Current Genetics

, Volume 29, Issue 6, pp 516–522 | Cite as

Molecular and functional characterization of a mutant allele of the mitogen-activated protein-kinase geneSLT2(MPK1) rescued from yeast autolytic mutants

  • H. Martín
  • M. C. Castellanos
  • R. Cenamor
  • M. Sánchez
  • M. Molina
  • C. Nombela
Original Paper


We have further characterized the functionality of theSaccharomyces cerevisiae geneSLT2(MPK1), coding for a MAP-kinase homolog essential for cell integrity, which is involved in the Pkc1p signalling pathway. This gene was isolated on the basis of its capacity to complement the thermosensitive-autolytic, osmotic-remediable phenotype oflyt2 mutants. Bothslt2A andlyt2 mutants displayed a caffeine-sensitive phenotype consisting of cell lysis that was not dependent on temperature. Caffeine concentrations affecting the growth of these mutant strains were dependent on the genetic background, theSSD1 allele being very significant in this regard. TheSLT2 allele of severallyt2 strains was both rescued and amplified by PCR. The recovered allele was shown to be non-functional as it could not complement the lytic phenotype of both deletion (slt2Δ) andlyt2 strains. After nucleotide sequencing of the recovered allele, we found that the defect oflyt2 mutants consists in a substitution of an aspartic acid for a glycine at position 35 of the amino-acid sequence of Slt2p. Gly35 is the third glycine of a glycine cluster (Gly-X-Gly-X-X-Gly), a conserved region in protein kinases and other nucleotide-binding proteins.


Yeast SLT2 MAP-kinase Caffeine 


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  1. Bossemeyer D (1994) The glycine-rich sequence of protein kinases: a multifunctional element. Trends Biochem Sci 19:201–205PubMedCrossRefGoogle Scholar
  2. Burgoyne RD, Cheek TR, Morgan A, O'Sullivan AJ, Moreton RB, Berridge MJ, Mata AM, Colyer J, Lee AG, East JM (1989) Distribution of two distinct Ca2+-ATPase-like proteins and their relationships to the agonist-sensitive calcium store in adrenal chromafin cells. Nature 342:72–74PubMedCrossRefGoogle Scholar
  3. Cid VJ, Durán A, del Rey F, Snyder MP, Nombela C, Sánchez M (1995) Molecular basis of cell integrity and morphogenesis inSaccharomyces cerevisiae. Microbiol Rev 59:345–386PubMedGoogle Scholar
  4. Costigan C, Gehrung S, Snyder M (1992) A synthetic lethal screen identifiesSLK1, a novel protein kinase homolog implicated in yeast morphogenesis and cell growth. Mol Cell Biol 12:1162–1178PubMedGoogle Scholar
  5. Costigan C, Kolodrubetz D, Snyder M (1994)NHP6A andNHP6B, which encode HMG1-like proteins, function downstream in the yeastSLT2 MAPK pathway. Mol Cell Biol 14:2391–2403PubMedGoogle Scholar
  6. De Bondt HL, Rosenblatt J, Jancarik J, Jones HD, Morgan DO, Kim SH (1993) Crystal structure of cyclin-dependent kinase 2. Nature 363:595–602PubMedCrossRefGoogle Scholar
  7. Dever TE, Glynias MJ, Merrick WC (1987) GTP-binding domain: three consensus sequence elements with distinct spacing. Proc Natl Acad Sci USA 84:1814–1818PubMedCrossRefGoogle Scholar
  8. Downes CS, Musk SRR, Watson JV, Johnson RT (1990) Caffeine overcomes a restriction point associated with DNA replication, but does not accelerate mitosis. J Cell Biol 110:1855–1859PubMedCrossRefGoogle Scholar
  9. Fuente JM de la, Alvarez A, Nombela C, Sanchez M (1992) Flow cytometric analysis ofSaccharomyces cerevisiae autolytic mutants and protoplasts. Yeast 8:39–45PubMedCrossRefGoogle Scholar
  10. Gietz RD, Sugino A (1988) New yeast-Escherichia coli shuttle vectors constructed with in vitro-mutagenized yeast genes lacking six-base pair restriction sites. Gene 74:527–534PubMedCrossRefGoogle Scholar
  11. Hanahan D (1985) Techniques for transformation ofE. coli. In: Glover DM (ed) DNA Cloning. IRL Press, Oxford, England, pp 120–121Google Scholar
  12. Hanks SK, Quinn AM, Hunter T (1988) The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science 241:42–52PubMedGoogle Scholar
  13. Iric K, Takase M, Lee KS, Levin DE, Araki H, Matsumoto K, Oshima Y (1993)MKK1 andMKK2, which encodeSaccharomyces cerevisiae mitogen-activated protein kinase-kinase homologs, function in the pathway mediated by protein kinase C. Mol Cell Biol 13:3076–3083Google Scholar
  14. Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations J Bacteriol 153:163–168PubMedGoogle Scholar
  15. Jäntti J, Kuismanen E (1993) Effect of caffeine and reduced temperature (20°C) on the organization of the pre-Golgi and the Golgi stack membranes. J Cell Biol 120:1321–1335PubMedCrossRefGoogle Scholar
  16. Kamada Y, Jung US, Piotrowski J, Levin DE (1995) The protein kinase C-activated MAP kinase pathway ofSaccharomyces cerevisiae mediates a novel aspect of the heat-shock response. Genes Dev 9:1559–1571PubMedGoogle Scholar
  17. Knighton DR, Zheng JH, Ten Eyck LF, Ashford VA, Xuong NH, Taylor SS, Sowadski JM (1991) Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science 253:407–414PubMedGoogle Scholar
  18. Lee KS, Levin DE (1992) Dominant mutations in a gene encoding a putative protein kinase (BCK1) bypass the requirement for aSaccharomyces cerevisiae protein kinase C homolog. Mol Cell Biol 12:172–182PubMedGoogle Scholar
  19. Lee KS, Hines LK, Levin DE (1993a) A pair of functionally redundant yeast genes (PPZ1 andPPZ2) encoding type 1-related protein phosphatase function within thePKC1-mediated pathway. Mol Cell Biol 13:5843–5853Google Scholar
  20. Lee KS, Irie K, Gotoh Y, Watanabe Y, Araki H, Nishida E, Matsumoto K, Levin DE (1993b) A yeast MAP kinase homolog (MPK1) mediates signaling by protein kinase C. Mol Cell Biol 13:3067–3075Google Scholar
  21. Levin DE, Fields FO, Kunisawa R, Bishop JM, Thorner J (1990) A candidate protein-kinase C gene,PKC1, is required for theS. cerevisiae cell cycle. Cell 62:213–224PubMedCrossRefGoogle Scholar
  22. Martin H, Arroyo J, Sanchez M, Molina M, Nombela C (1993) Activity of the yeast MAP-kinase homologue Slt2 is critically required for cell integrity at 37°C. Mol Gen Genet 241:177–184PubMedCrossRefGoogle Scholar
  23. Mazzoni C, Zarov P, Rambourg A, Mann C (1993) TheSLT2 (MPK1) MAP kinase homolog is involved in polarized cell growth inSaccharomyces cerevisiae. J Cell Biol 123:1821–1833PubMedCrossRefGoogle Scholar
  24. Mellor J, Dobson MJ, Roberts NA, Tuite MF, Emtage JS, White S, Lowe PA, Patel T, Kingsman AJ, Kingsman SM (1983) Efficient synthesis of enzimatically active calf chymosin inSaccharomyces cerevisiae. Gene 24:1–14PubMedCrossRefGoogle Scholar
  25. Odawara M, Kadowaki T, Yamamoto R, Shibasaki Y, Tobe K (1989) Human diabetes associated with a mutation in the tyrosine-kinase domain of the insulin receptor. Science 245:66–68PubMedGoogle Scholar
  26. Paravicini G, Cooper M, Friedli L, Smith DJ, Carpentier JL, Klig LS, Payton MA (1992) The osmotic integrity of the yeast cell requires a functionalPKC1 gene product. Mol Cell Biol 12:4896–4905PubMedGoogle Scholar
  27. Parsons WJ, Ramkumar V, Stiles GL (1988) Isobutyl-methilxanthine stimulates adenylate cyclase by blocking the inhibitory regulatory protein, Gi. Mol Pharmacol 34:37–41PubMedGoogle Scholar
  28. Pelley RJ, Maihle NJ, Boerkoel C, Shu HK, Carter TH (1989) Disease tropism of v-ebb-B: effects of carboxyl-terminal tyrosine and internal mutations on tissue-specific transformation. Proc Natl Acad Sci USA 86:7164–7168PubMedCrossRefGoogle Scholar
  29. Posas F, Casamayor A, Ariño J (1993) The PPZ protein phosphatases are involved in the manteinance of osmotic stability of yeast cells. FEBS Lett 318:282–286PubMedCrossRefGoogle Scholar
  30. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New YorkGoogle Scholar
  31. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467PubMedCrossRefGoogle Scholar
  32. Sherman F, Fink GR, Hicks JB (1983) Methods in yeast genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New YorkGoogle Scholar
  33. Sutton A, Immanuel D, Arndt KT (1991) The SIT4 protein phosphatase functions in late G1 for progression into S phase. Mol Cell Biol 11:2133–2148PubMedGoogle Scholar
  34. Taylor SS, Knighton DR, Zheng J, Ten Eyck LF, Sowadski JM (1992) Structural framework for the protein-kinase family. Annu Rev Cell Biol 8:429–462PubMedCrossRefGoogle Scholar
  35. 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 5:2845–2854PubMedGoogle Scholar
  36. Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of protein from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354PubMedCrossRefGoogle Scholar
  37. Uesono Y, Fujita A, Toh-e A, Kikuchi Y (1994), TheMCS1/SSD1/SRK1/SSL1 gene is involved in stable maintenance of the chromosome in yeast. Gene 143:135–138PubMedCrossRefGoogle Scholar
  38. Walker JE, Saraste M, Runswick MJ, Gay NJ (1982) Distantly related sequences in the a- and b-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide-binding fold. EMBO J 1:945–951PubMedGoogle Scholar
  39. Wilson RB, Brenner AA, White TB, Engler MJ, Gaughran JP, Tatchell K (1991) TheSaccharomyces cerevisiae SRK1 gene, a suppressor ofbcy1 andins1, may be involved in protein-phosphatase function. Mol Cell Biol 11:3369–3373PubMedGoogle Scholar
  40. Winston F, Chumley F, Fink GR (1983) Eviction and transplacement of mutant genes in yeast. Methods Enzymol 101:211–228PubMedCrossRefGoogle Scholar
  41. Zhang F, Strand A, Robbins D, Cobb MH, Goldsmith EJ (1994) Atomic structure of the MAP-kinase ERK2 at 2.3 Å resolution. Nature 367:704–711PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 1996

Authors and Affiliations

  • H. Martín
    • 1
  • M. C. Castellanos
    • 1
  • R. Cenamor
    • 1
  • M. Sánchez
    • 1
  • M. Molina
    • 1
  • C. Nombela
    • 1
  1. 1.Departamento de Microbiología II, Facultad de FarmaciaUniversidad ComplutenseMadridSpain

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