to the isolation ofEXG1,PBS2 andPTC1.EXG1 andPBS2 were isolated as genes that, when expressed from multicopy plasmids, led to a dominant killer toxin-resistant phenotype. ThePTC1 gene was cloned by functional complementation of the calcofluor white-hypersensitive mutantcwh47-1.PTC1/CWH47 is the structural gene for a type 2C serine/threonine phosphatase,EXG1 codes for an exo-β-glucanase, andPBS2 encodes a MAP kinase kinase in the Pbs2p-Hog1p signal transduction pathway. Overexpression ofEXG1 on a 2μ plasmid led to reduction in a cell wallβ1,6-glucan and caused killer resistance in wild type cells; while theexg1Δ mutant displayed modest increases in killer sensitivity andβ1,6-glucan levels. Disruption ofPTC1/CWH47 and overexpression ofPBS2 gave rise to similarβ-glucan related phenotypes, with higher levels ofEXG1 transcription, increased exo-β-glucanase activity, reducedβ1,6-glucan levels, and resistance to killer toxin. Genetic analysis revealed that loss of function of thePBS2 gene was epistatic toPTC1/CWH47 disruption, indicating a functional role for the Ptc1p/Cwh47p phosphatase in the Pbs2p-Hog1p signal transduction pathway. These results suggest that Ptc1p/Cwh47p and Pbs2p play opposing regulatory roles in cell wall glucan assembly, and that this is effected in part by modulating Exg1p activity.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Abd-el-al ATH, Phaff HJ (1968) Exo-β-glucanases in yeast. Biochem J 109:347–360

    PubMed  Google Scholar 

  • Avery L, Wasserman S (1992) Ordering gene function: the interpretation of epistasis in regulatory hierarchies. Trends Genet 8:312–316

    PubMed  Google Scholar 

  • Blomberg A, Adler L (1989) Roles of glycerol and glycerol-3-phosphate dehydrogenase (NAD+) in acquired osmotolerance ofSaccharomyces cerevisiae. J Bacteriol 171:1087–1092

    PubMed  Google Scholar 

  • Blomberg A, Larsson C, Gustafsson L (1988) Microcalorimetric monitoring of growth ofSaccharomyces cerevisiae osmotolerance in relation to physiological state. J Bacteriol 170:4562–4568

    PubMed  Google Scholar 

  • Boguslawski G, Polazzi JO (1987) Complete nucleotide sequence of a gene conferring polymyxin B resistance on yeast Similarity of the predicted polypeptide to protein kinases. Proc Natl Acad Sci USA 84:5848–5852

    PubMed  Google Scholar 

  • Boone C, Sommer SS, Hensel A, Bussey H (1990) Yeast KRE genes provide evidence for a pathway of cell wallβ-glucan assembly. J Cell Biol 110:1833–1843

    PubMed  Google Scholar 

  • Brewster JL, de Valoir T, Dwyer ND, Winter E, Gustin MC (1993) An osmosensing signal transduction pathway in yeast. Science 259:1760–1763

    PubMed  Google Scholar 

  • Brown JL, Bussey H (1993) The yeastKRE9 gene encodes an O-glycoprotein involved in cell surfaceβ-glucan assembly. Mol Cell Biol 13:6346–6356

    PubMed  Google Scholar 

  • Brown JL, Kossaczka Z, Jiang B, Bussey H (1993) A mutational analysis of killer toxin resistance inSaccharomyces cerevisiae identifies new genes involved in cell wall (1-6)-β-glucan synthesis. Genetics 133:837–849

    PubMed  Google Scholar 

  • Bussey, H (1991) K1 killer toxin, a pore-forming protein from yeast. Mol Microbiol 5:2339–2343

    PubMed  Google Scholar 

  • Bussey H, Sacks W, Galley D, Saville D (1982) Yeast killer plasmid mutations affecting toxin secretion and activity and toxin immunity function. Mol Cell Biol 2:346–354

    PubMed  Google Scholar 

  • Errede B, Levin DE (1993) A conserved kinase cascade for MAP kinase activation in yeast. Curr Opin Cell Biol 5:254–260

    PubMed  Google Scholar 

  • Farkas V, Biely P, Bauer S (1973) Extracellularβ-glucanases of the yeastSaccharomyces cerevisiae. Biochim Biophys Acta 321:246–255

    PubMed  Google Scholar 

  • Fleet GH, Phaff HJ (1981) Fungal glucans-structure and metabolism. In: Tanner W, Loewus FA (eds) Encyclopedia of plant physiology new series, vol. 13B. Plant carbohydrates II. Springer-Verlag, Berlin, pp 416–440

    Google Scholar 

  • Fourney RM, Miyakoshi J, Day III RS, Paterson MC (1988) Northern blotting: efficient RNA staining and transfer. Focus 10:5–6

    Google Scholar 

  • Hien NH, Fleet GH (1983) Separation and characterization of six (1-3)-β-glucanases fromSaccharomyces cerevisiae. J Bacteriol 156:1204–1213

    PubMed  Google Scholar 

  • Hoffman CS, Winston F (1987) A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation ofEscherichia coli. Gene 57:267–272

    PubMed  Google Scholar 

  • Hong Z, Mann P, Brown NH, Tran LE, Shaw KJ, Hare RS, DiDomenico B. (1994) Cloning and characterization ofKNR4, a yeast gene involved in (1,3)-β-glucan synthesis. Mol Cell Biol 14:1017–1025

    PubMed  Google Scholar 

  • Hutchins K, Bussey H (1983) Cell wall receptor for yeast killer toxin: involvement of (1-6)-β-D-glucan. J Bacteriol 154:161–169

    PubMed  Google Scholar 

  • Ito H, Fukuda Y, Murata M, Kimura A (1983) Transformations of intact yeast cells with alkali cations. J Bacteriol 153:163–168

    PubMed  Google Scholar 

  • Kaufer NF, Fried HM, Schwindinger WF, Jasin M, Warner JR (1983) Cycloheximide resistance in yeast: the gene and its protein. Nucleic Acids Res 11: 3123–3135

    PubMed  Google Scholar 

  • Klebl F, Tanner W (1989) Molecular cloning of a cell wall exo-β-1,3-glucanase fromSaccharomyces cerevisiae. J Bacteriol 171:6259–6264

    PubMed  Google Scholar 

  • Maeda T, Tsai AM, Saito H (1993) Mutations in a protein tyrosine phosphatase gene (PTP2) and a protein serine/threonine phosphatase gene (PTC1) cause a synthetic growth defect inSaccharomyces cerevisiae. Mol Cell Biol 13:5408–5417

    PubMed  Google Scholar 

  • Maeda T, Wurgler-Murphy SM, Saito H (1994) A two-component system that regulates an osmosensing MAP kinase cascade in yeast. Nature 369:242–245

    PubMed  Google Scholar 

  • Manners DJ, Masson AJ, Patterson JC (1973a) The structure of aβ-(1-3)-D-glucan from yeast cell walls. Biochem J 135:19–30

    PubMed  Google Scholar 

  • Manners DJ, Masson AJ, Patterson JC (1973b) The structure of aβ-(1-6)-D-glucan from yeast cell walls. Biochem J 135:31–36

    PubMed  Google Scholar 

  • Meaden P, Hill K, Wagner J, Slipetz D, Sommer SS, Bussey H (1990) The yeastKRE5 encodes a probable endoplasmic reticulum protein required for (1-6)-β-D-glucan synthesis and normal growth. Mol Cell Biol 10: 3013–3019

    PubMed  Google Scholar 

  • Mrsa V, Klebl F, Tanner W (1993) Purification and characterization of theSaccharomyces cerevisiae BGL2 gene product, a cell wall endo-β-1,3-glucanase. J Bacteriol 175:2102–2106

    PubMed  Google Scholar 

  • Muthukumar G, Suhng SH, Magee PT, Jewell RD, Primerano DA (1993) TheSaccharomyces cerevisiae SPR1 gene encodes a sporulation-specific exo-1,3-beta-glucanase which contributes to ascospore thermoresistance. J Bacteriol 175: 386–394

    PubMed  Google Scholar 

  • Nebreda AR, Villa TG, Villanueva JR, Rey F del (1986) Cloning of genes related to exo-β-glucanase production inSaccharomyces cerevisiae: characterization of an exo-β-glucanase structural gene Gene 47:245–259

    PubMed  Google Scholar 

  • Ram AFJ, Wolters A, ten Hoopen R, Klis FM (1994) A new approach for isolating cell wall mutants inSaccharomyces cerevisiae by screening for hypersensitivity to Calcofluor White. Yeast 10:1019–1030

    PubMed  Google Scholar 

  • Rine J (1991) Gene overexpression in studies ofSaccharomyces cerevisiae. Methods Enzymol 194:229–251

    Google Scholar 

  • Rine J, Hansen W, Hardeman E, Davis RW (1983) Targeted selection of recombinant clones through gene dosage effects. Proc Natl Acad Sci USA 80:6750–6754

    PubMed  Google Scholar 

  • Roemer T, Bussey H (1991) Yeastβ-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 USA 88:11295–11299

    PubMed  Google Scholar 

  • Roemer T, Delaney S, Bussey H (1993)SKN1 andKRE6 define a pair of functional homologs encoding putative membrane proteins involved inβ-glucan synthesis. Mol Cell Biol 13: 4039–4048

    PubMed  Google Scholar 

  • Roemer T, Paravicini G, Payton MA, Bussey H (1994) Characterization of the yeast (1-6)-β-glucan biosynthetic components, Kre6p and Sknlp; and genetic interactions between thePKC1 pathway and extracellular matrix assembly. J Cell Biol 127:567–579

    PubMed  Google Scholar 

  • Rose MD, Winston F, Hieter P (1990) Methods in yeast genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

    Google Scholar 

  • Rothstein RJ (1983) One step gene disruption in yeast. Methods Enzymol 101:202–211

    PubMed  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual (2nd ed), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

    Google Scholar 

  • Sanger FG, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467

    PubMed  Google Scholar 

  • San Segundo P, Correa J, Vazquez de Aldana CR, del Rey F (1993)SSG1, a gene encoding a sporulation-specific 1,3-β-glucanase inSaccharomyces cerevisiae. J Bacteriol 175: 3823–3837

    PubMed  Google Scholar 

  • Sanchez A, Villanueva JR, Villa TG (1982)Saccharomyces cerevisiae secretes 2 exo-β-glucanases. FEBS lett 138:209–212

    PubMed  Google Scholar 

  • Santos T, Rey F del, Conde J, Villanueva JR, Nombela C (1979)Saccharomyces cerevisiae mutant defective in exo-β-glucanase production. J Bacteriol 139: 333–338

    PubMed  Google Scholar 

  • Sherman F, Fink GR, Hicks JB (1982) Methods in yeast genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

    Google Scholar 

  • Shimizu J, Yoda K, Yamasaki M (1994) The hypo-osmolarity-sensitive phenotype of theSaccharomyces cerevisiae hpo2 mutant is due to a mutation inPKC1, which regulates expression ofβ-glucanase. Mol Gen Genet 242:641–648

    PubMed  Google Scholar 

  • Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517

    PubMed  Google Scholar 

  • Torres L, Martin H, Garcia-Saez MI, Arroyo J, Molina M, Sanchez M, Nombela C. (1991) A protein kinase gene complements the lytic phenotype ofSaccharomyces cerevisiae lyt2 mutants. Mol Microbiol 5:2845–2854

    PubMed  Google Scholar 

  • Vazquez de Aldana CR, Correa J, San Segundo P, Bueno A, Nebreda AR, Mendez E, del Rey F (1991) Nucleotide sequence of the exo-1,3-β-glucanase-encoding gene,EXG1, of the yeastSaccharomyces cerevisiae. Gene 97:173–182

    PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biology, McGill University, 1205 Dr. Penfield Avenue, H3A 1B1, Montreal, Quebec, Canada

    Bo Jiang, Jane Sheraton & Howard Bussey

  2. Institute for Molecular Cell Biology, BioCentrum Amsterdam, Kruislaan 318, 1098 SM, Amsterdam, The Netherlands

    Arthur F. J. Ram & Frans M. Klis

Authors
  1. Bo Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Arthur F. J. Ram
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jane Sheraton
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Frans M. Klis
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Howard Bussey
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Communicated by C. P. Hollenberg

Rights and permissions

Reprints and permissions

About this article

Cite this article

Jiang, B., Ram, A.F.J., Sheraton, J. et al. Regulation of cell wallβ-glucan assembly:PTC1 Negatively affectsPBS2 Action in a pathway that includes modulation ofEXG1 transcription. Molec. Gen. Genet. 248, 260–269 (1995). https://doi.org/10.1007/BF02191592

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02191592

Key word

Search

Navigation