Abstract
We developed a protein-proximity assay in yeast based on fusing a histone lysine methyltransferase onto a bait and its substrate onto a prey. Upon binding, the prey is stably methylated and detected by methylation-specific antibodies. We applied this approach to detect varying interaction affinities among proteins in a mitogen-activated protein kinase pathway and to detect short-lived interactions between protein phosphatase 2A and its substrates that have so far escaped direct detection.
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References
Piehler, J. Curr. Opin. Struct. Biol. 15, 4–14 (2005).
Bodenmiller, B. et al. Sci. Signal. 3, rs4 (2010).
Fernández-Suárez, M., Chen, T.S. & Ting, A.Y. J. Am. Chem. Soc. 130, 9251–9253 (2008).
Rea, S. et al. Nature 406, 593–599 (2000).
Choi, J., Chen, J., Schreiber, S.L. & Clardy, J. Science 273, 239–242 (1996).
Hohmann, S. FEBS Lett. 583, 4025–4029 (2009).
Marles, J.A., Dahesh, S., Haynes, J., Andrews, B.J. & Davidson, A.R. Mol. Cell 14, 813–823 (2004).
Hao, N. et al. Curr. Biol. 17, 659–667 (2007).
Shu, Y., Yang, H., Hallberg, E. & Hallberg, R. Mol. Cell. Biol. 17, 3242–3253 (1997).
Zhao, Y., Boguslawski, G., Zitomer, R.S. & DePaoli Roach, A.A. J. Biol. Chem. 272, 8256–8262 (1997).
Queralt, E., Lehane, C., Novak, B. & Uhlmann, F. Cell 125, 719–732 (2006).
Stark, C. et al. Nucleic Acids Res. 39, D698–D704 (2011).
Azzam, R. et al. Science 305, 516–519 (2004).
Chan, L.Y. & Amon, A. Genes Dev. 23, 1639–1649 (2009).
Ciaccio, M.F., Wagner, J.P., Chuu, C.P., Lauffenburger, D.A. & Jones, R.B. Nat. Methods 7, 148–155 (2010).
Kushnirov, V.V. Yeast 16, 857–860 (2000).
Fellner, T. et al. Genes Dev. 17, 2138–2150 (2003).
Perez-Burgos, L. et al. Methods Enzymol. 376, 234–254 (2004).
Köhler, G. & Milstein, C. Nature 256, 495–497 (1975).
Acknowledgements
We thank K. Nasmyth for scientific input; T. Jenuwein (Max Planck Institute of Immunobiology and Epigenetics, Freiburg) for materials crucial in setting up the assay; H. Charbonneau (Purdue University, West Lafayette), F. Uhlmann (London Research Institute, London), R. Deshaies (California Institute of Technology, Pasadena) and D. Brautigan (University of Virginia School of Medicine, Charlottesville) for providing plasmids and materials; and H. Hombauer, S. Kuderer and M. Roblek for technical assistance. This work was supported by grants from the Austrian Science Foundation (FWF P21712 and doctoral program APW01220FW) to E.O. G.A. was supported by FP6 (QUASI) and FP7 (UNICELLSYS) European Commission Program grants.
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A.Z., I.D., C.F. and G.A. conceived and designed the rapamycin-induced dimerization and Hog pathway experiments; T.K., B.B., I.E.F. and E.O. designed the PP2A and biotin ligase assay experiments. A.Z., I.D. and C.F. performed the rapamycin-induced dimerization and Hog pathway experiments; T.K., B.B., I.E.F. and I.M. performed the PP2A and biotin ligase assay experiments. A.Z., T.K., B.B., I.D., C.F., I.M., I.E.F., G.A. and E.O. analyzed the data. S.S. generated the monoclonal antibodies. A.Z., T.K., B.B., G.A. and E.O. wrote the paper.
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E.O. serves as a consultant to Merck/Millipore Corporation. The E.O. laboratory receives royalties from several biotech companies for monoclonal antibodies anti-me3K9H3 clone 6F12-H4 and anti-Myc clone 4A6.
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Zuzuarregui, A., Kupka, T., Bhatt, B. et al. M-Track: detecting short-lived protein-protein interactions in vivo. Nat Methods 9, 594–596 (2012). https://doi.org/10.1038/nmeth.2017
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DOI: https://doi.org/10.1038/nmeth.2017