Molecular Genetics and Genomics

, Volume 269, Issue 4, pp 562–573 | Cite as

Mutations that are synthetically lethal with a gas1Δ allele cause defects in the cell wall of Saccharomyces cerevisiae

  • N. Tomishige
  • Y. Noda
  • H. Adachi
  • H. Shimoi
  • A. Takatsuki
  • K. Yoda
Original Paper

Abstract

The GAS1 -related genes of fungi encode GPI-anchored proteins with β-1,3-glucanosyltransferase activity. Loss of this activity results in defects in the assembly of the cell wall. We isolated mutants that show a synthetic defect when combined with a gas1Δ allele in Saccharomyces cerevisiae, and identified nine wild-type genes that rescue this defect. The indispensability of BIG1 and KRE6 for the viability of gas1Δ cells confirmed the important role of β-1,6-glucan in cells that are defective in the processing of β-1,3-glucan. The identification of the Wsc1p hypo-osmotic stress sensor and components of the PKC signal transduction pathway in our screen also confirmed that the cell wall integrity response attenuates the otherwise lethal gas1Δ defect. Unexpectedly, we found that the KEX2 gene is also required for the viability of the gas1Δ mutant. Kex2p is a Golgi/endosome-membrane-anchored protease that processes secretory preproteins. A cell wall defect was also found in the kex2Δ mutant, which was suppressible by multiple copies of the MKC7 or YAP3 gene, both of which encode other GPI-anchored proteases. Therefore, normal cell wall assembly requires proteolytic processing of secretory preproteins. Furthermore, the genes CSG2 and IPT1 were found to be required for normal growth of gas1Δ cells in the presence of 1 M sorbitol. This finding suggests that complex sphingolipids play a role in the hyper-osmotic response.

Keywords

Cell wall defect  GAS1  KEX2 endoprotease  Saccharomyces cerevisiae Complex sphingolipids 

Notes

Acknowledgements

We would like to thank Akio Toh-e (University of Tokyo) for yeast strains and plasmids, Yves Bourbonnais (Université Laval) for antibody against Yap3p, Hiroshi Kitagaki (the National Research Institute of Brewing) for his valuable advice in determination of the Man/Glc ratio, and Ikuko Sato (Fujisawa Pharmaceutical Co.) for her contribution of K1 killer toxin and valuable advice. This work was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, grants for Bioarchitecture Research from the Institute of Physical and Chemical Research (RIKEN), a grant from the Kato Memorial Bioscience Foundation (to Y. N.), and a grant from the Nagase Science and Technology Foundation (to K. Y.)

References

  1. Ash J, Dominguez M, Bergeron JJ, Thomas DY, Bourbonnais Y (1995) The yeast proprotein convertase encoded by YAP3 is a glycosylphosphatidyl inositol-anchored protein that localizes to the plasma membrane. J Biol Chem 270:20847–20854CrossRefPubMedGoogle Scholar
  2. Azuma M, Levinson JN, Page N, Bussey H (2002) Saccharomyces cerevisiae Big1p, a putative endoplasmic reticulum membrane protein required for normal levels of cell wall β-1,6-glucan. Yeast 19:783–793CrossRefPubMedGoogle Scholar
  3. Bader O, Schaller M, Klein S, Kukula J, Haack K, Muhlschlegel F, Korting HC, Schafer W, Hube B (2001) The KEX2 gene of Candida glabrata is required for cell surface integrity. Mol Microbiol 41:1431–1444CrossRefPubMedGoogle Scholar
  4. Barz WP, Walter P (1999) Two endoplasmic reticulum (ER) membrane proteins that facilitate ER-to-Golgi transport of glycosylphosphatidylinositol-anchored proteins. Mol Biol Cell 10:1043–1059PubMedGoogle Scholar
  5. Beeler T, Gable K, Zhao C, Dunn T (1994) A novel protein, Csg2p, is required for Ca2+ regulation in Saccharomyces cerevisiae. J Biol Chem 269:7279–7284PubMedGoogle Scholar
  6. Bickle M, Delley PA, Schmidt A, Hall MN (1998) Cell wall integrity modulates RHO1 activity via the exchange factor ROM2. EMBO J 17:2235–2245CrossRefPubMedGoogle Scholar
  7. Cappellaro C, Mrša V, Tanner W (1998) New potential cell wall glucanases of Saccharomyces cerevisiae and their involvement in mating. J Bacteriol 180:5030–5037PubMedGoogle Scholar
  8. Caro LH, Tettelin H, Vossen JH, Ram AF, van den Ende H, Klis FM (1997) In silicio identification of glycosyl-phosphatidylinositol-anchored plasma-membrane and cell wall proteins of Saccharomyces cerevisiae. Yeast 13:1477–1489CrossRefPubMedGoogle Scholar
  9. Caro LH, Smits GJ, van Egmond P, Chapman JW, Klis FM (1998) Transcription of multiple cell wall protein-encoding genes in Saccharomyces cerevisiae is differentially regulated during the cell cycle. FEMS Microbiol Lett 161:345–349CrossRefPubMedGoogle Scholar
  10. Conzelmann A, Riezman H, Desponds C, Bron C (1988) A major 125-kd membrane glycoprotein of Saccharomyces cerevisiae is attached to the lipid bilayer through an inositol-containing phospholipid. EMBO J 7:2233–2240PubMedGoogle Scholar
  11. Dallies N, Francois J, Paquet V (1998) A new method for quantitative determination of polysaccharides in the yeast cell wall. Application to the cell wall defective mutants of Saccharomyces cerevisiae. Yeast 14:1297–1306CrossRefPubMedGoogle Scholar
  12. De Nobel H, Ruiz C, Martin H, Morris W, Brul S, Molina M, Klis FM (2000) Cell wall perturbation in yeast results in dual phosphorylation of the Slt2/Mpk1 MAP kinase and in an Slt2-mediated increase in FKS2-lacZ expression, glucanase resistance and thermotolerance. Microbiology 146:2121–2132PubMedGoogle Scholar
  13. Dickson RC (1998) Sphingolipid functions in Saccharomyces cerevisiae: comparison to mammals. Annu Rev Biochem 67:27–48CrossRefPubMedGoogle Scholar
  14. Dickson RC, Nagiec EE, Wells GB, Nagiec MM, Lester RL (1997) Synthesis of mannose-(inositol-P)2 -ceramide, the major sphingolipid in Saccharomyces cerevisiae, requires the IPT1 ( YDR072c) gene. J Biol Chem 272:29620–29625PubMedGoogle Scholar
  15. Douglas CM, Foor F, Marrinan JA, Morin N, Nilsen JB, Dahl AM, Mazur P, Baginsky W, Li W, el-Sherbeini M, Clemas JA, Mandala SM, Frommer BR, Kurtz MB (1994) The Saccharomyces cerevisiae FKS1 ( ETG1) gene encodes an integral membrane protein which is a subunit of 1,3-β-D-glucan synthase. Proc Natl Acad Sci USA 91:12907–12911PubMedGoogle Scholar
  16. Drgonová J, Drgon T, Tanaka K, Kollár R, Chen GC, Ford RA, Chan CS, Takai Y, Cabib E (1996) Rho1p, a yeast protein at the interface between cell polarization and morphogenesis. Science 272:277–279PubMedGoogle Scholar
  17. Egel-Mitani M, Flygenring HP, Hansen MT (1990) A novel aspartyl protease allowing KEX2 -independent MFα propheromone processing in yeast. Yeast 6:127–137PubMedGoogle Scholar
  18. Fraering P, Imhof I, Meyer U, Strub JM, van Dorsselaer A, Vionnet C, Conzelmann A (2001) The GPI transamidase complex of Saccharomyces cerevisiae contains Gaa1p, Gpi8p, and Gpi16p. Mol Biol Cell 12:3295–3306PubMedGoogle Scholar
  19. Fujii T, Shimoi H, Iimura Y (1999) Structure of the glucan-binding sugar chain of Tip1p, a cell wall protein of Saccharomyces cerevisiae. Biochim Biophys Acta 1427:133–144CrossRefPubMedGoogle Scholar
  20. Fujimuro M, Tanaka K, Yokosawa H, Toh-e A (1998) Son1p is a component of the 26S proteasome of the yeast Saccharomyces cerevisiae. FEBS Lett 423:149–154CrossRefPubMedGoogle Scholar
  21. Hamada K, Fukuchi S, Arisawa M, Baba M, Kitada K (1998) Screening for glycosylphosphatidylinositol (GPI)-dependent cell wall proteins in Saccharomyces cerevisiae. Mol Gen Genet 258:53–59PubMedGoogle Scholar
  22. Hashimoto H, Yoda K (1997) Novel membrane protein complexes for protein glycosylation in the yeast Golgi apparatus. Biochem Biophys Res Commun 241:682–686CrossRefPubMedGoogle Scholar
  23. Hashimoto H, Sakakibara A, Yamasaki M, Yoda K (1997) VIG9 encodes GDP-mannose pyrophosphorylase, which is essential for protein glycosylation. J Biol Chem 272:16308–16314CrossRefPubMedGoogle Scholar
  24. Hohman S (2002) Osmotic stress signaling and osmoadaptation in yeasts. Microbiol Mol Biol Rev 66:300–372PubMedGoogle Scholar
  25. Inoue SB, Takewaki N, Takasuka T, Mio T, Adachi M, Fujii Y, Miyamoto C, Arisawa M, Furuichi Y, Watanabe T (1995) Characterization and gene cloning of 1,3-β-D-glucan synthase from Saccharomyces cerevisiae. Eur J Biochem 231:845–854PubMedGoogle Scholar
  26. Jung US, Levin DE (1999) Genome-wide analysis of gene expression regulated by the yeast cell wall integrity signalling pathway. Mol Microbiol 34:1049–1057PubMedGoogle Scholar
  27. Kapteyn JC, Ram AF, Groos EM, Kollar R, Montijn RC, Van Den Ende H, Llobell A, Cabib E, Klis FM (1997) Altered extent of cross-linking of β1,6-glucosylated mannoproteins to chitin in Saccharomyces cerevisiae mutants with reduced cell wall β1,3-glucan content. J Bacteriol 179:6279–6284PubMedGoogle Scholar
  28. Kapteyn JC, van den Ende H, Klis FM (1999) The contribution of cell wall proteins to the organization of the yeast cell wall. Biochem Biophys Acta 1426:373–383CrossRefPubMedGoogle Scholar
  29. Ketela T, Green R, Bussey H (1999) Saccharomyces cerevisiae Mid2p is a potential cell wall stress sensor and upstream activator of the PKC1-MPK1 cell integrity pathway. J Bacteriol 181:3330–3340PubMedGoogle Scholar
  30. Klis FM (1994) Cell wall assembly in yeast. Yeast 10:851–869PubMedGoogle Scholar
  31. Klis FM, Mol P, Hellingwerf K, Brul S (2002) Dynamics of cell wall structure in Saccharomyces cerevisiae. FEMS Microbiol Rev 26:239–256CrossRefPubMedGoogle Scholar
  32. Kollar R, Petrakova E, Ashwell G, Robbins PW, Cabib E (1995) Architecture of the yeast cell wall. The linkage between chitin and β(1→3)-glucan. J Biol Chem 270:1170–1178PubMedGoogle Scholar
  33. Kollar R, Reinhold BB, Petrakova E, Yeh HJ, Ashwell G, Drgonova J, Kapteyn JC, Klis FM, Cabib E (1997) Architecture of the yeast cell wall. β(1→6)-glucan interconnects mannoprotein, β(1→3)-glucan, and chitin. J Biol Chem 272:17762–17775CrossRefPubMedGoogle Scholar
  34. Komano H, Fuller RS (1995) Shared functions in vivo of a glycosyl-phosphatidylinositol-linked aspartyl protease, Mkc7, and the proprotein processing protease Kex2 in yeast. Proc Natl Acad Sci USA 92:10752–10756PubMedGoogle Scholar
  35. Kopecka M, Gabriel M (1992) The influence of Congo red on the cell wall and (1→3)-β-D-glucan microfibril biogenesis in Saccharomyces cerevisiae. Arch Microbiol 158:115–126PubMedGoogle Scholar
  36. Koshland D, Kent JC, Hartwell LH (1985) Genetic analysis of the mitotic transmission of minichromosomes. Cell 40:393–403PubMedGoogle Scholar
  37. Kranz JE, Holm C (1990) Cloning by function: an alternative approach for identifying yeast homologs of genes from other organisms. Proc Natl Acad Sci USA 87:6629–6633PubMedGoogle Scholar
  38. Lu CF, Montijn RC, Brown JL, Klis F, Kurjan J, Bussey H, Lipke PN (1995) Glycosyl phosphatidylinositol-dependent cross-linking of α-agglutinin and β1,6-glucan in the Saccharomyces cerevisiae cell wall. J Cell Biol 128:333–340PubMedGoogle Scholar
  39. Lussier M, et al (1997) Large scale identification of genes involved in cell surface biosynthesis and architecture in Saccharomyces cerevisiae. Genetics 147:435–450PubMedGoogle Scholar
  40. Mouyna I, Fontaine T, Vai M, Monod M, Fonzi WA, Diaquin M, Popolo L, Hartland RP, Latge JP (2000) Glycosylphosphatidylinositol-anchored glucanosyltransferases play an active role in the biosynthesis of the fungal cell wall. J Biol Chem 275:14882–14889CrossRefPubMedGoogle Scholar
  41. Mrša V, Tanner W (1999) Role of NaOH-extractable cell wall proteins Ccw5p, Ccw6p, Ccw7p and Ccw8p (members of the Pir protein family) in stability of the Saccharomyces cerevisiae cell wall. Yeast 15:813–820CrossRefPubMedGoogle Scholar
  42. Mrša V, Seidl T, Gentzsch M, Tanner W (1997) Specific labelling of cell wall proteins by biotinylation. Identification of four covalently linked O -mannosylated proteins of Saccharomyces cerevisiae. Yeast 13:1145–1154PubMedGoogle Scholar
  43. Nakazawa T, Horiuchi H, Ohta A, Takagi M (1998) Isolation and characterization of EPD1, an essential gene for pseudohyphal growth of a dimorphic yeast, Candida maltosa. J Bacteriol 180:2079–2086PubMedGoogle Scholar
  44. Oluwatosin YE, Kane PM (1998) Mutations in the yeast KEX2 gene cause a Vma--like phenotype: a possible role for the Kex2 endoprotease in vacuolar acidification. Mol Cell Biol 18:1534–1543PubMedGoogle Scholar
  45. Philip B, Levin DE (2001) Wsc1 and Mid2 are cell surface sensors for cell wall integrity signaling that act through Rom2, a guanine nucleotide exchange factor for Rho1. Mol Cell Biol 21:271–280CrossRefPubMedGoogle Scholar
  46. Popolo L, Vai M, Gatti E, Porello S, Bonfante P, Balestrini R, Alberghina L (1993) Physiological analysis of mutants indicates involvement of the Saccharomyces cerevisiae GPI-anchored protein gp115 in morphogenesis and cell separation. J Bacteriol 175:1879–1885PubMedGoogle Scholar
  47. Popolo L, Gilardelli D, Bonfante P, Vai M (1997) Increase in chitin as an essential response to defects in assembly of cell wall polymers in the ggp1Δ mutant of Saccharomyces cerevisiae. J Bacteriol 179:463–469PubMedGoogle Scholar
  48. Qadota H, Python CP, Inoue SB, Arisawa M, Anraku Y, Zheng Y, Watanabe T, Levin DE, Ohya Y (1996) Identification of yeast Rho1p GTPase as a regulatory subunit of 1,3-β-glucan synthase. Science 272:279–281PubMedGoogle Scholar
  49. Ram AF, Wolters A, Ten Hoopen R, Klis FM (1994) A new approach for isolating cell wall mutants in Saccharomyces cerevisiae by screening for hypersensitivity to Calcofluor white. Yeast 10:1019–1030PubMedGoogle Scholar
  50. Ram AF, Kapteyn JC, Montijn RC, Caro LH, Douwes JE, Baginsky W, Mazur P, van den Ende H, Klis FM (1998) Loss of the plasma membrane-bound protein Gas1p in Saccharomyces cerevisiae results in the release of β1,3-glucan into the medium and induces a compensation mechanism to ensure cell wall integrity. J Bacteriol 180:1418–1424PubMedGoogle Scholar
  51. Rep M, Krantz M, Thevelein JM, Hohmann S (2000) The transcriptional response of Saccharomyces cerevisiae to osmotic shock. J Biol Chem 275:8290–8300PubMedGoogle Scholar
  52. Rockwell NC, Fuller RS (1998) Interplay between S1 and S4 subsites in Kex2 protease: Kex2 exhibits dual specificity for the P4 side chain. Biochemistry 37:3386–3391CrossRefPubMedGoogle Scholar
  53. Roemer T, Delaney S, Bussey H (1993) SKN1 and KRE6 define a pair of functional homologs encoding putative membrane proteins involved in β-glucan synthesis. Mol Cell Biol 13:4039–4048PubMedGoogle Scholar
  54. Saporito-Irwin SM, Birse CE, Sypherd PS, Fonzi WA (1995) PHR1, a pH-regulated gene of Candida albicans, is required for morphogenesis. Mol Cell Biol 15:601–613Google Scholar
  55. Schmelzle T, Helliwell SB, Hall MN (2002) Yeast protein kinases and the RHO1 exchange factor TUS1 are novel components of the cell integrity pathway in yeast. Mol Cell Biol 22:1329–1339CrossRefPubMedGoogle Scholar
  56. Sherman F (1991) Getting started with yeast. Methods Enzymol 194:21–37PubMedGoogle Scholar
  57. Shimoi H, Iimura Y, Obata T (1995) Molecular cloning of CWP1: a gene encoding a Saccharomyces cerevisiae cell wall protein solubilized with Rarobacter faecitabidus protease I. J Biochem (Tokyo) 118:302–311Google Scholar
  58. Sikorski RS, Boeke JD (1991) In vitro mutagenesis and plasmid shuffling: from cloned gene to mutant yeast. Methods Enzymol 194:302–318PubMedGoogle Scholar
  59. Sikorski RS, Hieter P (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122:19–27PubMedGoogle Scholar
  60. Simons K, Ikonen E (1997) Functional rafts in cell membranes. Nature 387:569–572PubMedGoogle Scholar
  61. Smits GJ, Kapteyn JC, van den Ende H, Klis FM (1999) Cell wall dynamics in yeast. Curr Opin Microbiol 2:348–352PubMedGoogle Scholar
  62. Smits GJ, van den Ende H, Klis FM (2001) Differential regulation of cell wall biogenesis during growth and development in yeast. Microbiology 147:781–794PubMedGoogle Scholar
  63. Spellman PT, Sherlock G, Zhang MQ, Iyer VR, Anders K, Eisen MB, Brown PO, Botstein D, Futcher B (1998) Comprehensive identification of cell cycle-regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization. Mol Biol Cell 9:3273–3297PubMedGoogle Scholar
  64. Stock SD, Hama H, Radding JA, Young DA, Takemoto JY (2000) Syringomycin E inhibition of Saccharomyces cerevisiae: requirement for biosynthesis of sphingolipids with very-long-chain fatty acids and mannose- and phosphoinositol-containing head groups. Antimicrob Agents Chemother 44:1174–1180PubMedGoogle Scholar
  65. Sun Y, Taniguchi R, Tanoue D, Yamaji T, Takematsu H, Mori K, Fujita T, Kawasaki T, Kozutsumi Y (2000) Sli2 (Ypk1), a homologue of mammalian protein kinase SGK, is a downstream kinase in the sphingolipid-mediated signaling pathway of yeast. Mol Cell Biol 20:4411–4419CrossRefPubMedGoogle Scholar
  66. Turchini A, Ferrario L, Popolo L (2000) Increase of external osmolarity reduces morphogenetic defects and accumulation of chitin in a gas1 mutant of Saccharomyces cerevisiae. J Bacteriol 182:1167–1171CrossRefPubMedGoogle Scholar
  67. Vai M, Gatti E, Lacana E, Popolo L, Alberghina L (1991) Isolation and deduced amino acid sequence of the gene encoding gp115, a yeast glycophospholipid-anchored protein containing a serine-rich region. J Biol Chem 266:12242–12248PubMedGoogle Scholar
  68. Van der Vaart JM, Caro LH, Chapman JW, Klis FM, Verrips CT (1995) Identification of three mannoproteins in the cell wall of Saccharomyces cerevisiae. J Bacteriol 177:3104–3110PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  • N. Tomishige
    • 1
  • Y. Noda
    • 1
  • H. Adachi
    • 1
  • H. Shimoi
    • 2
  • A. Takatsuki
    • 3
  • K. Yoda
    • 1
  1. 1.Department of BiotechnologyUniversity of TokyoTokyoJapan
  2. 2.National Research Institute of BrewingHigashi-HiroshimaJapan
  3. 3.Institute of Physical and Chemical Research (RIKEN)WakoJapan

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