Abstract
Proteins are generally organized into molecular complexes, in which multiple interaction partners collaborate to carry out cellular processes. Thus, techniques to map protein–protein interactions have become pivotal for biological studies of as yet uncharacterized proteins. Investigation of interaction partners of membrane proteins is of special interest, as they play a major role in cellular processes and are often directly linked to human diseases. Owing to their hydrophobic nature, however, it has proven difficult to study their interaction partners. To circumvent this problem, a yeast-based genetic technology for the in vivo detection of membrane protein interactions, the split-ubiquitin membrane yeast two-hybrid (MYTH) system, has been developed. MYTH allows for detection of both stable and transient interactions and can be applied to large- and small-scale screens. It uses the split-ubiquitin approach, in which the reconstitution of two ubiquitin halves is mediated by a specific protein–protein interaction. Briefly, the bait membrane protein is fused to the C-terminal half of ubiquitin and an artificial transcription factor. The mutated N-terminal moiety of ubiquitin is fused to the prey protein. Upon interaction of bait and prey proteins, ubiquitin is reconstituted and further recognized by ubiquitin-specific proteases, which subsequently cleave off the transcription factor, thus resulting in reporter gene activation. To date, MYTH has been successfully applied to study interactions of membrane proteins from various organisms and has only recently been adapted for the identification of interaction partners of mammalian receptor tyrosine kinases.
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Fetchko, M., Auerbach, D., and Stagljar, I. (2003) Yeast genetic methods for the detection of membrane protein interactions: potential use in drug discovery, BioDrugs 17, 413–424.
Auerbach, D., Thaminy, S., Hottiger, M. O., and Stagljar, I. (2002) The post-genomic era of interactive proteomics: facts and perspectives, Proteomics 2, 611–623.
Suter, B., Kittanakom, S., and Stagljar, I. (2008) Two-hybrid technologies in proteomics research, Curr Opin Biotechnol 19, 316–323.
Suter, B., Kittanakom, S., and Stagljar, I. (2008) Interactive proteomics: what lies ahead?, Biotechniques 44, 681–691.
Fields, S., and Song, O. (1989) A novel genetic system to detect protein-protein interactions, Nature 340, 245–246.
Stagljar, I., and Fields, S. (2002) Analysis of membrane protein interactions using yeast-based technologies, Trends Biochem Sci 27, 559–563.
Iyer, K., Burkle, L., Auerbach, D., Thaminy, S., Dinkel, M., Engels, K., and Stagljar, I. (2005) Utilizing the split-ubiquitin membrane yeast two-hybrid system to identify protein-protein interactions of integral membrane proteins, Sci STKE 2005, pl3.
Stagljar, I., Korostensky, C., Johnsson, N., and te Heesen, S. (1998) A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo, Proc Natl Acad Sci USA 95, 5187–5192.
Johnsson, N., and Varshavsky, A. (1994) Split ubiquitin as a sensor of protein interactions in vivo, Proc Natl Acad Sci USA 91, 10340–10344.
Kittanakom, S., Chuk, M., Wong, V., Snyder, J., Edmonds, D., Lydakis, A., Zhang, Z., Auerbach, D., and Stagljar, I. (2009) Analysis of membrane protein complexes using the split-ubiquitin membrane yeast two-hybrid (MYTH) system, Methods Mol Biol 548, 247–271.
Snider, J., Kittanakom, S., Curak, J., and Stagljar, I. (2010) Split-ubiquitin based membrane yeast two-hybrid (MYTH) system: a powerful tool for identifying protein-protein interactions, J Vis Exp. 36. pii: 1698
Thaminy, S., Miller, J., and Stagljar, I. (2004) The split-ubiquitin membrane-based yeast two-hybrid system, Methods Mol Biol 261, 297–312.
Paumi, C. M., Menendez, J., Arnoldo, A., Engels, K., Iyer, K. R., Thaminy, S., Georgiev, O., Barral, Y., Michaelis, S., and Stagljar, I. (2007) Mapping protein-protein interactions for the yeast ABC transporter Ycf1p by integrated split-ubiquitin membrane yeast two-hybrid analysis, Mol Cell 26, 15–25.
Deribe, Y. L., Wild, P., Chandrashaker, A., Curak, J., Schmidt, M. H., Kalaidzidis, Y., Milutinovic, N., Kratchmarova, I., Buerkle, L., Fetchko, M. J., Schmidt, P., Kittanakom, S., Brown, K. R., Jurisica, I., Blagoev, B., Zerial, M., Stagljar, I., and Dikic, I. (2009) Regulation of epidermal growth factor receptor trafficking by lysine deacetylase HDAC6, Sci Signal 2 (102), p. ra84.
Fetchko, M., and Stagljar, I. (2004) Application of the split-ubiquitin membrane yeast two-hybrid system to investigate membrane protein interactions, Methods 32, 349–362.
Gisler, S. M., Kittanakom, S., Fuster, D., Wong, V., Bertic, M., Radanovic, T., Hall, R. A., Murer, H., Biber, J., Markovich, D., Moe, O. W., and Stagljar, I. (2008) Monitoring protein-protein interactions between the mammalian integral membrane transporters and PDZ-interacting partners using a modified split-ubiquitin membrane yeast two-hybrid system, Mol Cell Proteomics 7, 1362–1377.
Paumi, C. M., Chuk, M., Chevelev, I., Stagljar, I., and Michaelis, S. (2008) Negative regulation of the yeast ABC transporter Ycf1p by phosphorylation within its N-terminal extension, J Biol Chem 283, 27079–27088.
Thaminy, S., Auerbach, D., Arnoldo, A., and Stagljar, I. (2003) Identification of novel ErbB3-interacting factors using the split-ubiquitin membrane yeast two-hybrid system, Genome Res 13, 1744–1753.
Ullrich, A., and Schlessinger, J. (1990) Signal transduction by receptors with tyrosine kinase activity, Cell 61, 203–212.
Inoue, H., Nojima, H., and Okayama, H. (1990) High efficiency transformation of Escherichia coli with plasmids. Gene 96, 23–28.
Acknowledgments
The Stagljar lab is supported by grants from the Canadian Foundation for Innovation (CFI), the Canadian Institute for Health Research (CIHR), the Canadian Cancer Society Research Institute (CCSRI), the Heart and Stroke Foundation, the Cystic Fibrosis Foundation, the Ontario Genomics Institute, and Novartis. J.P. is a recipient of an FWF-Erwin-Schrödinger postdoctoral fellowship.
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Petschnigg, J., Wong, V., Snider, J., Stagljar, I. (2012). Investigation of Membrane Protein Interactions Using the Split-Ubiquitin Membrane Yeast Two-Hybrid System. In: Suter, B., Wanker, E. (eds) Two Hybrid Technologies. Methods in Molecular Biology, vol 812. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-455-1_13
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DOI: https://doi.org/10.1007/978-1-61779-455-1_13
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