Advertisement

Antonie van Leeuwenhoek

, Volume 65, Issue 3, pp 211–216 | Cite as

Protein targeting and secretion in filamentous fungi

A progress report
  • Peter J. Punt
  • G. Veldhuisen
  • Cees A. M. J. J. van den Hondel
Article

Abstract

Although the application of filamentous fungi, such asAspergillus niger for the production of extracellular proteins is well established for several decades, hardly any information is available about the molecular mechanisms of the process of protein secretion in these organisms.

Two lines of research initiated towards a systematic analysis of the mechanism of protein targeting and secretion are presented in this paper.

1 — To study routing and targeting of proteins in filamentous fungi the availability of a versatile reporter/carrier protein will be of considerable importance. Experiments towards the identification of such a protein are presented.

2 — In analogy to the situation inSaccharomyces cerevisiae, the availability of defined (conditional) mutations in the secretion pathway will provide very important information about the organisation of the pathway. Therefore, based on results obtained inS. cerevisiae, the cloning of several fungal ‘secretion’ genes was started. The results of the cloning and characterisation of one of these genes is presented.

Key words

Aspergillus niger filamentous fungi (conditional) secretion mutants reporter/carrier proteins subcellular compartments 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Bielefeld M & Hollenberg CP (1992) Bacterial β-lactamase is efficiently secreted inSaccharomyces cerevisiae under control of the invertase signal sequence. Curr. Genet. 21: 265–268PubMedGoogle Scholar
  2. Broekhuijsen MP, Mattern IE, Contreras R, Kinghorn JR, van den Hondel CAMJJ (1993) Secretion of heterologous proteins byAspergillus niger: Production of active human interleukin-6 in a protease-deficient mutant by KEX2-like processing of a glucoamylase-hIL6 fusion protein. J. Biotech. 31: 135–145Google Scholar
  3. Casadaban MJ, Martinez-Arias A, Shapira SK & Chou J (1983) β-Galactosidase gene fusions for analyzing gene expression inE. coli and yeast. Meth. Enzymol. 100: 293–308PubMedGoogle Scholar
  4. Chapman CM, Loewenberg JR, Schaller MJ & Piechura JE (1983) Ultrastructural localization of cellulase inTrichoderma reesei using immunocytochemistry and enzyme cytochemistry. J. Histochem. Cytochem. 31: 1363–1366PubMedGoogle Scholar
  5. Denecke J, De Rycke R & Botterman J (1992) Plant and mammalian sorting signals for protein retention in the endoplasmatic reticulum contain a conserved epitope. EMBO J. 11: 2345–2355PubMedGoogle Scholar
  6. d'Enfert C, Gensse M & Gaillardin C (1992) Fission yeast and a plant have functional homologues of the Sar1 and Sec12 proteins involved in ER to Golgi traffic in budding yeast. EMBO J. 11: 4205–4211PubMedGoogle Scholar
  7. Den Herder, IF, Mateo-Rosell AM, van Zuilen CM, Punt PJ & van den Hondel CAMJJ (1992) Cloning and expression of a member of theAspergillus niger gene family encoding α-galactosidase. Mol. Gen. Genet. 233: 404–410PubMedGoogle Scholar
  8. Gems DH & Clutterbuck AJ (1993) Co-transformation with autonomously-replicating helper plasmids facilitates gene cloning from anAspergillus nidulans gene library. Curr. Genet. 24: 520–524PubMedGoogle Scholar
  9. Goldman GH, Demolder J, Dewaele S, Herrera-Estrella A, Geremia RA, Van Montagu M & Contreras R (1992) Molecular cloning of the imidazoleglycerolphosphate dehydratase gene ofTrichoderma harzianum by genetic complementation inSaccharomyces cerevisiae using a direct expression vector. Mol. Gen. Genet. 234: 481–488PubMedGoogle Scholar
  10. Gould SJ & Subramani S (1988) Firefly luciferase as a tool in molecular and cell biology. Anal. Biochem. 175: 5–13PubMedGoogle Scholar
  11. Iturriaga G, Jefferson RA & Bevan MW (1989) Endoplasmic reticulum targeting ands glycosylation of hybrid proteins in transgenic tobacco. Plant Cell 1: 381–390CrossRefPubMedGoogle Scholar
  12. Jefferson RA, Kavanagh TA & Bevan MW (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gen fusion marker in higher plants. EMBO J. 6: 3901–3907PubMedGoogle Scholar
  13. Klionsky DJ, Banta LM & Emr SD (1988) Intracellular sorting and processing of a yeast vacuolar hydrolase: Proteinase A propeptide contains vacuolar targeting information. Mol. Cell. Biol. 8: 2105–2116PubMedGoogle Scholar
  14. Kumar V, Ramakrishnan S, Teeri TT, Knowles JKC & Hartley BS (1992)Saccharomyces cerevisiae cells secreting anAspergillus niger β-galactosidase grow on whey permeate. Biotechnol. 10: 82–85Google Scholar
  15. Nakano A & Muramatsu M (1989) A novel GTP-binding protein, Sar1p, is involved in transport from the endoplasmatic reticulum to the Golgi apparatus. J. Cell Biol. 109: 2677–2691PubMedGoogle Scholar
  16. Normington K, Kohno K, Kozutsumi Y, Gething M-J, & Sambrook J (1989)S. cerevisiae encodes an essential protein homologous in sequence and function to mammalian BiP. Cell 57: 1223–1236CrossRefPubMedGoogle Scholar
  17. Punt PJ, Zegers ND, Busscher M, Pouwels PH & van den Hondel CAMJJ (1991) Intracellular and extracellular production of proteins inAspergillus under the control of expression signals of the highly expressedA. nidulans gpdA gene. J. Biotechnol. 17: 19–34PubMedGoogle Scholar
  18. Roberts IN, Oliver RP, Punt PJ & Van den Hondel CAMJJ (1989) Expression of theEscherichia coli β-glucuronidase gene in industrial and phytopathogenic filamentous fungi. Curr. Genet. 15: 177–180PubMedGoogle Scholar
  19. Salminen A & Novick PJ (1987) Aras like protein is required for a post-Golgi event in yeast secretion. Cell 49: 527–538PubMedGoogle Scholar
  20. Spee JH, de Vos WM & Kuipers OP (1993) Efficient random mutagenesis method with adjustable mutation frequency by use of PCR and dITP. Nucl. Acid Res. 21: 777–778Google Scholar
  21. Van den Hondel CAMJJ & Punt PJ (1991) Gene-transfer systems and vector development for filamentous fungi. In: Peberdy JF, Caten CE, Ogden JE & Bennett JW (Eds) BSM Symposium Series Volume 18, Applied Molecular Genetics of Fungi (pp 1–29). British Mycological Society, Cambridge University Press, CambridgeGoogle Scholar
  22. Van den Hondel CAMJJ, Punt PJ & Van Gorcom RFM (1991) Heterologous gene expression in filamentous fungi. In: Bennett JW & Lasure LL (Eds) More gene manipulation in fungi (pp 396–428). Academic Press, San Diego, CAGoogle Scholar
  23. Van Gorcom RFM, Pouwels PH, Goosen T, Visser J, Van den Broek HWJ, Hamer JE, Timberlake WE & Van den Hondel CAMJJ (1985) Expression of anEscherichia coli β-galactosidase fusion gene inAspergillus nidulans. Gene 40: 99–106PubMedGoogle Scholar
  24. Verdoes JC (1994) Molecular genetic studies of glucoamylase over-producing strains ofAspergillus niger. Ph.D. Thesis, Free University, AmsterdamGoogle Scholar
  25. Verdoes JC, Calil MR, Punt PJ, Debets F, Swart K, Stouthamer AH & van den Hondel CAMJJ (1994a) The complete karyotype ofAspergillus niger: The use of introduced electrophoretic mobility variation of chromosomes for chromosome-localization studies. Mol. Gen. Genet., in pressGoogle Scholar
  26. Verdoes JC, Punt PJ, van der Berg P, Debets F, Stouthamer AH & van den Hondel CAMJJ (1994b) Characterization of an efficient gene cloning strategy forAspergillus niger based on an autonomously replicating plasmid: Cloning of thenicB gene ofA. niger. Gene, in pressGoogle Scholar
  27. Ward M, Wilson LJ, Kodama KH, Rey MW, Berka RM (1990) Improved production of chymosin inAspergillus by expression as a glucoamylase-chymosin fusion. Biotechnol. 8: 435–440Google Scholar
  28. Wiebe MG, Robson, GD, Cunliffe B, Trinci APJ & Oliver SG (1992) Nutrient-dependent selection of morphological mutants ofFusarium graminearum A3/5 isolated from long-term continuous flow cultures. Biotech. Bioengin. 40: 1181–1189Google Scholar
  29. Withers JM, Wiebe MG, Robson GD & Trinci APJ (1994) Development of morphological heterogeneity in glucose-limited chemostat cultures ofAspergillus oryzae. Mycol. Res. 98: 95–100Google Scholar
  30. Wösten HAB, Moukha SM, Sietsma JH & Wessels JGH (1991) Lacalization of growth and secretion of proteins inAspergillus niger. J. Gen. Microbiol. 137: 2017–2023PubMedGoogle Scholar

Copyright information

© Kluwer Academic Publishers 1994

Authors and Affiliations

  • Peter J. Punt
    • 1
  • G. Veldhuisen
    • 1
  • Cees A. M. J. J. van den Hondel
    • 1
  1. 1.Department of Molecular Genetics and Gene TechnologyTNO Nutrition and Food research InstituteRijswijkThe Netherlands

Personalised recommendations