Nucleic Acids Research Advance Access published online on March 4, 2008
Nucleic Acids Research, doi:10.1093/nar/gkm1163
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Methods Online |
An optimized split-ubiquitin cDNA-library screening system to identify novel interactors of the human Frizzled1 receptor
1Bio3/Bioinformatics and Molecular Genetics (Faculty of Biology), Center for Biochemistry and Molecular Cell Research (ZBMZ, Faculty of Medicine), University of Freiburg, ZBSA (Center for Systems Biology) and 2GPC Biotech AG, D-82152 Martinsried, Germany
*To whom correspondence should be addressed. Tel: +49-761-203 2799; Fax: +49-761-203 8351; Email: baumeister{at}celegans.de
Received August 3, 2007. Revised November 29, 2007. Accepted December 17, 2007.
The yeast split-ubiquitin system has previously been shown to be suitable to detect protein interactions of membrane proteins and of transcription factors in vivo. Therefore, this technology complements the classical split-transcription factor based yeast two-hybrid system (Y2H). Success or failure of the Y2H depends primarily on the ability to avoid false-negative and false-positive hits that become a limiting factor for the value of the system, especially in large scale proteomic analyses. We provide here a systematic assessment of parameters to help improving the quality of split-ubiquitin cDNA-library screenings. We experimentally defined the optimal 5-fluoroorotic acid (5-FOA) concentration as a key parameter to increase the reproducibility of interactions and, at the same time, to keep non-specific background growth low. Furthermore, we show that the efficacy of the 5-FOA selection is modulated by the plating density of the yeast clones. Moreover, a reporter-specific class of false-positive hits was identified, and a simple phenotypic assay for efficient de-selection was developed. We demonstrate the application of this improved system to identify novel interacting proteins of the human Frizzled 1 receptor. We identified several novel interactors with components of the Wnt-Frizzled signalling pathways and discuss their potential roles as direct mediators of Frizzled receptor signalling. The present work is the first example of a split-ubiquitin interaction screen using an in-situ expressed receptor of the serpentine class, emphasizing the suitability of the described improvements in the screening protocol.
Present address: Dietmar Dirnberger, Sandoz International GmbH, Industriestraße 25, D-83607 Holzkirchen, Germany.