Advertisement

Identification of Protein Complexes from Filamentous Fungi with Tandem Affinity Purification

  • Özgür Bayram
  • Özlem Sarikaya Bayram
  • Oliver Valerius
  • Bastian Jöhnk
  • Gerhard H. Braus
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 944)

Abstract

Fungal molecular biology has benefited from the enormous advances in understanding protein–protein interactions in prokaryotic or eukaryotic organisms of the past decade. Tandem affinity purification (TAP) allows the enrichment of native protein complexes from cell extracts under mild conditions. We codon-optimized tags and established TAP, previously not applicable to filamentous fungi, for the model organism Aspergillus nidulans. We could identify by this method the trimeric Velvet complex VelB/VeA/LaeA or the eight subunit COP9 signalosome. Here, we describe an optimized protocol for A. nidulans which can also be adapted to other filamentous fungi.

Key words

Aspergillus nidulans Tandem affinity purification Filamentous fungi Protein complexes Interaction partners Fungal biochemistry 

Notes

Acknowledgments

This work has been funded by grants from the Deutsche Forschungsgemeinschaft (DFG), the Volkswagen-Stiftung, and the Fonds der Chemischen Industrie. Özlem Sarikaya Bayram is supported by the excellence stipend of Göttingen Graduate School for Neurosciences and Molecular Biosciences (GGNB) and Bastian Jöhnk by the German-Mexican DFG Research Unit 1334.

References

  1. 1.
    Puig O, Caspary F, Rigaut G, Rutz B, Bouveret E, Bragado-Nilsson E, Wilm M, Seraphin B (2001) The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24:218–229PubMedCrossRefGoogle Scholar
  2. 2.
    Rigaut G, Shevchenko A, Rutz B, Wilm M, Mann M, Seraphin B (1999) A generic protein purification method for protein complex characterization and proteome exploration. Nat Biotechnol 17:1030–1032PubMedCrossRefGoogle Scholar
  3. 3.
    Butland G, Zhang JW, Yang W, Sheung A, Wong P, Greenblatt JF, Emili A, Zamble DB (2006) Interactions of the Escherichia coli hydrogenase biosynthetic proteins: HybG complex formation. FEBS Lett 580:677–681PubMedCrossRefGoogle Scholar
  4. 4.
    Rohila JS, Chen M, Cerny R, Fromm ME (2004) Improved tandem affinity purification tag and methods for isolation of protein ­heterocomplexes from plants. Plant J 38:172–181PubMedCrossRefGoogle Scholar
  5. 5.
    Rohila JS, Chen M, Chen S, Chen J, Cerny R, Dardick C, Canlas P, Xu X, Gribskov M, Kanrar S, Zhu JK, Ronald P, Fromm ME (2006) Protein-protein interactions of tandem affinity purification-tagged protein kinases in rice. Plant J 46:1–13PubMedCrossRefGoogle Scholar
  6. 6.
    Burckstummer T, Bennett KL, Preradovic A, Schutze G, Hantschel O, Superti-Furga G, Bauch A (2006) An efficient tandem affinity purification procedure for interaction proteomics in mammalian cells. Nat Methods 3:1013–1019PubMedCrossRefGoogle Scholar
  7. 7.
    Gregan J, Riedel CG, Petronczki M, Cipak L, Rumpf C, Poser I, Buchholz F, Mechtler K, Nasmyth K (2007) Tandem affinity purification of functional TAP-tagged proteins from human cells. Nat Protoc 2:1145–1151PubMedCrossRefGoogle Scholar
  8. 8.
    Gyuris J, Golemis E, Chertkov H, Brent R (1993) Cdi1, a human G1 and S phase protein phosphatase that associates with Cdk2. Cell 75:791–803PubMedCrossRefGoogle Scholar
  9. 9.
    Hoff B, Kuck U (2005) Use of bimolecular fluorescence complementation to demonstrate transcription factor interaction in nuclei of ­living cells from the filamentous fungus Acremonium chrysogenum. Curr Genet 47:132–138PubMedCrossRefGoogle Scholar
  10. 10.
    Hu CD, Chinenov Y, Kerppola TK (2002) Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. Mol Cell 9:789–798PubMedCrossRefGoogle Scholar
  11. 11.
    Busch S, Schwier EU, Nahlik K, Bayram O, Helmstaedt K, Draht OW, Krappmann S, Valerius O, Lipscomb WN, Braus GH (2007) An eight-subunit COP9 signalosome with an intact JAMM motif is required for fungal fruit body formation. Proc Natl Acad Sci USA 104:8089–8094PubMedCrossRefGoogle Scholar
  12. 12.
    Helmstaedt K, Laubinger K, Vosskuhl K, Bayram O, Busch S, Hoppert M, Valerius O, Seiler S, Braus GH (2008) The nuclear migration protein NUDF/LIS1 forms a complex with NUDC and BNFA at spindle pole bodies. Eukaryot Cell 7:1041–1052PubMedCrossRefGoogle Scholar
  13. 13.
    Bayram O, Krappmann S, Ni M, Bok JW, Helmstaedt K, Valerius O, Braus-Stromeyer S, Kwon NJ, Keller NP, Yu JH, Braus GH (2008) VelB/VeA/LaeA complex coordinates light signal with fungal development and secondary metabolism. Science 320:1504–1506PubMedCrossRefGoogle Scholar
  14. 14.
    Braus GH, Irniger S, Bayram O (2010) Fungal development and the COP9 signalosome. Curr Opin Microbiol 13:672–676PubMedCrossRefGoogle Scholar
  15. 15.
    Sarikaya Bayram O, Bayram O, Valerius O, Park H, Irniger S, Gerke J, Ni M, Han K, Yu JH, Braus GH (2010) LaeA control of velvet family regulatory proteins for light-dependent development and fungal cell-type specificity. PLoS Genet 6:e1001226PubMedCrossRefGoogle Scholar
  16. 16.
    James GT (1978) Inactivation of the protease inhibitor phenylmethylsulfonyl fluoride in buffers. Anal Biochem 86:574–579PubMedCrossRefGoogle Scholar
  17. 17.
    Neuhoff V, Arold N, Taube D, Ehrhardt W (1988) Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 25:1327–1333Google Scholar
  18. 18.
    Blum H, Hildburg B, Hans JG (1987) Improved silver staining of plant proteins. RNA and DNA in polyacrylamide gels. Electrophoresis 8:93–99CrossRefGoogle Scholar
  19. 19.
    Shevchenko A, Wilm M, Vorm O, Mann M (1996) Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 68:850–858PubMedCrossRefGoogle Scholar
  20. 20.
    Valerius O, Kleinschmidt M, Rachfall N, Schulze F, Lopez Marin S, Hoppert M, Streckfuss-Bomeke K, Fischer C, Braus GH (2007) The Saccharomyces homolog of mammalian RACK1, Cpc2/Asc1p, is required for FLO11-dependent adhesive growth and dimorphism. Mol Cell Proteomics 6:1968–1979PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Özgür Bayram
    • 1
  • Özlem Sarikaya Bayram
    • 1
  • Oliver Valerius
    • 2
  • Bastian Jöhnk
    • 2
  • Gerhard H. Braus
    • 3
  1. 1.Abteilung Molekulare Mikrobiologie und Genetik, Institut für Mikrobiologie und Genetik, DFG Research Center for Molecular Physiology of the Brain (CMPB)Georg-August-Universität GöttingenGöttingenGermany
  2. 2.Abteilung Molekulare Mikrobiologie und Genetik, Institut für Mikrobiologie und Genetik, and DFG Research Center for Molecular Physiology of the Brain (CMPB)Georg-August-Universität GöttingenGöttingenGermany
  3. 3.Institut für Mikrobiologie und Genetik, Abteilung Molekulare Mikrobiologie und Genetik, DFG Research Center for Molecular Physiology of the Brain (CMPB)Georg-August-Universität GöttingenGöttingenGermany

Personalised recommendations