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. 2003 Mar;163(3):875–894. doi: 10.1093/genetics/163.3.875

A Saccharomyces cerevisiae genome-wide mutant screen for altered sensitivity to K1 killer toxin.

Nicolas Pagé 1, Manon Gérard-Vincent 1, Patrice Ménard 1, Maude Beaulieu 1, Masayuki Azuma 1, Gerrit J P Dijkgraaf 1, Huijuan Li 1, José Marcoux 1, Thuy Nguyen 1, Tim Dowse 1, Anne-Marie Sdicu 1, Howard Bussey 1
PMCID: PMC1462477  PMID: 12663529

Abstract

Using the set of Saccharomyces cerevisiae mutants individually deleted for 5718 yeast genes, we screened for altered sensitivity to the antifungal protein, K1 killer toxin, that binds to a cell wall beta-glucan receptor and subsequently forms lethal pores in the plasma membrane. Mutations in 268 genes, including 42 in genes of unknown function, had a phenotype, often mild, with 186 showing resistance and 82 hypersensitivity compared to wild type. Only 15 of these genes were previously known to cause a toxin phenotype when mutated. Mutants for 144 genes were analyzed for alkali-soluble beta-glucan levels; 63 showed alterations. Further, mutants for 118 genes with altered toxin sensitivity were screened for SDS, hygromycin B, and calcofluor white sensitivity as indicators of cell surface defects; 88 showed some additional defect. There is a markedly nonrandom functional distribution of the mutants. Many genes affect specific areas of cellular activity, including cell wall glucan and mannoprotein synthesis, secretory pathway trafficking, lipid and sterol biosynthesis, and cell surface signal transduction, and offer new insights into these processes and their integration.

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Selected References

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  1. Ahmed A., Sesti F., Ilan N., Shih T. M., Sturley S. L., Goldstein S. A. A molecular target for viral killer toxin: TOK1 potassium channels. Cell. 1999 Oct 29;99(3):283–291. doi: 10.1016/s0092-8674(00)81659-1. [DOI] [PubMed] [Google Scholar]
  2. Albertyn J., Hohmann S., Thevelein J. M., Prior B. A. GPD1, which encodes glycerol-3-phosphate dehydrogenase, is essential for growth under osmotic stress in Saccharomyces cerevisiae, and its expression is regulated by the high-osmolarity glycerol response pathway. Mol Cell Biol. 1994 Jun;14(6):4135–4144. doi: 10.1128/mcb.14.6.4135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Azuma Masayuki, Levinson Joshua N., Pagé Nicolas, Bussey Howard. Saccharomyces cerevisiae Big1p, a putative endoplasmic reticulum membrane protein required for normal levels of cell wall beta-1,6-glucan. Yeast. 2002 Jun 30;19(9):783–793. doi: 10.1002/yea.873. [DOI] [PubMed] [Google Scholar]
  4. Benachour A., Sipos G., Flury I., Reggiori F., Canivenc-Gansel E., Vionnet C., Conzelmann A., Benghezal M. Deletion of GPI7, a yeast gene required for addition of a side chain to the glycosylphosphatidylinositol (GPI) core structure, affects GPI protein transport, remodeling, and cell wall integrity. J Biol Chem. 1999 May 21;274(21):15251–15261. doi: 10.1074/jbc.274.21.15251. [DOI] [PubMed] [Google Scholar]
  5. Bennett C. B., Lewis L. K., Karthikeyan G., Lobachev K. S., Jin Y. H., Sterling J. F., Snipe J. R., Resnick M. A. Genes required for ionizing radiation resistance in yeast. Nat Genet. 2001 Dec;29(4):426–434. doi: 10.1038/ng778. [DOI] [PubMed] [Google Scholar]
  6. Bianchi M. M., Sartori G., Vandenbol M., Kaniak A., Uccelletti D., Mazzoni C., Di Rago J. P., Carignani G., Slonimski P. P., Frontali L. How to bring orphan genes into functional families. Yeast. 1999 Apr;15(6):513–526. doi: 10.1002/(SICI)1097-0061(199904)15:6<513::AID-YEA370>3.0.CO;2-P. [DOI] [PubMed] [Google Scholar]
  7. Bickle M., Delley P. A., Schmidt A., Hall M. N. Cell wall integrity modulates RHO1 activity via the exchange factor ROM2. EMBO J. 1998 Apr 15;17(8):2235–2245. doi: 10.1093/emboj/17.8.2235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bilsland-Marchesan E., Ariño J., Saito H., Sunnerhagen P., Posas F. Rck2 kinase is a substrate for the osmotic stress-activated mitogen-activated protein kinase Hog1. Mol Cell Biol. 2000 Jun;20(11):3887–3895. doi: 10.1128/mcb.20.11.3887-3895.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Brachmann C. B., Davies A., Cost G. J., Caputo E., Li J., Hieter P., Boeke J. D. Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast. 1998 Jan 30;14(2):115–132. doi: 10.1002/(SICI)1097-0061(19980130)14:2<115::AID-YEA204>3.0.CO;2-2. [DOI] [PubMed] [Google Scholar]
  10. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  11. Breinig Frank, Tipper Donald J., Schmitt Manfred J. Kre1p, the plasma membrane receptor for the yeast K1 viral toxin. Cell. 2002 Feb 8;108(3):395–405. doi: 10.1016/s0092-8674(02)00634-7. [DOI] [PubMed] [Google Scholar]
  12. Bussey H. K1 killer toxin, a pore-forming protein from yeast. Mol Microbiol. 1991 Oct;5(10):2339–2343. doi: 10.1111/j.1365-2958.1991.tb02079.x. [DOI] [PubMed] [Google Scholar]
  13. Bussey H., Saville D., Hutchins K., Palfree R. G. Binding of yeast killer toxin to a cell wall receptor on sensitive Saccharomyces cerevisiae. J Bacteriol. 1979 Dec;140(3):888–892. doi: 10.1128/jb.140.3.888-892.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dijkgraaf Gerrit J. P., Abe Mitsuhiro, Ohya Yoshikazu, Bussey Howard. Mutations in Fks1p affect the cell wall content of beta-1,3- and beta-1,6-glucan in Saccharomyces cerevisiae. Yeast. 2002 Jun 15;19(8):671–690. doi: 10.1002/yea.866. [DOI] [PubMed] [Google Scholar]
  15. Errede B., Levin D. E. A conserved kinase cascade for MAP kinase activation in yeast. Curr Opin Cell Biol. 1993 Apr;5(2):254–260. doi: 10.1016/0955-0674(93)90112-4. [DOI] [PubMed] [Google Scholar]
  16. García-Rodriguez L. J., Durán A., Roncero C. Calcofluor antifungal action depends on chitin and a functional high-osmolarity glycerol response (HOG) pathway: evidence for a physiological role of the Saccharomyces cerevisiae HOG pathway under noninducing conditions. J Bacteriol. 2000 May;182(9):2428–2437. doi: 10.1128/jb.182.9.2428-2437.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Giaever Guri, Chu Angela M., Ni Li, Connelly Carla, Riles Linda, Véronneau Steeve, Dow Sally, Lucau-Danila Ankuta, Anderson Keith, André Bruno. Functional profiling of the Saccharomyces cerevisiae genome. Nature. 2002 Jul 25;418(6896):387–391. doi: 10.1038/nature00935. [DOI] [PubMed] [Google Scholar]
  18. Gustin M. C., Albertyn J., Alexander M., Davenport K. MAP kinase pathways in the yeast Saccharomyces cerevisiae. Microbiol Mol Biol Rev. 1998 Dec;62(4):1264–1300. doi: 10.1128/mmbr.62.4.1264-1300.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hong Z., Mann P., Shaw K. J., Didomenico B. Analysis of beta-glucans and chitin in a Saccharomyces cerevisiae cell wall mutant using high-performance liquid chromatography. Yeast. 1994 Aug;10(8):1083–1092. doi: 10.1002/yea.320100810. [DOI] [PubMed] [Google Scholar]
  20. Hunter T., Plowman G. D. The protein kinases of budding yeast: six score and more. Trends Biochem Sci. 1997 Jan;22(1):18–22. doi: 10.1016/s0968-0004(96)10068-2. [DOI] [PubMed] [Google Scholar]
  21. Jiang B., Brown J. L., Sheraton J., Fortin N., Bussey H. A new family of yeast genes implicated in ergosterol synthesis is related to the human oxysterol binding protein. Yeast. 1994 Mar;10(3):341–353. doi: 10.1002/yea.320100307. [DOI] [PubMed] [Google Scholar]
  22. Ketela T., Green R., Bussey H. Saccharomyces cerevisiae mid2p is a potential cell wall stress sensor and upstream activator of the PKC1-MPK1 cell integrity pathway. J Bacteriol. 1999 Jun;181(11):3330–3340. doi: 10.1128/jb.181.11.3330-3340.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kollár R., Reinhold B. B., Petráková E., Yeh H. J., Ashwell G., Drgonová J., Kapteyn J. C., Klis F. M., Cabib E. Architecture of the yeast cell wall. Beta(1-->6)-glucan interconnects mannoprotein, beta(1-->)3-glucan, and chitin. J Biol Chem. 1997 Jul 11;272(28):17762–17775. doi: 10.1074/jbc.272.28.17762. [DOI] [PubMed] [Google Scholar]
  24. Levin D. E., Bartlett-Heubusch E. Mutants in the S. cerevisiae PKC1 gene display a cell cycle-specific osmotic stability defect. J Cell Biol. 1992 Mar;116(5):1221–1229. doi: 10.1083/jcb.116.5.1221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Levin D. E., Errede B. The proliferation of MAP kinase signaling pathways in yeast. Curr Opin Cell Biol. 1995 Apr;7(2):197–202. doi: 10.1016/0955-0674(95)80028-x. [DOI] [PubMed] [Google Scholar]
  26. Li Huijuan, Pagé Nicolas, Bussey Howard. Actin patch assembly proteins Las17p and Sla1p restrict cell wall growth to daughter cells and interact with cis-Golgi protein Kre6p. Yeast. 2002 Sep 30;19(13):1097–1112. doi: 10.1002/yea.904. [DOI] [PubMed] [Google Scholar]
  27. Li Y., Moir R. D., Sethy-Coraci I. K., Warner J. R., Willis I. M. Repression of ribosome and tRNA synthesis in secretion-defective cells is signaled by a novel branch of the cell integrity pathway. Mol Cell Biol. 2000 Jun;20(11):3843–3851. doi: 10.1128/mcb.20.11.3843-3851.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lipke P. N., Ovalle R. Cell wall architecture in yeast: new structure and new challenges. J Bacteriol. 1998 Aug;180(15):3735–3740. doi: 10.1128/jb.180.15.3735-3740.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lussier M., Sdicu A. M., Shahinian S., Bussey H. The Candida albicans KRE9 gene is required for cell wall beta-1, 6-glucan synthesis and is essential for growth on glucose. Proc Natl Acad Sci U S A. 1998 Aug 18;95(17):9825–9830. doi: 10.1073/pnas.95.17.9825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lussier M., White A. M., Sheraton J., di Paolo T., Treadwell J., Southard S. B., Horenstein C. I., Chen-Weiner J., Ram A. F., Kapteyn J. C. Large scale identification of genes involved in cell surface biosynthesis and architecture in Saccharomyces cerevisiae. Genetics. 1997 Oct;147(2):435–450. doi: 10.1093/genetics/147.2.435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Magnelli Paula, Cipollo John F., Abeijon Claudia. A refined method for the determination of Saccharomyces cerevisiae cell wall composition and beta-1,6-glucan fine structure. Anal Biochem. 2002 Feb 1;301(1):136–150. doi: 10.1006/abio.2001.5473. [DOI] [PubMed] [Google Scholar]
  32. Martin H., Dagkessamanskaia A., Satchanska G., Dallies N., François J. KNR4, a suppressor of Saccharomyces cerevisiae cwh mutants, is involved in the transcriptional control of chitin synthase genes. Microbiology. 1999 Jan;145(Pt 1):249–258. doi: 10.1099/13500872-145-1-249. [DOI] [PubMed] [Google Scholar]
  33. Martinac B., Zhu H., Kubalski A., Zhou X. L., Culbertson M., Bussey H., Kung C. Yeast K1 killer toxin forms ion channels in sensitive yeast spheroplasts and in artificial liposomes. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6228–6232. doi: 10.1073/pnas.87.16.6228. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ni L., Snyder M. A genomic study of the bipolar bud site selection pattern in Saccharomyces cerevisiae. Mol Biol Cell. 2001 Jul;12(7):2147–2170. doi: 10.1091/mbc.12.7.2147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Nierras C. R., Warner J. R. Protein kinase C enables the regulatory circuit that connects membrane synthesis to ribosome synthesis in Saccharomyces cerevisiae. J Biol Chem. 1999 May 7;274(19):13235–13241. doi: 10.1074/jbc.274.19.13235. [DOI] [PubMed] [Google Scholar]
  36. Otte S., Belden W. J., Heidtman M., Liu J., Jensen O. N., Barlowe C. Erv41p and Erv46p: new components of COPII vesicles involved in transport between the ER and Golgi complex. J Cell Biol. 2001 Feb 5;152(3):503–518. doi: 10.1083/jcb.152.3.503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Paravicini G., Cooper M., Friedli L., Smith D. J., Carpentier J. L., Klig L. S., Payton M. A. The osmotic integrity of the yeast cell requires a functional PKC1 gene product. Mol Cell Biol. 1992 Nov;12(11):4896–4905. doi: 10.1128/mcb.12.11.4896. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Philip B., Levin D. E. 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. 2001 Jan;21(1):271–280. doi: 10.1128/MCB.21.1.271-280.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Planta R. J., Mager W. H. The list of cytoplasmic ribosomal proteins of Saccharomyces cerevisiae. Yeast. 1998 Mar 30;14(5):471–477. doi: 10.1002/(SICI)1097-0061(19980330)14:5<471::AID-YEA241>3.0.CO;2-U. [DOI] [PubMed] [Google Scholar]
  40. Posas F., Takekawa M., Saito H. Signal transduction by MAP kinase cascades in budding yeast. Curr Opin Microbiol. 1998 Apr;1(2):175–182. doi: 10.1016/s1369-5274(98)80008-8. [DOI] [PubMed] [Google Scholar]
  41. Raitt D. C., Posas F., Saito H. Yeast Cdc42 GTPase and Ste20 PAK-like kinase regulate Sho1-dependent activation of the Hog1 MAPK pathway. EMBO J. 2000 Sep 1;19(17):4623–4631. doi: 10.1093/emboj/19.17.4623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. 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]
  43. Robinson J. S., Klionsky D. J., Banta L. M., Emr S. D. Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases. Mol Cell Biol. 1988 Nov;8(11):4936–4948. doi: 10.1128/mcb.8.11.4936. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. 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]
  45. Romero P. A., Dijkgraaf G. J., Shahinian S., Herscovics A., Bussey H. The yeast CWH41 gene encodes glucosidase I. Glycobiology. 1997 Oct;7(7):997–1004. doi: 10.1093/glycob/7.7.997. [DOI] [PubMed] [Google Scholar]
  46. Ross-Macdonald P., Coelho P. S., Roemer T., Agarwal S., Kumar A., Jansen R., Cheung K. H., Sheehan A., Symoniatis D., Umansky L. Large-scale analysis of the yeast genome by transposon tagging and gene disruption. Nature. 1999 Nov 25;402(6760):413–418. doi: 10.1038/46558. [DOI] [PubMed] [Google Scholar]
  47. Sacher M., Barrowman J., Wang W., Horecka J., Zhang Y., Pypaert M., Ferro-Novick S. TRAPP I implicated in the specificity of tethering in ER-to-Golgi transport. Mol Cell. 2001 Feb;7(2):433–442. doi: 10.1016/s1097-2765(01)00190-3. [DOI] [PubMed] [Google Scholar]
  48. Shahinian S., Bussey H. beta-1,6-Glucan synthesis in Saccharomyces cerevisiae. Mol Microbiol. 2000 Feb;35(3):477–489. doi: 10.1046/j.1365-2958.2000.01713.x. [DOI] [PubMed] [Google Scholar]
  49. Shahinian S., Dijkgraaf G. J., Sdicu A. M., Thomas D. Y., Jakob C. A., Aebi M., Bussey H. Involvement of protein N-glycosyl chain glucosylation and processing in the biosynthesis of cell wall beta-1,6-glucan of Saccharomyces cerevisiae. Genetics. 1998 Jun;149(2):843–856. doi: 10.1093/genetics/149.2.843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Sherman F. Getting started with yeast. Methods Enzymol. 1991;194:3–21. doi: 10.1016/0076-6879(91)94004-v. [DOI] [PubMed] [Google Scholar]
  51. Suzuki C., Shimma Y. I. P-type ATPase spf1 mutants show a novel resistance mechanism for the killer toxin SMKT. Mol Microbiol. 1999 May;32(4):813–823. doi: 10.1046/j.1365-2958.1999.01400.x. [DOI] [PubMed] [Google Scholar]
  52. Tokai M., Kawasaki H., Kikuchi Y., Ouchi K. Cloning and characterization of the CSF1 gene of Saccharomyces cerevisiae, which is required for nutrient uptake at low temperature. J Bacteriol. 2000 May;182(10):2865–2868. doi: 10.1128/jb.182.10.2865-2868.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Van Rinsum J., Klis F. M., van den Ende H. Cell wall glucomannoproteins of Saccharomyces cerevisiae mnn9. Yeast. 1991 Oct;7(7):717–726. doi: 10.1002/yea.320070707. [DOI] [PubMed] [Google Scholar]
  54. Verna J., Lodder A., Lee K., Vagts A., Ballester R. A family of genes required for maintenance of cell wall integrity and for the stress response in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1997 Dec 9;94(25):13804–13809. doi: 10.1073/pnas.94.25.13804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Wickner R. B. Double-stranded RNA viruses of Saccharomyces cerevisiae. Microbiol Rev. 1996 Mar;60(1):250–265. doi: 10.1128/mr.60.1.250-265.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. de Groot P. W., Ruiz C., Vázquez de Aldana C. R., Duenas E., Cid V. J., Del Rey F., Rodríquez-Peña J. M., Pérez P., Andel A., Caubín J., Arroyo J., García J. C., Gil C., Molina M., García L. J., Nombela C., Klis F. M. A genomic approach for the identification and classification of genes involved in cell wall formation and its regulation in Saccharomyces cerevisiae. Comp Funct Genomics. 2001;2(3):124–142. doi: 10.1002/cfg.85. [DOI] [PMC free article] [PubMed] [Google Scholar]

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