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
This is a preview of subscription content, access via your institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
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 24:218–229
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 17:1030–1032
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 580:677–681
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 38:172–181
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 46:1–13
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 3:1013–1019
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 2:1145–1151
Gyuris J, Golemis E, Chertkov H, Brent R (1993) Cdi1, a human G1 and S phase protein phosphatase that associates with Cdk2. Cell 75:791–803
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 47:132–138
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 9:789–798
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 104:8089–8094
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 7:1041–1052
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 320:1504–1506
Braus GH, Irniger S, Bayram O (2010) Fungal development and the COP9 signalosome. Curr Opin Microbiol 13:672–676
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:e1001226
James GT (1978) Inactivation of the protease inhibitor phenylmethylsulfonyl fluoride in buffers. Anal Biochem 86:574–579
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 25:1327–1333
Blum H, Hildburg B, Hans JG (1987) Improved silver staining of plant proteins. RNA and DNA in polyacrylamide gels. Electrophoresis 8:93–99
Shevchenko A, Wilm M, Vorm O, Mann M (1996) Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 68:850–858
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 6:1968–1979
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.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Bayram, Ö., Bayram, Ö.S., Valerius, O., Jöhnk, B., Braus, G.H. (2012). Identification of Protein Complexes from Filamentous Fungi with Tandem Affinity Purification. In: Keller, N., Turner, G. (eds) Fungal Secondary Metabolism. Methods in Molecular Biology, vol 944. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-122-6_14
Download citation
DOI: https://doi.org/10.1007/978-1-62703-122-6_14
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-121-9
Online ISBN: 978-1-62703-122-6
eBook Packages: Springer Protocols