Summary
The yeast two-hybrid system (Y2H) is a powerful tool to identify protein–protein interactions. Here we describe array-based two-hybrid methods that use defined libraries of open reading frames (ORFs) as opposed to random genomic or cDNA libraries. The array-based Y2H system is well suited for interactome studies of existing ORFeomes or subsets thereof, preferentially in a recombination-based cloning system. Array-based Y2H screens efficiently reduce false positives by using built-in controls, retesting, and evaluation of background activation. Hands-on time and the amount of used resources grow exponentially with the number of tested proteins; this is a disadvantage for large genome sizes. For large genomes, random library screen may be more efficient in terms of time and resources, but not as comprehensive as array screens, and they require an efficient sequencing facility. However, large array screens require some extent of automation although they can be carried out manually on smaller scales. Future-generation Y2H plasmid constructs including tightly regulated expression systems and features that facilitate biochemical characterization will provide more efficient and powerful tools to identify interacting proteins.
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References
Uetz, P., Giot, L., Cagney, G., Mansfield, T.A., Judson, R.S., Knight, J.R., et al (2000). A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Nature 403, 623–627.
Ho, Y., Gruhler, A., Heilbut, A., Bader, G.D., Moore, L., Adams, S.L., et al (2002). Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature 415, 180–183.
Gavin, A.C., Bosche, M., Krause, R., Grandi, P., Marzioch, M., Bauer, A., et al (2002). Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415, 141–147.
Fu, H. (2004). Protein-Protein Interactions. Methods and Applications., Volume 261 (Totowa, NJ: Humana Press).
Fields, S., and Song, O. (1989). A novel genetic system to detect protein-protein interactions. Nature 340, 245–246.
Schwartz, H., Alvares, C.P., White, M.B., and Fields, S. (1998). Mutation detection by a two-hybrid assay. Hum Mol Genet 7, 1029–1032.
Vidal, M., and Endoh, H. (1999). Prospects for drug screening using the reverse two-hybrid system. Trends Biotechnol 17, 374–381.
Vidal, M., and Legrain, P. (1999). Yeast forward and reverse ‘n’-hybrid systems. Nucleic Acids Res 27, 919–929.
SenGupta, D.J., Zhang, B., Kraemer, B., Pochart, P., Fields, S., and Wickens, M. (1996). A three-hybrid system to detect RNA-protein interactions in vivo. Proc Natl Acad Sci U S A 93, 8496–8501.
Estojak, J., Brent, R., and Golemis, E.A. (1995). Correlation of two-hybrid affinity data with in vitro measurements. Mol Cell Biol 15, 5820–5829.
Rain, J.C., Selig, L., De Reuse, H., Battaglia, V., Reverdy, C., Simon, S., et al (2001). The protein-protein interaction map of Helicobacter pylori. Nature 409, 211–215.
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.
Cagney, G., Uetz, P., and Fields, S. (2001). Two-hybrid analysis of the Saccharomyces cerevisiae 26S proteasome. Physiol Genomics 7, 27–34.
Rajagopala, S.V., Titz, B., Goll, J., Parrish, J.R., Wohlbold, K., McKevitt, M.T., et al (2007). The protein network of bacterial motility. Mol Syst Biol 3, 128.
Ito, T., Chiba, T., Ozawa, R., Yoshida, M., Hattori, M., and Sakaki, Y. (2001). A comprehensive two-hybrid analysis to explore the yeast protein interactome. Proc Natl Acad Sci U S A 98, 4569–4574.
Li, S., Armstrong, C.M., Bertin, N., Ge, H., Milstein, S., Boxem, M., et al (2004). A map of the interactome network of the metazoan C. elegans. Science 303, 540–543.
Giot, L., Bader, J.S., Brouwer, C., Chaudhuri, A., Kuang, B., Li, Y., et al (2003). A protein interaction map of Drosophila melanogaster. Science 302, 1727–1736.
Rual, J.F., Venkatesan, K., Hao, T., Hirozane-Kishikawa, T., Dricot, A., Li, N., et al (2005). Towards a proteome-scale map of the human protein-protein interaction network. Nature 437, 1173–1178.
Stelzl, U., Worm, U., Lalowski, M., Haenig, C., Brembeck, F.H., Goehler, H., et al (2005). A human protein-protein interaction network: a resource for annotating the proteome. Cell 122, 957–968.
Schwikowski, B., Uetz, P., and Fields, S. (2000). A network of protein-protein interactions in yeast. Nat Biotechnol 18, 1257–1261.
Serebriiskii, I., Estojak, J., Berman, M., and Golemis, E.A. (2000). Approaches to detecting false positives in yeast two-hybrid systems. Biotechniques 28, 328–336.
Serebriiskii, I.G., and Golemis, E.A. (2001). Two-hybrid system and false positives. Approaches to detection and elimination. Methods Mol Biol 177, 123–134.
Koegl, M., and Uetz, P. (2007). Improving yeast two-hybrid screening systems. Brief Funct Genomic Proteomic 6, 302–312.
Uetz, P., Dong, Y.A., Zeretzke, C., Atzler, C., Baiker, A., Berger, B., et al (2006). Herpesviral protein networks and their interaction with the human proteome. Science 311, 239–242.
Parrish, J.R., Yu, J., Liu, G., Hines, J.A., Chan, J.E., Mangiola, B.A., et al (2007). A proteome-wide protein interaction map for Campylobacter jejuni. Genome Biol 8, R130.
Zhong, J., Zhang, H., Stanyon, C.A., Tromp, G., and Finley, R.L., Jr. (2003). A strategy for constructing large protein interaction maps using the yeast two-hybrid system: regulated expression arrays and two-phase mating. Genome Res 13, 2691–2699.
Jin, F., Avramova, L., Huang, J., and Hazbun, T. (2007). A yeast two-hybrid smart-pool-array system for protein-interaction mapping. Nat Methods 4, 405–407.
Cormack, R.S., Hahlbrock, K., and Somssich, I.E. (1998). Isolation of putative plant transcriptional coactivators using a modified two-hybrid system incorporating a GFP reporter gene. Plant J 14, 685–692.
Rossi, F., Charlton, C.A., and Blau, H.M. (1997). Monitoring protein-protein interactions in intact eukaryotic cells by beta-galactosidase complementation. Proc Natl Acad Sci U S A 94, 8405–8410.
Drees, B.L. (1999). Progress and variations in two-hybrid and three-hybrid technologies. Curr Opin Chem Biol 3, 64–70.
Frederickson, R.M. (1998). Macromolecular matchmaking: advances in two-hybrid and related technologies. Curr Opin Biotechnol 9, 90–96.
Walhout, A.J., Temple, G.F., Brasch, M.A., Hartley, J.L., Lorson, M.A., van den Heuvel, S., et al (2000). GATEWAY recombinational cloning: application to the cloning of large numbers of open reading frames or ORFeomes. Methods Enzymol 328, 575–592.
Cagney, G., Uetz, P., and Fields, S. (2000). High-throughput screening for protein-protein interactions using two-hybrid assay. Methods Enzymol 328, 3–14.
Landy, A. (1989). Dynamic, structural, and regulatory aspects of lambda site-specific recombination. Annu Rev Biochem 58, 913–949.
Lamesch, P., Milstein, S., Hao, T., Rosenberg, J., Li, N., Sequerra, R., et al (2004). C. elegans ORFeome version 3.1: increasing the coverage of ORFeome resources with improved gene predictions. Genome Res 14, 2064–2069.
Rual, J.F., Hirozane-Kishikawa, T., Hao, T., Bertin, N., Li, S., Dricot, A., et al (2004). Human ORFeome version 1.1: a platform for reverse proteomics. Genome Res 14, 2128–2135.
Gong, W., Shen, Y.P., Ma, L.G., Pan, Y., Du, Y.L., Wang, D.H., et al (2004). Genome-wide ORFeome cloning and analysis of Arabidopsis transcription factor genes. Plant Physiol 135, 773–782.
Uetz, P. (2002). Two-hybrid arrays. Curr Opin Chem Biol 6, 57–62.
Harper, J.W., Adami, G.R., Wei, N., Keyomarsi, K., and Elledge, S.J. (1993). The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell 75, 805–816.
James, P., Halladay, J., and Craig, E.A. (1996). Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics 144, 1425–1436.
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Rajagopala, S.V., Uetz, P. (2009). Analysis of Protein–Protein Interactions Using Array-Based Yeast Two-Hybrid Screens. In: Stagljar, I. (eds) Yeast Functional Genomics and Proteomics. Methods in Molecular Biology, vol 548. Humana Press. https://doi.org/10.1007/978-1-59745-540-4_13
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