An optimized split-ubiquitin cDNA-library screening system to identify novel interactors of the human Frizzled 1 receptor.
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.
Dirnberger
Dietmar
D
Bio3/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), Martinsried, Germany.
Messerschmid
Monika
M
Baumeister
Ralf
R
D001921:Brain / Q000196:embryology / Q000378:metabolism; D003001:Cloning, Molecular; D051157:Frizzled Receptors / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics; D015723:Gene Library; D006801:Humans; D009963:Orotic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D025941:Protein Interaction Mapping / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000032:analysis; D025801:Ubiquitin / Q000235:genetics*; D015003:Yeasts / Q000187:drug effects / Q000235:genetics
C489740:FZD1 protein, human; D051157:Frizzled Receptors; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D009963:Orotic Acid; C001242:5-fluoroorotic acid