Note to users. If you're seeing this message, it means that your browser cannot find this page's style/presentation instructions -- or possibly that you are using a browser that does not support current Web standards. Find out more about why this message is appearing, and what you can do to make your experience of our site the best it can be.


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


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}

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).

Read the Full Text

Identification of Novel Host Factors via Conserved Domain Search: Cns1 Cochaperone Is a Novel Restriction Factor of Tombusvirus Replication in Yeast.
J.-Y. Lin and P. D. Nagy (2013)
J. Virol. 87, 12600-12610
   Abstract »    Full Text »    PDF »
Osmotic Stress Responses and Plant Growth Controlled by Potassium Transporters in Arabidopsis.
Y. Osakabe, N. Arinaga, T. Umezawa, S. Katsura, K. Nagamachi, H. Tanaka, H. Ohiraki, K. Yamada, S.-U. Seo, M. Abo, et al. (2013)
PLANT CELL 25, 609-624
   Abstract »    Full Text »    PDF »
Identification of novel ATP13A2 interactors and their role in {alpha}-synuclein misfolding and toxicity.
M. Usenovic, A. L. Knight, A. Ray, V. Wong, K. R. Brown, G. A. Caldwell, K. A. Caldwell, I. Stagljar, and D. Krainc (2012)
Hum. Mol. Genet. 21, 3785-3794
   Abstract »    Full Text »    PDF »
Identification of Novel Host Cell Binding Partners of Oas1b, the Protein Conferring Resistance to Flavivirus-Induced Disease in Mice.
S. C. Courtney, H. Di, B. M. Stockman, H. Liu, S. V. Scherbik, and M. A. Brinton (2012)
J. Virol. 86, 7953-7963
   Abstract »    Full Text »    PDF »
A Role for Nyctalopin, a Small Leucine-Rich Repeat Protein, in Localizing the TRP Melastatin 1 Channel to Retinal Depolarizing Bipolar Cell Dendrites.
J. N. Pearring, P. Bojang Jr, Y. Shen, C. Koike, T. Furukawa, S. Nawy, and R. G. Gregg (2011)
J. Neurosci. 31, 10060-10066
   Abstract »    Full Text »    PDF »
Lumenal interactions in nuclear pore complex assembly and stability.
W. T. Yewdell, P. Colombi, T. Makhnevych, and C. P. Lusk (2011)
Mol. Biol. Cell 22, 1375-1388
   Abstract »    Full Text »    PDF »
Regulation of M3 Muscarinic Receptor Expression and Function by Transmembrane Protein 147.
E. Rosemond, M. Rossi, S. M. McMillin, M. Scarselli, J. G. Donaldson, and J. Wess (2011)
Mol. Pharmacol. 79, 251-261
   Abstract »    Full Text »    PDF »
Regulation of Epidermal Growth Factor Receptor Trafficking by Lysine Deacetylase HDAC6.
Y. Lissanu Deribe, P. Wild, A. Chandrashaker, J. Curak, M. H. H. Schmidt, Y. Kalaidzidis, N. Milutinovic, I. Kratchmarova, L. Buerkle, M. J. Fetchko, et al. (2009)
Science Signaling 2, ra84
   Abstract »    Full Text »    PDF »
ABC Transporters in Saccharomyces cerevisiae and Their Interactors: New Technology Advances the Biology of the ABCC (MRP) Subfamily.
C. M. Paumi, M. Chuk, J. Snider, I. Stagljar, and S. Michaelis (2009)
Microbiol. Mol. Biol. Rev. 73, 577-593
   Abstract »    Full Text »    PDF »
The SPX domain of the yeast low-affinity phosphate transporter Pho90 regulates transport activity.
H. C. Hurlimann, B. Pinson, M. Stadler-Waibel, S. C. Zeeman, and F. M. Freimoser (2009)
EMBO Rep. 10, 1003-1008
   Abstract »    Full Text »    PDF »
Monitoring Protein-Protein Interactions between the Mammalian Integral Membrane Transporters and PDZ-interacting Partners Using a Modified Split-ubiquitin Membrane Yeast Two-hybrid System.
S. M. Gisler, S. Kittanakom, D. Fuster, V. Wong, M. Bertic, T. Radanovic, R. A. Hall, H. Murer, J. Biber, D. Markovich, et al. (2008)
Mol. Cell. Proteomics 7, 1362-1377
   Abstract »    Full Text »    PDF »
G Protein-coupled Receptor Gpr4 Senses Amino Acids and Activates the cAMP-PKA Pathway in Cryptococcus neoformans.
C. Xue, Y.-S. Bahn, G. M. Cox, and J. Heitman (2006)
Mol. Biol. Cell 17, 667-679
   Abstract »    Full Text »    PDF »

To Advertise     Find Products

Science Signaling. ISSN 1937-9145 (online), 1945-0877 (print). Pre-2008: Science's STKE. ISSN 1525-8882