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Protein interaction platforms: visualization of interacting proteins in yeast

Nature Methods volume 6, pages 500–502 (2009)Cite this article

Abstract

Here we describe the protein interaction platform assay, a method for identifying interacting proteins in Saccharomyces cerevisiae. This assay relies on the reovirus scaffolding protein μNS, which forms large focal inclusions in living cells. When a query protein is fused to μNS and potential interaction partners are fused to a fluorescent reporter, interactors can be identified by screening for yeast that display fluorescent foci.

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Figure 1: Development of PIP assay in yeast.
Figure 2: Detection of previously characterized chaperone-effector interactions via PIP.
Figure 3: S. flexneri effector-chaperone interactions by PIP.

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Acknowledgements

We thank S. Alberti and S. Lindquist (Whitehead Institute, Massachusetts Institute of Technology) for providing the pAG destination clones; A. Gray, K. Fixen and M. Goldberg (Massachusetts General Hospital, Harvard Medical School) for providing plasmids pNG162 and pDSW206 and antibodies to IcsA and isocitrate dehydrogenase; J. Heindl (Massachusetts General Hospital, Harvard Medical School) for providing the IpgB2 W62A construct; T. Hao, D. Hill and M. Vidal (Dana Farber Cancer Institute, Harvard Medical School) for assistance in designing primers to create the S. flexneri Gateway entry clones and for providing the Gateway-compatible Y2H vectors; and R. Levy and C. Koser (Massachusetts General Hospital, Harvard Medical School) for cloning and expressing S. typhimurium effectors in yeast. We also thank the US National Institute of Allergy and Infectious Diseases and the J. Craig Venter Institute for supplying the S. typhimurium Gateway entry clones. Partial support for this work was provided by US National Institutes of Health grants R56 AI067445 to M.L.N. and R01 AI064285 to C.F.L., and by a Charles E. Culpeper Medical Scholarship from the Rockefeller Brothers Fund and Goldman Philanthropic Partnerships to C.F.L.

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Authors and Affiliations

  1. Department of Medicine (Microbiology and Molecular Genetics), Division of Infectious Diseases, Massachusetts General Hospital and Harvard Medical School, Cambridge, Massachusetts, USA

    Alexa M Schmitz, Monica F Morrison, Akochi O Agunwamba & Cammie F Lesser

  2. Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts, USA

    Max L Nibert & Cammie F Lesser

Authors
  1. Alexa M Schmitz
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  2. Monica F Morrison
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  3. Akochi O Agunwamba
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  4. Max L Nibert
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  5. Cammie F Lesser
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Contributions

A.M.S., M.F.M. and A.O.A. performed and analyzed the experiments. A.M.S., M.L.N. and C.F.L. designed the experiments. M.L.N. and C.F.L. wrote the manuscript.

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Correspondence to Cammie F Lesser.

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Supplementary Figures 1–7 and Supplementary Tables 1 and 2 (PDF 7447 kb)

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Schmitz, A., Morrison, M., Agunwamba, A. et al. Protein interaction platforms: visualization of interacting proteins in yeast. Nat Methods 6, 500–502 (2009). https://doi.org/10.1038/nmeth.1337

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