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Sci. STKE, 15 March 2005
Vol. 2005, Issue 275, p. pl3
[DOI: 10.1126/stke.2752005pl3]

PROTOCOLS

Utilizing the Split-Ubiquitin Membrane Yeast Two-Hybrid System to Identify Protein-Protein Interactions of Integral Membrane Proteins

Kavitha Iyer1, Lukas Bürkle1, Daniel Auerbach2, Safia Thaminy1, Martin Dinkel1, Kim Engels1, and Igor Stagljar1*

1 Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich, CH-8057 Zurich, Switzerland.
2 DUALSYSTEMS Biotech, CH-8057 Zurich, Switzerland.

Abstract: Various modifications of the conventional yeast two-hybrid system have played an essential role in confirming or detecting protein-protein interactions among nuclear and cytoplasmic proteins. These approaches have permitted the identification of novel interaction partners, as well as provided hints as to their function. However, membrane proteins, such as receptor tyrosine kinases, G protein–coupled receptors, membrane-bound phosphatases, and transporters, which represent important classes of signaling molecules, are difficult to study using classical protein interaction assays because of their hydrophobic nature. Here, we describe a genetic system that allows the identification of integral membrane-interacting proteins. This so-called "split-ubiquitin membrane-based yeast two-hybrid assay" involves fusing the halves of ubiquitin to two interacting proteins, at least one of which is membrane bound. Upon interaction of these two proteins, the halves of ubiquitin are brought together, and the transcription factor that is fused to a membrane protein of interest is cleaved and released. The free transcription factor then enters the nucleus and activates transcription of reporter genes. We also describe how this technology is used to screen complementary DNA libraries to identify novel binding partners of a membrane protein of interest.

*Corresponding author. Telephone, +41-1-635 54 74; fax, +41-1-635 68 40; e-mail, stagljar{at}vetbio.unizh.ch

Citation: K. Iyer, L. Bürkle, D. Auerbach, S. Thaminy, M. Dinkel, K. Engels, I. Stagljar, Utilizing the Split-Ubiquitin Membrane Yeast Two-Hybrid System to Identify Protein-Protein Interactions of Integral Membrane Proteins. Sci. STKE 2005, pl3 (2005).

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