Molecular Genetics and Genomics

, Volume 274, Issue 4, pp 410–418 | Cite as

UPR-independent dithiothreitol stress-induced genes in Aspergillus niger

  • D. A. MacKenzie
  • T. Guillemette
  • H. Al-Sheikh
  • A. J. Watson
  • D. J. Jeenes
  • P. Wongwathanarat
  • N. S. Dunn-Coleman
  • N. van. Peij
  • D. B. Archer
Original Paper

Abstract

A subtraction library was prepared from cultures of Aspergillus niger that had or had not been exposed to dithiothreitol (DTT), in order to identify genes involved in the unfolded protein response (UPR) or in the response to reductive stress. A large fraction of the clones in the library (40%) encoded two putative methyltransferases (MTs) whose function has yet to be determined. Other stress-responsive genes included a homologue of the Mn2+ -containing superoxide dismutase gene (sodB) and a number of genes predicted to code for products that function in protein turnover and in intra- and extracellular transport of molecules. Transcriptional microarray analysis was carried out with a group of 15 genes, comprising 11 from the cDNA library, two genes linked to the putative MT genes but not represented in the library, and two UPR control genes (bipA and pdiA). Eleven of the 15 genes were inducible with DTT. This was either reflected by the presence of transcripts in cells subjected to DTT stress compared to absence under control conditions, or by an induction ratio of between 1.4 and 8.0 in cases where transcripts were already detectable under control conditions. The MT genes were among the four most highly induced. None of the genes, apart from bipA and pdiA, showed significant induction in response to other stresses that are known to induce the UPR in fungi. We conclude that DTT alone does not provide for specific induction of UPR genes and that other stress conditions must also be examined.

Keywords

PCR subtraction Reducing agent Protein secretion Methyltransferases Filamentous fungi 

References

  1. Aina R, Sgorbati S, Santagostino A, Labra M, Ghiani A, Citterio S (2004) Specific hypomethylation of DNA is induced by heavy metals in white clover and industrial hemp. Physiol Plant 121:472–480CrossRefGoogle Scholar
  2. Al-Sheikh H, Watson AJ, Lacey GA, Punt PJ, MacKenzie DA, Jeenes DJ, Pakula T, Penttilä M, Alcocer MJC, Archer DB (2004) Endoplasmic reticulum stress leads to the selective transcriptional down-regulation of the glucoamylase gene in Aspergillus niger. Mol Microbiol 53:1731–1742CrossRefPubMedGoogle Scholar
  3. Archer DB, Jeenes DJ, MacKenzie DA, Brightwell G, Lambert N, Lowe G, Radford SE, Dobson CM (1990) Hen egg white lysozyme expressed in, and secreted from, Aspergillus niger is correctly processed and folded. Biotechnology 8:741–745CrossRefPubMedGoogle Scholar
  4. Baba SW, Belogrudov GI, Lee JC, Lee PT, Strahan J, Shepherd JN, Clarke CF (2004) Yeast Coq5 C-methyltransferase is required for stability of other polypeptides involved in coenzyme Q biosynthesis. J Biol Chem 279:10052–10059CrossRefPubMedGoogle Scholar
  5. Bujnicki JM, Feder M, Ayres CL, Redman KL (2004) Sequence-structure-function studies of tRNA:m5 C methyltransferase Trm4p and its relationship to DNA:m5 C and RNA:m5 U methyltransferases. Nucleic Acid Res 32:2453–2463CrossRefPubMedGoogle Scholar
  6. Cleves AE (1997) Protein transport: the nonclassical ins and outs. Curr Biol 7:R318–R320CrossRefPubMedGoogle Scholar
  7. Cleves AE, Cooper DNW, Barondes SH, Kelly RB (1996) A new pathway for protein export in Saccharomyces cerevisiae. J Cell Biol 133:1017–1026CrossRefPubMedGoogle Scholar
  8. Conesa A, Punt PJ, van den Hondel CAMJJ (2001) The secretion pathway in filamentous fungi: a biotechnological view. Fung Genet Biol 33:155–171CrossRefGoogle Scholar
  9. Conesa A, Jeenes D, Archer DB, van den Hondel CAMJJ, Punt PJ (2002) Calnexin overexpression increases manganese peroxidase production in Aspergillus niger. Appl Environ Microbiol 68:846–851CrossRefPubMedGoogle Scholar
  10. Esteves TC, Echtay KS, Jonassen T, Clarke CF, Brand MD (2004) Ubiquinone is not required for proton conductance by uncoupling protein 1 in yeast mitochondria. Biochem J 379:309–315CrossRefPubMedGoogle Scholar
  11. Giot L, Demattei C, Konopka JB (1999) Combining mutations in the incoming and outgoing pheromone signal pathways causes a synergistic mating defect in Saccharomyces cerevisiae. Yeast 15:765–780CrossRefPubMedGoogle Scholar
  12. Goller SP, Gorfer M, Kubicek CP (1998) Trichoderma reesei prs12 encodes a stress- and unfolded-protein-response-inducible regulatory subunit of the fungal 26S proteasome. Curr Genet 33:284–290CrossRefPubMedGoogle Scholar
  13. Groll M, Clausen T (2003) Molecular shredders: how proteasomes fulfil their role. Curr Opin Struct Biol 13:665–673CrossRefPubMedGoogle Scholar
  14. Iwanyshyn WM, Han G-S, Carman GM (2004) Regulation of phospholipid synthesis in Saccharomyces cerevisiae by zinc. J Biol Chem 279:21976–21983CrossRefPubMedGoogle Scholar
  15. Ju D, Wang L, Mao X, Xie Y (2004) Homeostatic regulation of the proteasome via an Rpn4-dependent feedback circuit. Biochem Biophys Res Commun 321:51–57CrossRefPubMedGoogle Scholar
  16. Katz JE, Dumlao DS, Wasserman JI, Lansdown MG, Jung ME, Faull KF, Clarke S (2004) 3-isopropylmalate is the major endogenous substrate of the Saccharomyces cerevisiae trans-aconitate methyltransferase. Biochem 43:5976–5986CrossRefGoogle Scholar
  17. Lombraña M, Moralejo FJ, Pinto R, Martín JF (2004) Modulation of Aspergillus awamori thaumatin secretion by modification of bipA gene expression. Appl Environ Microbiol 70:5145–5152CrossRefPubMedGoogle Scholar
  18. MacKenzie DA, Jeenes DJ, Archer DB (2004) Filamentous fungi as expression systems for heterologous proteins. In: Kück U (ed) The mycota II: genetics and biotechnology, 2nd edn. Springer, Berlin Heidelberg New York, pp 289–315Google Scholar
  19. Moralejo FJ, Watson AJ, Jeenes DJ, Archer DB, Martín JF (2001) A defined level of protein disulfide isomerase expression is required for optimal secretion of thaumatin by Aspergillus awamori. Mol Genet Genomics 266:246–253CrossRefPubMedGoogle Scholar
  20. Mulder HJ, Saloheimo M, Penttilä M, Madrid SM (2004) The transcription factor HACA mediates the unfolded protein response in Aspergillus niger, and up-regulates its own transcription. Mol Genet Genomics 271:130–140CrossRefPubMedGoogle Scholar
  21. Müllner H, Zweytick D, Leber R, Turnowsky F, Daum G (2004) Targeting of proteins involved in sterol biosynthesis to lipid particles of the yeast Saccharomyces cerevisiae. Biochim Biophys Acta 1663:9–13PubMedCrossRefGoogle Scholar
  22. Ngiam C, Jeenes DJ, Punt PJ, van den Hondel CAMJJ, Archer DB (2000) Characterization of a foldase, protein disulfide isomerase A, in the protein secretory pathway of Aspergillus niger. Appl Environ Microbiol 66:775–782CrossRefPubMedGoogle Scholar
  23. Omori K, Idei A (2003) Gram-negative bacterial ATP-binding cassette protein exporter family and diverse secretory proteins. J Biosci Bioeng 95:1–12PubMedGoogle Scholar
  24. Pak FE, Gropper S, Dai WD, Havkin-Frenkel D, Belanger FC (2004) Characterization of a multifunctional methyltransferase from the orchid Vanilla planifolia. Plant Cell Rep 22:959–966CrossRefPubMedGoogle Scholar
  25. Saloheimo M, Valkonen M, Penttilä M (2003) Activation mechanisms of the HACI-mediated unfolded protein response in filamentous fungi. Mol Microbiol 47:1149–1161CrossRefPubMedGoogle Scholar
  26. Schübeler D, MacAlpine DM, Scalzo D, Wirbelauer C, Kooperberg C, van Leeuwen F, Gottschling DE, O’Neill LP, Turner BM, Delrow J, Bell SP, Groudine M (2004) The histone modification pattern of active genes revealed through genome-wide chromatin analysis of a higher eukaryote. Genes Dev 18:1263–1271CrossRefPubMedGoogle Scholar
  27. Sims AH, Gent ME, Lanthaler K, Dunn-Coleman NS, Oliver SG, Robson GD (2005) Transcriptome analysis of recombinant protein secretion by Aspergillus nidulans and the unfolded-protein response in vivo. Appl Environ Microbiol 71:2737–2747CrossRefPubMedGoogle Scholar
  28. Travers KJ, Patil CK, Wodicka L, Lockhart DJ, Weissman JS, Walter P (2000) Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER-associated degradation. Cell 101:249–258CrossRefPubMedGoogle Scholar
  29. Van Hartingsveldt W, Mattern IE, van Zeijl CMJ, Pouwels PH, van den Hondel CAMJJ (1987) Development of a homologous transformation system for Aspergillus niger based on the pyrG gene. Mol Gen Genet 206:71–75CrossRefPubMedGoogle Scholar
  30. Wang H, Entwistle J, Morlon E, Archer DB, Peberdy JF, Ward M, Jeenes DJ (2003) Isolation and characterisation of a calnexin homologue, clxA, from Aspergillus niger. Mol Gen Genom 268:684–691Google Scholar
  31. Watson AJ, Worley J, Elliott RM, Jeenes DJ, Archer DB (2000) Cloning stress-induced genes from Aspergillus niger using polymerase chain reaction-augmented subtractive hybridization. Anal Biochem 277:162–165CrossRefPubMedGoogle Scholar
  32. Wein S, Fauroux M, Laffitte J, de Nadaï P, Guaïni C, Pons F, Coméra C (2004) Mediation of annexin 1 secretion by a probenecid-sensitive ABC-transporter in rat inflamed mucosa. Biochem Pharmacol 67:1195–1202CrossRefPubMedGoogle Scholar
  33. Wiebe MG, Karandikar A, Robson GD, Trinci APJ, Candia JLF, Trappe S, Wallis G, Rinas U, Derkx PMF, Madrid SM, Sisniega H, Faus I, Montijn R, van den Hondel CAMJJ, Punt PJ (2001) Production of tissue plasminogen activator (t-PA) in Aspergillus niger. Biotechnol Bioeng 76:164–174CrossRefPubMedGoogle Scholar
  34. Wu CC, MacCoss MJ, Mardones G, Finnigan C, Mogelsvang S, Yates (III) JR, Howell KE (2004) Organellar proteomics reveals Golgi arginine dimethylation. Mol Biol Cell 15:2907–2919CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • D. A. MacKenzie
    • 1
  • T. Guillemette
    • 2
  • H. Al-Sheikh
    • 2
  • A. J. Watson
    • 2
    • 6
  • D. J. Jeenes
    • 1
  • P. Wongwathanarat
    • 3
  • N. S. Dunn-Coleman
    • 4
  • N. van. Peij
    • 5
  • D. B. Archer
    • 2
  1. 1.Department of Food Safety ScienceInstitute of Food ResearchColney, NorwichUK
  2. 2.School of BiologyUniversity of NottinghamNottinghamUK
  3. 3.Department of Biotechnology, Faculty of Science and TechnologyThammasat UniversityKhlong Luang, Pathum ThaniThailand
  4. 4.Genencor International Inc.Palo AltoUSA
  5. 5.Research and DevelopmentDSM Food SpecialtiesDelftThe Netherlands
  6. 6.School of BiologyUniversity of Newcastle-upon-TyneNewcastle-upon-TyneUK

Personalised recommendations