Abstract
Dynamic and reversible protein-protein interactions have a pivotal function in all living cells. For instance, protein-protein interactions are involved in the assembly and regulation of multimeric enzymes and transcription factors, various signal response pathways, intracellular sorting and movement of proteins and membrane vesicles, cell-to-cell protein transport, and many others. Here we provide a detailed protocol for the mating-based split-ubiquitin system (mbSUS), which is a sensitive and user-friendly alternative to the classical yeast two-hybrid system in particular. mbSUS relies on the ubiquitin-degradation pathway as a sensor for protein-protein interactions. Thus, mbSUS is predominantly suitable for the determination of full-length proteins localized in the cytoplasm and in or at membrane compartments, without the need for their truncation and nuclear mislocation. In addition, we present a set of Gateway®-compatible mbSUS vectors that allow the rapid generation of constructs for fast and efficient interaction studies. An additional vector is introduced that allows the extension of mbSUS for the analysis of oligomeric protein complex formation and competition assays in vivo. In summary, mbSUS provides an additional versatile tool for protein-protein interaction studies, which is complementary to in planta assays such as BiFC and FRET.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Preview
Unable to display preview. Download preview PDF.
References
Phizicky, E.M. and Fields, S. (1995) Protein-protein interactions: methods for detection and analysis. Microbiol. Rev. 59, 94–123.
Fields, S. and Song, O. (1989) A novel genetic system to detect protein-protein interactions. Nature 340, 245–246.
Hu, C.-D., Chinenov, Y., and Kerppola, T. (2002) Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. Molecular Cell. 9, 789–798.
Bracha-Drori, K., Shichrur, K., Katz, A., Oliva, M., Angelovici, R, Yalovsky, S., and Ohad, N. (2004) Detection of protein-protein interactions in plants using bimolecular fluorescence complementation. Plant J. 40, 419–427.
Walter, M., Chaban, C., Schiitze, K, Batistic, O., Weckermann, K., Nàke, C., Blazevic, D. , Grefen, C., Schumacher, K., Oecking, C. , Harter, K, and Kudla, J. (2004) Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J. 40, 428–438.
Tsien, R.Y., Bacskai, B.J., and Adams, S.R. (1993) FRET for studying intracellular signalling. Trends Cell Biol. 3, 242–245.
Obrdlik, P., El-Bakkoury, M., Hamacher, T., Cappellaro, C., Vilarino, C., Fleischer, C. , Ellerbrok, H., Kamuzinzi, R, Ledent, V., Blaudez, D., Sanders, D., Revuelta, J.L., Boles, E., Andre, B., and Frommer, W.B. (2004) K + channel interactions detected by a genetic system optimized for systematic studies of membrane protein interactions. Proc. Natl. Acad. Sci. U S A 101, 12242–12247.
Johnsson, N. and Varshavsky, A. (1994) Split ubiquitin as a sensor of protein interactions in vivo. Proc. Natl. Acad. Sci. USA 91,10340–10344
Grefen, C., Stadele, K, Ruzizka, K, Obrdlik, P., Harter, K, and Horak, J. (2008) Subcellular localization and in vivo interaction of the Arabidopsis thaliana ethylene receptor family. Molecular Plant, Mol Plant; 1:308–320
Schaller, G.E., Ladd, A.N., Lanahan, M.B., Spanbauer, J.M., and Bleecker, A.B. (1995) The ethylene response mediator ETR1 from Arabidopsis forms a disulfide-linked dimer. J. Biol. Chem. 270, 12526–12530.
Chen, Y.F., Randlett, M.D., Findell, J.L., and Schaller, G.E. (2002) Localization of the ethylene receptor ETR1 to the endoplasmic reticulum of Arabidopsis. J. Biol. Chem. 277,19861–19866.
Raquet, X., Eckert, J.H., Muller, S., and Johnsson, N. (2001) Detection of altered protein conformations in living cells. J. Mol. Biol. 305, 927–938.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Grefen, C., Obrdlik, P., Harter, K. (2009). The Determination of Protein-protein Interactions by the Mating-based Split-ubiquitin system (mbSUS). In: Pfannschmidt, T. (eds) Plant Signal Transduction. Methods in Molecular Biology, vol 479. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-289-2_14
Download citation
DOI: https://doi.org/10.1007/978-1-59745-289-2_14
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-58829-943-7
Online ISBN: 978-1-59745-289-2
eBook Packages: Springer Protocols