Dirnberger Dietmar, Messerschmid Monika, Baumeister Ralf
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.
Nucleic Acids Res. 2008 Apr;36(6):e37. doi: 10.1093/nar/gkm1163. Epub 2008 Mar 4.
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.
酵母分裂泛素系统此前已被证明适用于在体内检测膜蛋白和转录因子的蛋白质相互作用。因此，该技术补充了基于经典分裂转录因子的酵母双杂交系统（Y2H）。Y2H的成败主要取决于避免假阴性和假阳性结果的能力，而这已成为该系统价值的限制因素，尤其是在大规模蛋白质组分析中。我们在此提供对参数的系统评估，以帮助提高分裂泛素cDNA文库筛选的质量。我们通过实验确定了最佳5-氟乳清酸（5-FOA）浓度，这是提高相互作用重现性并同时保持非特异性背景生长较低的关键参数。此外，我们表明5-FOA选择的效果受酵母克隆铺板密度的调节。此外，我们鉴定出一类报告基因特异性的假阳性结果，并开发了一种用于高效去除假阳性的简单表型分析方法。我们展示了这种改进系统在鉴定人卷曲蛋白1受体新相互作用蛋白方面的应用。我们鉴定出了几种与Wnt-卷曲蛋白信号通路成分相互作用的新蛋白，并讨论了它们作为卷曲蛋白受体信号直接介导因子的潜在作用。目前的工作是使用蛇形类原位表达受体进行分裂泛素相互作用筛选的首个实例，强调了所描述的筛选方案改进的适用性。
