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M-Track: detecting short-lived protein-protein interactions in vivo

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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Figure 1: The M-Track assay for detection of stable and transient PPIs in the HOG pathway.
Figure 2: M-Track detection of the short-lived PPI between PP2A-Cdc55 and its substrate Net1.

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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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Authors and Affiliations

Authors

Contributions

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.

Corresponding author

Correspondence to Egon Ogris.

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Competing interests

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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Supplementary Figures 1–7, Supplementary Tables 1–3, Supplementary Notes 1,2 (PDF 557 kb)

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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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