Current Genetics

, Volume 28, Issue 5, pp 458–466 | Cite as

Inactivation of a single type-2A phosphoprotein phosphatase is lethal in Neurospora crassa

  • E. Yatzkan
  • O. Yarden
Original Paper

Abstract

A PCR approach, employing the use of degenerate oligonucleotide mixtures, was used to isolate pph-1, a type-2A protein phosphatase (catalytic subunit)-encoding gene, from Neurospora crassa. The isolated single-copy gene is 1327 nucleotides in length, contains four putative introns and encodes a 310 amino-acid polypeptide. pph-1 is located between pdx-1 and col-4 on the right arm of N. crassa linkage group IV. pph-1 transcript levels are highest during the first hours of conidial germination. Failure to obtain viable progeny in which pph-1 had been inactivated via the repeat-induced point (RIP) mutation process, and evidence that nuclei harboring a disrupted pph-1 gene could only be maintained in a hererokaryon, indicated that a functional pph-1 gene is essential for fungal growth. This is the first report providing evidence that inactivation of a single type-2A protein phosphatase gene results in a lethal phenotype in fungi.

Key words

Cantharidin Phosphoprotein phosphatase Neurospora crassa RIP 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Arino J, Wai Woon C, Brautigan DL, Miller TB, Johnson GL (1988) Human liver phosphatase 2A: cDNA and amino-acid sequence of two catalytic subunit isotypes. Proc Natl Acad Sci USA 85:4252–4256Google Scholar
  2. Arino J, Perez-Callejon E, Cunillera N, Camps M, Posas F, Ferrer A (1993) Protein phosphatases in higher plants: multiplicity of type 2A phosphatases in Arabidopsis thaliana. Plant Mol Biol 21:475–485Google Scholar
  3. Bialojan C, Takai A (1988) Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases. Biochem J 256:283–290Google Scholar
  4. Bruchez JJP, Eberle J, Russo VEA (1993) Regulatory sequences in the transcription of Neurospora crassa genes: CAAT box, TATA box, introns, poly(A) tail formation sequences. Fungal Genet Newslett 40:89–96Google Scholar
  5. Cairns J, Qin S, Philp R, Tan YH, Guy GR (1994) Dephosphorylation of the small heat-shock protein Hsp27 in vivo by protein phosphatase 2A. J Biol Chem 269:9176–9183Google Scholar
  6. Chen J, Martin BL, Brautigan DL (1992) Regulation of protein serine-threonine phosphatase type-2A by tyrosine phosphorylation. Science 257:1261–1264Google Scholar
  7. Cohen P (1989) The structure and regulation of protein phosphatases. Annu Rev Biochem 58:453–508Google Scholar
  8. Cohen PTW, Cohen P (1989) Discovery of a protein phosphatase activity encoded in the genome of bacteriophage λ. Biochem J 260:931–934Google Scholar
  9. Cohen P, Holmes CFB, Tsukitani Y (1990a) Okadaic acid: a new probe for the study of cellular regulation. Trends Biochem Sci 15:98–102Google Scholar
  10. Cohen PTW, Brewis ND, Hughes V, Mann DJ (1990b) Protein serine/threonine phosphatases: an expanding family. FEBS Lett 268:355–359Google Scholar
  11. Cormier P, Osborne HB, Bassez T, Poulhe R, Belle R, Mulner-Lorillon O (1991) Protein phosphatase 2A from Xenopus oocytes, characterization during meiotic cell division. FEBS Lett 295:185–188Google Scholar
  12. Davis RH, de Serres FJ (1970) Genetic and microbiological research techniques for Neurospora crassa. Methods Enzymol 17A:79–143Google Scholar
  13. Devereux J, Haeberli P, Smithies O (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12:387–395Google Scholar
  14. Doonan JH, Morris NR (1989) The bimG gene of Aspergillus nidulans, required for completion of anaphase, encodes a homologue of mammalian phosphoprotein phosphatases. Cell 57:987–996Google Scholar
  15. Fincham JRS (1989) Transformation in fungi. Microbiol Rev 53:148–170Google Scholar
  16. Haystead TAJ, Sim ATR, Carling D, Honnor RC, Tsukitani Y, Cohen P, Hardie DG (1989) Effect of the tumour promoter okadic on intracellular protein phosphorylation and metabolism. Nature 337:78–81Google Scholar
  17. Healy AM, Zolnierowicz A, Stapelton E, Goebel M, dePaoli-Roach AA, Pringle JR (1991) CDC55 a Saccharomyces cerevisiae gene involved in cellular morphogenesis: identification, characterization, and homology to the B subunit of mammalian type-2A protein phosphatase. Mol Cell Biol 11:5767–5780Google Scholar
  18. Higuchi S, Tamura J, Giri PR, Polli JW, Kincaid RL (1991) Calmodulin-dependent protein phosphatase from Neurospora crassa, molecular cloning and expression of recombinant catalytic subunit. J Biol Chem 266:18104–18112Google Scholar
  19. Honkanen RE (1993) Cantharidin, another natural toxin that inhibits the activity of serine/threonine protein phosphatases type 1 and 2 A. FEBS Lett 330:283–286Google Scholar
  20. Hu G, Ronne H (1994) Overexpression of the yeast PAM1 gene perimits survival without protein phosphatase 2A and induces a filamentous phenotype. J Biol Chem 269:3429–3435Google Scholar
  21. Inagaki N, Ito M, Nakano T, Inagaki M (1994) Spatiotemporal distribution of protein kinase and phosphatase activities. Trends Biochem Sci 19:448–452Google Scholar
  22. Khew-Goodall Y, Mayer RE, Maurer F, Stone SR, Hemmings BA (1991) Structure and transcriptional regulation of protein phosphatase-2A catalytic subunit genes. Biochemistry 30:89–97Google Scholar
  23. Kinoshita N, Ohkura H, Yanagida M (1990) Distinct, essential roles of type-1 and-2A protein phosphatases in the control of the fission-yeast cell-division cycle. Cell 63:405–415Google Scholar
  24. Kinoshita N, Yamano H, Niwa H, Yoshida T, Yanagida M (1993) Negative regulation of mitosis by the fission yeast protein phosphatase ppa2. Genes Dev 7:1059–1071Google Scholar
  25. Kitagawa Y, Sasaki K, Shima H, Shibuya M, Sugimura T, Nagao M (1990) Protein phosphatases possibility involved in rat spermatogenesis. Biochem Biophys Res Commun 171:230–235Google Scholar
  26. Kozak M (1987) An analyses of 5′-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res 15:8125–8148Google Scholar
  27. Li Y, Casida JE (1992) Cantharidin-binding protein: identification as protein phosphatase 2A. Proc Natl Acad Sci USA 89:11867–11870Google Scholar
  28. Li Y, MacKintosh C, Casida JE (1993) Protein phosphatase 2A and its [3H]cantharidin/[3H]endothall thioanhydride binding site. Biochem Pharm 46:1435–1443Google Scholar
  29. MacKintosh C, MacKintosh PW (1994) Inhibitors of kinases and phosphatases. Trends Biochem Sci 19:444–448Google Scholar
  30. Mayer-Jaekel RE, Hemmings BA (1994) Protein phosphatase 2A —a ‘menage a trois’. Trends Cell Biol 4:287–291Google Scholar
  31. Metzenberg RL, Grotelueschen J (1994) Restriction polymorphism maps of Neurospora crassa: update. Fungal Genet Newslett 40:130–138Google Scholar
  32. Metzenberg RL, Stevens JN, Selker EU, Morzycka-Wroblewska E (1985) Identification and chromosomal distribution of 5s rRNA genes in Neurospora crassa. Proc Natl Acad Sci USA 82:2067–2071Google Scholar
  33. Mumby MC, Walter G (1993) Protein serine/threonine phosphatases: structure, regulation, and functions in cell growth. Physiol Rev 73:673–699Google Scholar
  34. Orgad S, Brewis ND, Alphey L, Axton JM, Dudai Y, Cohen PTW (1990) The structure of protein phosphatase 2A is as highly conserved as that of protein phosphatase 1. FEBS Lett 275:44–48Google Scholar
  35. Rambosek J, Leach JU (1987) Recombinant DNA in filamentous fungi: progress and prospects. Rev Biotechnol 6:357–393Google Scholar
  36. Ronne H, Carlberg M, Hu G, Nehlin JO (1991) Protein phosphatase 2A in Saccharomyces cerevisiae: effects on cell growth and bud morphogenesis. Mol Cell Biol 11:4876–4884Google Scholar
  37. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Larboratory, Cold Spring Harbor, New YorkGoogle Scholar
  38. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467Google Scholar
  39. Selker E (1990) Premeiotic instability of repeated sequences in Neurospora crassa. Annu Rev Genet 24:579–613Google Scholar
  40. Shenolikar S (1994) Protein serine/threonine phosphatases — new avenues for cell regulation. Annu Rev Cell Biol 10:55–86Google Scholar
  41. Sneddon AA, Cohen PTW, Stark MJR (1990) Saccharomyces cerevisiae protein phosphatase 2A performs an essential cellular function and is encoded by two genes. EMBO J 9:4339–4346Google Scholar
  42. Staben C, Jensen B, Singer M, Pollock J, Schectman M, Kinsey J, Selker E (1989) Use of a bacterial hygromycin B resistance gene as a dominant selectable marker in Neurospora crassa transformation. Fungal Genet Newslett 36:79–81Google Scholar
  43. Stone SR, Mayer R, Wernet W, Maurer F, Hofsteenge J, Hemmings BA (1988) The nucleotide sequence of the c-DNA encoding the human lung protein phosphatase 2Aα catalytic subunit. Nucleic Acids Rev 16:11365Google Scholar
  44. Sutton A, Immanuel D, Arndt KT (1991) The SIT4 protein phosphatase functions in the late G1 for progression into S phase. Mol Cell Biol 11:2133–2148Google Scholar
  45. Szoor B, Feher Z, Bako E, Szabo G, Gergely P, Dombradi V (1994) Isolation and characterization of the catalytic subunit of protein phosphatase 2A from Neurospora crassa. Fungal Genet Newslett 41:82–84Google Scholar
  46. Vogel HJ (1956) A convenient growth medium for Neurospora crassa (medium N). Microb Genet Bull 13:42–43Google Scholar

Copyright information

© Springer-Verlag 1995

Authors and Affiliations

  • E. Yatzkan
    • 1
  • O. Yarden
    • 1
  1. 1.Department of Plant Pathology and Microbiology, Faculty of AgricultureThe Hebrew University of JerusalemRehovotIsrael

Personalised recommendations