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. 2004 Jul;5(5):419–431. doi: 10.1002/cfg.415

Genome-Wide Analysis of the Effects of Heat Shock on a Saccharomyces cerevisiae Mutant With a Constitutively Activated cAMP-Dependent Pathway

Dawn L Jones 1, June Petty 2, David C Hoyle 3,5, Andrew Hayes 2, Stephen G Oliver 2, Isabel Riba-Garcia 4, Simon J Gaskell 4, Lubomira Stateva 1,✉
PMCID: PMC2447466  PMID: 18629174

Abstract

We have used DNA microarray technology and 2-D gel electrophoresis combined with mass spectrometry to investigate the effects of a drastic heat shock from 30℃ to 50℃ on a genome-wide scale. This experimental condition is used to differentiate between wild-type cells and those with a constitutively active cAMP-dependent pathway in Saccharomyces cerevisiae. Whilst more than 50% of the former survive this shock, almost all of the latter lose viability. We compared the transcriptomes of the wildtype and a mutant strain deleted for the gene PDE2, encoding the high-affinity cAMP phosphodiesterase before and after heat shock treatment. We also compared the two heat-shocked samples with one another, allowing us to determine the changes that occur in the pde2Δ mutant which cause such a dramatic loss of viability after heat shock. Several genes involved in ergosterol biosynthesis and carbon source utilization had altered expression levels, suggesting that these processes might be potential factors in heat shock survival. These predictions and also the effect of the different phases of the cell cycle were confirmed by biochemical and phenotypic analyses. 146 genes of previously unknown function were identified amongst the genes with altered expression levels and deletion mutants in 13 of these genes were found to be highly sensitive to heat shock. Differences in response to heat shock were also observed at the level of the proteome, with a higher level of protein degradation in the mutant, as revealed by comparing 2-D gels of wild-type and mutant heat-shocked samples and mass spectrometry analysis of the differentially produced proteins.

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Selected References

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  1. Andre B. An overview of membrane transport proteins in Saccharomyces cerevisiae. Yeast. 1995 Dec;11(16):1575–1611. doi: 10.1002/yea.320111605. [DOI] [PubMed] [Google Scholar]
  2. Arkinstall S. J., Papasavvas S. G., Payton M. A. Yeast alpha-mating factor receptor-linked G-protein signal transduction suppresses Ras-dependent activity. FEBS Lett. 1991 Jun 17;284(1):123–128. doi: 10.1016/0014-5793(91)80777-z. [DOI] [PubMed] [Google Scholar]
  3. Arthington-Skaggs B. A., Jradi H., Desai T., Morrison C. J. Quantitation of ergosterol content: novel method for determination of fluconazole susceptibility of Candida albicans. J Clin Microbiol. 1999 Oct;37(10):3332–3337. doi: 10.1128/jcm.37.10.3332-3337.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baroni M. D., Monti P., Alberghina L. Repression of growth-regulated G1 cyclin expression by cyclic AMP in budding yeast. Nature. 1994 Sep 22;371(6495):339–342. doi: 10.1038/371339a0. [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. Boorstein W. R., Craig E. A. Transcriptional regulation of SSA3, an HSP70 gene from Saccharomyces cerevisiae. Mol Cell Biol. 1990 Jun;10(6):3262–3267. doi: 10.1128/mcb.10.6.3262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Borges-Walmsley M. I., Walmsley A. R. cAMP signalling in pathogenic fungi: control of dimorphic switching and pathogenicity. Trends Microbiol. 2000 Mar;8(3):133–141. doi: 10.1016/s0966-842x(00)01698-x. [DOI] [PubMed] [Google Scholar]
  8. Boy-Marcotte E., Tadi D., Perrot M., Boucherie H., Jacquet M. High cAMP levels antagonize the reprogramming of gene expression that occurs at the diauxic shift in Saccharomyces cerevisiae. Microbiology. 1996 Mar;142(Pt 3):459–467. doi: 10.1099/13500872-142-3-459. [DOI] [PubMed] [Google Scholar]
  9. Broach J. R., Deschenes R. J. The function of ras genes in Saccharomyces cerevisiae. Adv Cancer Res. 1990;54:79–139. doi: 10.1016/s0065-230x(08)60809-x. [DOI] [PubMed] [Google Scholar]
  10. Broach J. R. Ras-regulated signaling processes in Saccharomyces cerevisiae. Curr Opin Genet Dev. 1991 Oct;1(3):370–377. doi: 10.1016/s0959-437x(05)80302-8. [DOI] [PubMed] [Google Scholar]
  11. Cannon J. F., Tatchell K. Characterization of Saccharomyces cerevisiae genes encoding subunits of cyclic AMP-dependent protein kinase. Mol Cell Biol. 1987 Aug;7(8):2653–2663. doi: 10.1128/mcb.7.8.2653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Causton H. C., Ren B., Koh S. S., Harbison C. T., Kanin E., Jennings E. G., Lee T. I., True H. L., Lander E. S., Young R. A. Remodeling of yeast genome expression in response to environmental changes. Mol Biol Cell. 2001 Feb;12(2):323–337. doi: 10.1091/mbc.12.2.323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Drees B. L., Sundin B., Brazeau E., Caviston J. P., Chen G. C., Guo W., Kozminski K. G., Lau M. W., Moskow J. J., Tong A. A protein interaction map for cell polarity development. J Cell Biol. 2001 Aug 6;154(3):549–571. doi: 10.1083/jcb.200104057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gasch A. P., Huang M., Metzner S., Botstein D., Elledge S. J., Brown P. O. Genomic expression responses to DNA-damaging agents and the regulatory role of the yeast ATR homolog Mec1p. Mol Biol Cell. 2001 Oct;12(10):2987–3003. doi: 10.1091/mbc.12.10.2987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gasch A. P., Spellman P. T., Kao C. M., Carmel-Harel O., Eisen M. B., Storz G., Botstein D., Brown P. O. Genomic expression programs in the response of yeast cells to environmental changes. Mol Biol Cell. 2000 Dec;11(12):4241–4257. doi: 10.1091/mbc.11.12.4241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ghaemmaghami Sina, Huh Won-Ki, Bower Kiowa, Howson Russell W., Belle Archana, Dephoure Noah, O'Shea Erin K., Weissman Jonathan S. Global analysis of protein expression in yeast. Nature. 2003 Oct 16;425(6959):737–741. doi: 10.1038/nature02046. [DOI] [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. Gimeno C. J., Ljungdahl P. O., Styles C. A., Fink G. R. Unipolar cell divisions in the yeast S. cerevisiae lead to filamentous growth: regulation by starvation and RAS. Cell. 1992 Mar 20;68(6):1077–1090. doi: 10.1016/0092-8674(92)90079-r. [DOI] [PubMed] [Google Scholar]
  19. Hauser N. C., Vingron M., Scheideler M., Krems B., Hellmuth K., Entian K. D., Hoheisel J. D. Transcriptional profiling on all open reading frames of Saccharomyces cerevisiae. Yeast. 1998 Sep 30;14(13):1209–1221. doi: 10.1002/(SICI)1097-0061(19980930)14:13<1209::AID-YEA311>3.0.CO;2-N. [DOI] [PubMed] [Google Scholar]
  20. Hegde P., Qi R., Abernathy K., Gay C., Dharap S., Gaspard R., Hughes J. E., Snesrud E., Lee N., Quackenbush J. A concise guide to cDNA microarray analysis. Biotechniques. 2000 Sep;29(3):548-50, 552-4, 556 passim. doi: 10.2144/00293bi01. [DOI] [PubMed] [Google Scholar]
  21. Jones D. L., Petty J., Hoyle D. C., Hayes A., Ragni E., Popolo L., Oliver S. G., Stateva L. I. Transcriptome profiling of a Saccharomyces cerevisiae mutant with a constitutively activated Ras/cAMP pathway. Physiol Genomics. 2003 Dec 16;16(1):107–118. doi: 10.1152/physiolgenomics.00139.2003. [DOI] [PubMed] [Google Scholar]
  22. Kruckeberg A. L. The hexose transporter family of Saccharomyces cerevisiae. Arch Microbiol. 1996 Nov;166(5):283–292. doi: 10.1007/s002030050385. [DOI] [PubMed] [Google Scholar]
  23. Ma P., Wera S., Van Dijck P., Thevelein J. M. The PDE1-encoded low-affinity phosphodiesterase in the yeast Saccharomyces cerevisiae has a specific function in controlling agonist-induced cAMP signaling. Mol Biol Cell. 1999 Jan;10(1):91–104. doi: 10.1091/mbc.10.1.91. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Martínez-Pastor M. T., Marchler G., Schüller C., Marchler-Bauer A., Ruis H., Estruch F. The Saccharomyces cerevisiae zinc finger proteins Msn2p and Msn4p are required for transcriptional induction through the stress response element (STRE). EMBO J. 1996 May 1;15(9):2227–2235. [PMC free article] [PubMed] [Google Scholar]
  25. Nelissen B., De Wachter R., Goffeau A. Classification of all putative permeases and other membrane plurispanners of the major facilitator superfamily encoded by the complete genome of Saccharomyces cerevisiae. FEMS Microbiol Rev. 1997 Sep;21(2):113–134. doi: 10.1111/j.1574-6976.1997.tb00347.x. [DOI] [PubMed] [Google Scholar]
  26. Nikawa J., Sass P., Wigler M. Cloning and characterization of the low-affinity cyclic AMP phosphodiesterase gene of Saccharomyces cerevisiae. Mol Cell Biol. 1987 Oct;7(10):3629–3636. doi: 10.1128/mcb.7.10.3629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pan X., Heitman J. Cyclic AMP-dependent protein kinase regulates pseudohyphal differentiation in Saccharomyces cerevisiae. Mol Cell Biol. 1999 Jul;19(7):4874–4887. doi: 10.1128/mcb.19.7.4874. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Piper P. W. The heat shock and ethanol stress responses of yeast exhibit extensive similarity and functional overlap. FEMS Microbiol Lett. 1995 Dec 15;134(2-3):121–127. doi: 10.1111/j.1574-6968.1995.tb07925.x. [DOI] [PubMed] [Google Scholar]
  29. Reifenberger E., Boles E., Ciriacy M. Kinetic characterization of individual hexose transporters of Saccharomyces cerevisiae and their relation to the triggering mechanisms of glucose repression. Eur J Biochem. 1997 Apr 15;245(2):324–333. doi: 10.1111/j.1432-1033.1997.00324.x. [DOI] [PubMed] [Google Scholar]
  30. Reifenberger E., Freidel K., Ciriacy M. Identification of novel HXT genes in Saccharomyces cerevisiae reveals the impact of individual hexose transporters on glycolytic flux. Mol Microbiol. 1995 Apr;16(1):157–167. doi: 10.1111/j.1365-2958.1995.tb02400.x. [DOI] [PubMed] [Google Scholar]
  31. Ruis H., Schüller C. Stress signaling in yeast. Bioessays. 1995 Nov;17(11):959–965. doi: 10.1002/bies.950171109. [DOI] [PubMed] [Google Scholar]
  32. Sass P., Field J., Nikawa J., Toda T., Wigler M. Cloning and characterization of the high-affinity cAMP phosphodiesterase of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9303–9307. doi: 10.1073/pnas.83.24.9303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Schmitt A. P., McEntee K. Msn2p, a zinc finger DNA-binding protein, is the transcriptional activator of the multistress response in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1996 Jun 11;93(12):5777–5782. doi: 10.1073/pnas.93.12.5777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Shevchenko A., Wilm M., Vorm O., Mann M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem. 1996 Mar 1;68(5):850–858. doi: 10.1021/ac950914h. [DOI] [PubMed] [Google Scholar]
  35. Steinmetz Lars M., Scharfe Curt, Deutschbauer Adam M., Mokranjac Dejana, Herman Zelek S., Jones Ted, Chu Angela M., Giaever Guri, Prokisch Holger, Oefner Peter J. Systematic screen for human disease genes in yeast. Nat Genet. 2002 Jul 22;31(4):400–404. doi: 10.1038/ng929. [DOI] [PubMed] [Google Scholar]
  36. Suoranta K., Londesborough J. Purification of intact and nicked forms of a zinc-containing, Mg2+-dependent, low Km cyclic AMP phosphodiesterase from bakers' yeast. J Biol Chem. 1984 Jun 10;259(11):6964–6971. [PubMed] [Google Scholar]
  37. Swan T. M., Watson K. Stress tolerance in a yeast sterol auxotroph: role of ergosterol, heat shock proteins and trehalose. FEMS Microbiol Lett. 1998 Dec 1;169(1):191–197. doi: 10.1111/j.1574-6968.1998.tb13317.x. [DOI] [PubMed] [Google Scholar]
  38. Thevelein J. M. Signal transduction in yeast. Yeast. 1994 Dec;10(13):1753–1790. doi: 10.1002/yea.320101308. [DOI] [PubMed] [Google Scholar]
  39. Thevelein J. M. The RAS-adenylate cyclase pathway and cell cycle control in Saccharomyces cerevisiae. Antonie Van Leeuwenhoek. 1992 Aug;62(1-2):109–130. doi: 10.1007/BF00584466. [DOI] [PubMed] [Google Scholar]
  40. Thevelein J. M., de Winde J. H. Novel sensing mechanisms and targets for the cAMP-protein kinase A pathway in the yeast Saccharomyces cerevisiae. Mol Microbiol. 1999 Sep;33(5):904–918. doi: 10.1046/j.1365-2958.1999.01538.x. [DOI] [PubMed] [Google Scholar]
  41. Thevelein JM, Cauwenberg L, Colombo S, De Winde JH, Donation M, Dumortier F, Kraakman L, Lemaire K, Ma P, Nauwelaers D. Nutrient-induced signal transduction through the protein kinase A pathway and its role in the control of metabolism, stress resistance, and growth in yeast. Enzyme Microb Technol. 2000 Jun 1;26(9-10):819–825. doi: 10.1016/s0141-0229(00)00177-0. [DOI] [PubMed] [Google Scholar]
  42. Toda T., Cameron S., Sass P., Zoller M., Scott J. D., McMullen B., Hurwitz M., Krebs E. G., Wigler M. Cloning and characterization of BCY1, a locus encoding a regulatory subunit of the cyclic AMP-dependent protein kinase in Saccharomyces cerevisiae. Mol Cell Biol. 1987 Apr;7(4):1371–1377. doi: 10.1128/mcb.7.4.1371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Toda T., Cameron S., Sass P., Zoller M., Wigler M. Three different genes in S. cerevisiae encode the catalytic subunits of the cAMP-dependent protein kinase. Cell. 1987 Jul 17;50(2):277–287. doi: 10.1016/0092-8674(87)90223-6. [DOI] [PubMed] [Google Scholar]
  44. Toda T., Uno I., Ishikawa T., Powers S., Kataoka T., Broek D., Cameron S., Broach J., Matsumoto K., Wigler M. In yeast, RAS proteins are controlling elements of adenylate cyclase. Cell. 1985 Jan;40(1):27–36. doi: 10.1016/0092-8674(85)90305-8. [DOI] [PubMed] [Google Scholar]
  45. Tokiwa G., Tyers M., Volpe T., Futcher B. Inhibition of G1 cyclin activity by the Ras/cAMP pathway in yeast. Nature. 1994 Sep 22;371(6495):342–345. doi: 10.1038/371342a0. [DOI] [PubMed] [Google Scholar]
  46. Wanke V., Vavassori M., Thevelein J. M., Tortora P., Vanoni M. Regulation of maltose utilization in Saccharomyces cerevisiae by genes of the RAS/protein kinase A pathway. FEBS Lett. 1997 Feb 3;402(2-3):251–255. doi: 10.1016/s0014-5793(97)00009-4. [DOI] [PubMed] [Google Scholar]
  47. Wiederkehr A., Meier K. D., Riezman H. Identification and characterization of Saccharomyces cerevisiae mutants defective in fluid-phase endocytosis. Yeast. 2001 Jun;18(8):759–773. doi: 10.1002/yea.726. [DOI] [PubMed] [Google Scholar]
  48. Wilson R. B., Renault G., Jacquet M., Tatchell K. The pde2 gene of Saccharomyces cerevisiae is allelic to rca1 and encodes a phosphodiesterase which protects the cell from extracellular cAMP. FEBS Lett. 1993 Jul 5;325(3):191–195. doi: 10.1016/0014-5793(93)81071-7. [DOI] [PubMed] [Google Scholar]
  49. Wilson R. B., Tatchell K. SRA5 encodes the low-Km cyclic AMP phosphodiesterase of Saccharomyces cerevisiae. Mol Cell Biol. 1988 Jan;8(1):505–510. doi: 10.1128/mcb.8.1.505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Winston F., Dollard C., Ricupero-Hovasse S. L. Construction of a set of convenient Saccharomyces cerevisiae strains that are isogenic to S288C. Yeast. 1995 Jan;11(1):53–55. doi: 10.1002/yea.320110107. [DOI] [PubMed] [Google Scholar]
  51. Xiao H., Lis J. T. Germline transformation used to define key features of heat-shock response elements. Science. 1988 Mar 4;239(4844):1139–1142. doi: 10.1126/science.3125608. [DOI] [PubMed] [Google Scholar]

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