Abstract
Using bimolecular fluorescence complementation assays, we were able to demonstrate protein–protein interaction of the transcription factors AcFKH1 and CPCR1 in living cells from the filamentous fungus Acremonium chrysogenum. This was accomplished by splitting the gene for the enhanced yellow fluorescent protein (EYFP) into two parts encoding the N– and C-terminus. Both fragments were fused to different gene derivatives of the fungal transcription factors. The recombinant plasmids were used to generate transgenic fungal strains for subsequent confocal laser microscopy. Only when the full-length transcription factors were fused to EYFP fragments yellow fluorescence was observed due to the bimolecular complementation of both chimeric proteins. The nuclear localization of the protein–protein interaction was verified by staining fungal cells with the nucleic acid dye TOTO-3.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Atmakuri K, Ding Z, Christie PJ (2003) VirE2, a type IV secretion substrate, interacts with the VirD4 transfer protein at cell poles of Agrobacterium tumefaciens. Mol Microbiol 49:1699–1713
Bullock WO, Fernandez JM, Short JM (1987) XL1-Blue: a high efficiency plasmid transforming recA Escherichia coli strain with beta-galactosidase selection. Biotechniques 5:376–378
Dingwall C, Laskey RA (1991) Nuclear targeting sequences—a consensus? Trends Biochem Sci 16:478–481
Grinberg AV, Hu CD, Kerppola TK (2004) Visualization of Myc/Max/Mad family dimers and the competition for dimerization in living cells. Mol Cell Biol 24:4294–4308
Hellqvist M, Mahlapuu M, Blixt A, Enerback S, Carlsson P (1998) The human forkhead protein FREAC-2 contains two functionally redundant activation domains and interacts with TBP and TFIIB. J Biol Chem 273:23335–23343
Hink MA, Bisselin T, Visser AJ (2002) Imaging protein–protein interactions in living cells. Plant Mol Biol 50:871–883
Hu CD, Kerppola TK (2003) Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis. Nat Biotechnol 21:539–545
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
Hynes TR, Tang L, Mervine SM, Sabo JL, Yost EA, Devreotes PN, Berlot CH (2004) Visualization of G protein beta gamma dimers using bimolecular fluorescence complementation demonstrates roles for both beta and gamma in subcellular targeting. J Biol Chem 279:30279–30286
Immink RG, Gadella TW Jr, Ferrario S, Busscher M, Angenent GC (2002) Analysis of MADS box protein–protein interactions in living plant cells. Proc Natl Acad Sci USA 99:2416–2421
Ito T, Chiba T, Ozawa R, Yoshida M, Hattori M, Sakaki Y (2001) A comprehensive two-hybrid analysis to explore the yeast protein interactome. Proc Natl Acad Sci USA 98:4569–4574
Jares-Erijman EA, Jovin TM (2003) FRET imaging. Nat Biotechnol 21:1387–1395
Katan-Khaykovich Y, Shaul Y (2001) Nuclear import and DNA-binding activity of RFX1. Evidence for an autoinhibitory mechanism. Eur J Biochem 268:3108–3116
Kück U, Pöggeler S (2004) pZHK2, a bifunctional transformation vector, suitable for two step gene targeting. Fungal Genet Newsl 51:4–6
LaCasse EC, Lefebvre YA (1995) Nuclear localization signals overlap DNA- or RNA-binding domains in nucleic acid-binding proteins. Nucleic Acids Res 23:1647–1656
Mullaney EJ, Hamer JE, Roberti KA, Yelton MM, Timberlake WE (1985) Primary structure of the trpC gene from Aspergillus nidulans. Mol Gen Genet 199:37–45
Peretti M, Villard J, Barras E, Zufferey M, Reith W (2001) Expression of the three human major histocompatibility complex class II isotypes exhibits a differential dependence on the transcription factor RFXAP. Mol Cell Biol 21:5699–5709
Pöggeler S, Masloff S, Hoff B, Mayrhofer S, Kück U (2003) Versatile EGFP reporter plasmids for cellular localization of recombinant gene products in filamentous fungi. Curr Genet 43:54–61
Prein B, Natter K, Kohlwein SD (2000) A novel strategy for constructing N-terminal chromosomal fusions to green fluorescent protein in the yeast Saccharomyces cerevisiae. FEBS Lett 485:29–34
Punt PJ, Dingemanse MA, Kuyvenhoven A, Soede RD, Pouwels PH, Hondel CA van den (1990) Functional elements in the promoter region of the Aspergillus nidulans gpdA gene encoding glyceraldehyde-3-phosphate dehydrogenase. Gene 93:101–109
Romanelli MG, Tato L, Lorenzi P, Morandi C (2003) Nuclear localization domains in human thyroid transcription factor 2. Biochim Biophys Acta 1643:55–64
Schmitt EK, Kück U (2000) The fungal CPCR1 protein, which binds specifically to beta-lactam biosynthesis genes, is related to human regulatory factor X transcription factors. J Biol Chem 275:9348–9357
Schmitt EK, Hoff B, Kück U (2004a) Regulation of cephalosporin biosynthesis. In: Brakhage AA (eds) Advances in biochemical engineering biotechnology. Springer, Berlin Heidelberg New York, pp 1–43
Schmitt EK, Hoff B, Kück U (2004b) AcFKH1, a novel member of the forkhead family, associates with RFX transcription factor CPCR1 in the cephalosporin C-producing fungus Acremonium chrysogenum. Gene 342:269–281
Schmitt EK, Bunse A, Janus D, Hoff B, Friedlin E, Kürnsteiner H, Kück U (2004c) Winged helix transcription factor CPCR1 is involved in regulation of beta-lactam biosynthesis in the fungus Acremonium chrysogenum. Eukaryot Cell 3:121–134
Serebriiskii I, Estojak J, Berman M, Golemis EA (2000) Approaches to detecting false positives in yeast two-hybrid systems. Biotechniques 28:328–336
Tsuchisaka A, Theologis A (2004) Heterodimeric interactions among the 1-amino-cyclopropane-1-carboxylate synthase polypeptides encoded by the Arabidopsis gene family. Proc Natl Acad Sci USA 101:2275–2280
Uetz P, Giot L, Cagney G, Mansfield TA, Judson RS, Knight JR, Lockshon D, Narayan V, Srinivasan M, Pochart P, Qureshi-Emili A, Li Y, Godwin B, Conover D, Kalbfleisch T, Vijayadamodar G, Yang M, Johnston M, Fields S, Rothberg JM (2000) A comprehensive analysis of protein–protein interactions in Saccharomyces cerevisiae. Nature 403:623–627
Walz M, Kück U (1993) Targeted integration into the Acremonium chrysogenum genome: disruption of the pcbC gene. Curr Genet 24:421–427
Yin X, Landay MF, Han W, Levitan ES, Watkins SC, Levenson RM, Farkas DL, Prochownik EV (2001) Dynamic in vivo interactions among Myc network members. Oncogene 20:4650–4664
Ylikomi T, Bocquel MT, Berry M, Gronemeyer H, Chambon P (1992) Cooperation of proto-signals for nuclear accumulation of estrogen and progesterone receptors. EMBO J 11:3681–3694
Zhao X, Gan L, Pan H, Kan D, Majeski M, Adam SA, Unterman TG (2004) Multiple elements regulate nuclear/cytoplasmic shuttling of FOXO1: characterization of phosphorylation- and 14-3-3-dependent and -independent mechanisms. Biochem J 378:839–849
Acknowledgements
We thank Ms. Kerstin Kalkreuter and Ms. Ingeborg Godehardt for their excellent technical assistance, Drs. C. Theiß and H.-G. Mannherz (Ruhr-University, Medical Faculty) for their support with CLSM, Drs. S. Pöggeler and M. Nowrousian for reading the manuscript and Drs. H. Kürnsteiner and E. Friedlin (Sandoz GmbH, Kundl, Austria) for their interest and discussion. This work was supported by Sandoz GmbH (Kundl, Austria) and the Fonds der Chemischen Industrie (FCI).
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by S. Hohmann
Rights and permissions
About this article
Cite this article
Hoff, B., Kück, U. 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 (2005). https://doi.org/10.1007/s00294-004-0546-0
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00294-004-0546-0