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Detecting interactions with membrane proteins using a membrane two-hybrid assay in yeast

Nature Protocols volume 5, pages 1281–1293 (2010)Cite this article

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Abstract

The biological function of proteins may be predicted by identification of their interacting partners, and one of the major goals of the postgenomic era is the mapping of protein interaction networks. Membrane proteins are of particular interest because of their role in disease and because of their prevalence as major pharmaceutical targets. Unfortunately, because of their hydrophobic nature, they have long been difficult to study in a high-throughput format. A powerful technology recently developed to facilitate the characterization of membrane protein interactions is the membrane yeast two-hybrid (MYTH) assay. MYTH adapts the principle of split ubiquitin for use as a potent in vivo sensor of protein–protein interactions, allowing large-scale screening for interactors of full-length membrane proteins, from a range of organisms, using Saccharomyces cerevisiae as a host. In this article, we describe a protocol for MYTH bait generation, validation and library screening. The entire MYTH procedure can generally be completed in 4–6 weeks.

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Figure 1: Important elements of the MYTH system.
Figure 2
Figure 3: MYTH system vectors.
Figure 4: Generation of tMYTH bait constructs.
Figure 5: Generation of iMYTH bait strains.
Figure 6: Sample results for key MYTH validation steps including bait localization and NubGI control and bait dependency tests.
Figure 7

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Acknowledgements

The Stagljar laboratory was supported by the Canadian Foundation for Innovation (CFI), the Canadian Institute for Health Research (CIHR), the Canadian Cancer Society Research Institute (CCSRI), the Canadian Heart and Stroke Foundation and by Novartis.

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Authors and Affiliations

  1. Department of Biochemistry and Department of Molecular Genetics, Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada

    Jamie Snider, Saranya Kittanakom, Dunja Damjanovic, Jasna Curak, Victoria Wong & Igor Stagljar

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  1. Jamie Snider
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  2. Saranya Kittanakom
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  3. Dunja Damjanovic
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  4. Jasna Curak
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  5. Victoria Wong
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Contributions

J.S. carried out the experiments, provides the results illustrated in the protocol and prepared the bulk of the article. S.K., D.D., V.W. and J.C. assisted in writing the article, figure preparation and critical review of the document. I.S. played a major role in the development of the MYTH technology, supervised MYTH-related projects and provided critical review of the document. All the authors played an active role in the application and further development of MYTH and its variants.

Corresponding author

Correspondence to Igor Stagljar.

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Competing interests

The authors declare competing financial interests. Igor Stagljar is co-founder of Dualsystems Biotech, Switzerland.

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Snider, J., Kittanakom, S., Damjanovic, D. et al. Detecting interactions with membrane proteins using a membrane two-hybrid assay in yeast. Nat Protoc 5, 1281–1293 (2010). https://doi.org/10.1038/nprot.2010.83

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