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Rewiring the Budding Yeast Proteome using Synthetic Physical Interactions

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Genome Instability

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1672))

Abstract

Artificially tethering two proteins or protein fragments together is a powerful method to query molecular mechanisms. However, this approach typically relies upon a prior understanding of which two proteins, when fused, are most likely to provide a specific function and is therefore not readily amenable to large-scale screening. Here, we describe the Synthetic Physical Interaction (SPI) method to create proteome-wide forced protein associations in the budding yeast Saccharomyces cerevisiae. This method allows thousands of protein–protein associations to be screened for those that affect either normal growth or sensitivity to drugs or specific conditions. The method is amenable to proteins, protein domains, or any genetically encoded peptide sequence.

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References

  1. Coudreuse D, Nurse P (2010) Driving the cell cycle with a minimal CDK control network. Nature 468:1074–1079

    Article  CAS  PubMed  Google Scholar 

  2. Lau DT, Murray AW (2012) Mad2 and Mad3 cooperate to arrest budding yeast in mitosis. Curr Biol 22:180–190

    Article  CAS  PubMed  Google Scholar 

  3. Hagan IM, Grallert A (2013) Spatial control of mitotic commitment in fission yeast. Biochem Soc Trans 41:1766–1771

    Article  CAS  PubMed  Google Scholar 

  4. Scott JD, Pawson T (2009) Cell signaling in space and time: where proteins come together and when they’re apart. Science 326:1220–1224

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Rothbauer U, Zolghadr K, Muyldermans S, Schepers A, Cardoso MC, Leonhardt H (2008) A versatile nanotrap for biochemical and functional studies with fluorescent fusion proteins. Mol Cell Proteomics 7:282–289

    Article  CAS  PubMed  Google Scholar 

  6. Rothbauer U, Zolghadr K, Tillib S, Nowak D, Schermelleh L, Gahl A, Backmann N, Conrath K, Muyldermans S, Cardoso MC, Leonhardt H (2006) Targeting and tracing antigens in live cells with fluorescent nanobodies. Nat Methods 3:887–889

    Article  CAS  PubMed  Google Scholar 

  7. Kubala MH, Kovtun O, Alexandrov K, Collins BM (2010) Structural and thermodynamic analysis of the GFP:GFP-nanobody complex. Protein Sci 19:2389–2401

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Helma J, Cardoso MC, Muyldermans S, Leonhardt H (2015) Nanobodies and recombinant binders in cell biology. J Cell Biol 209:633–644

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Fridy PC, Li Y, Keegan S, Thompson MK, Nudelman I, Scheid JF, Oeffinger M, Nussenzweig MC, Fenyo D, Chait BT, Rout MP (2014) A robust pipeline for rapid production of versatile nanobody repertoires. Nat Methods 11:1253–1260

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R (1993) Naturally occurring antibodies devoid of light chains. Nature 363:446–448

    Article  CAS  PubMed  Google Scholar 

  11. Muyldermans S (2013) Nanobodies: natural single-domain antibodies. Annu Rev Biochem 82:775–797

    Article  CAS  PubMed  Google Scholar 

  12. Grallert A, Chan KY, Alonso-Nunez ML, Madrid M, Biswas A, Alvarez-Tabares I, Connolly Y, Tanaka K, Robertson A, Ortiz JM, Smith DL, Hagan IM (2013) Removal of centrosomal PP1 by NIMA kinase unlocks the MPF feedback loop to promote mitotic commitment in S. pombe. Curr Biol 23:213–222

    Article  CAS  PubMed  Google Scholar 

  13. Olafsson G, Thorpe PH (2015) Synthetic physical interactions map kinetochore regulators and regions sensitive to constitutive Cdc14 localization. Proc Natl Acad Sci U S A 112:10413–10418

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Huh WK, Falvo JV, Gerke LC, Carroll AS, Howson RW, Weissman JS, O’Shea EK (2003) Global analysis of protein localization in budding yeast. Nature 425:686–691

    Article  CAS  PubMed  Google Scholar 

  15. Reid RJ, Gonzalez-Barrera S, Sunjevaric I, Alvaro D, Ciccone S, Wagner M, Rothstein R (2011) Selective ploidy ablation, a high-throughput plasmid transfer protocol, identifies new genes affecting topoisomerase I-induced DNA damage. Genome Res 21:477–486

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Butt TR, Sternberg EJ, Gorman JA, Clark P, Hamer D, Rosenberg M, Crooke ST (1984) Copper metallothionein of yeast, structure of the gene, and regulation of expression. Proc Natl Acad Sci U S A 81:3332–3336

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Brachmann CB, Davies A, Cost GJ, Caputo E, Li J, Hieter P, Boeke JD (1998) Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14:115–132

    Article  CAS  PubMed  Google Scholar 

  18. Hill A, Bloom K (1989) Acquisition and processing of a conditional dicentric chromosome in Saccharomyces cerevisiae. Mol Cell Biol 9:1368–1370

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Dittmar JC, Reid RJ, Rothstein R (2010) ScreenMill: a freely available software suite for growth measurement, analysis and visualization of high-throughput screen data. BMC Bioinformatics 11:353

    Article  PubMed  PubMed Central  Google Scholar 

  20. Baryshnikova A, Costanzo M, Kim Y, Ding H, Koh J, Toufighi K, Youn JY, Ou J, San Luis BJ, Bandyopadhyay S, Hibbs M, Hess D, Gingras AC, Bader GD, Troyanskaya OG, Brown GW, Andrews B, Boone C, Myers CL (2010) Quantitative analysis of fitness and genetic interactions in yeast on a genome scale. Nat Methods 7:1017–1024

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Collins SR, Schuldiner M, Krogan NJ, Weissman JS (2006) A strategy for extracting and analyzing large-scale quantitative epistatic interaction data. Genome Biol 7:R63

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

This work was supported by a Medical Research Council (MRC) UK centenary award and grant to P.T. (MC_UP_A252_1027). The Francis Crick Institute is funded by the MRC UK, Cancer Research UK, the Wellcome Trust, Imperial College London, University College London and Kings College London. We thank Lisa Berry for comments on the chapter.

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Authors and Affiliations

  1. Mitotic Control Laboratory, The Francis Crick Institute, 1 Midland Road, London, NW1 1AT, UK

    Guðjón Ólafsson & Peter H. Thorpe

Authors
  1. Guðjón Ólafsson
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  2. Peter H. Thorpe
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Corresponding author

Correspondence to Peter H. Thorpe .

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Editors and Affiliations

  1. Dipartimento di Bioscienze, Università degli Studi di Milano, Milano, Milano, Italy

    Marco Muzi-Falconi

  2. Donnelly Centre and Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada

    Grant W Brown

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Ólafsson, G., Thorpe, P.H. (2018). Rewiring the Budding Yeast Proteome using Synthetic Physical Interactions. In: Muzi-Falconi, M., Brown, G. (eds) Genome Instability. Methods in Molecular Biology, vol 1672. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7306-4_39

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  • DOI: https://doi.org/10.1007/978-1-4939-7306-4_39

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7305-7

  • Online ISBN: 978-1-4939-7306-4

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