Identification of novel ErbB3-interacting factors using the split-ubiquitin membrane yeast two-hybrid system
Identification of novel ErbB3-interacting factors using the split-ubiquitin membrane yeast two-hybrid system
复制标题
DOI：
10.1101/gr.1276503
复制
发表时间：
2003-07-01
期刊：
影响因子：
7
通讯作者：
Stagljar, I
中科院分区：
文献类型：
--
作者：
Thaminy, S;Auerbach, D;Stagljar, I
膜蛋白相互作用的分析是困难的，因为这些蛋白的疏水性质，这往往使传统的生化和遗传分析无济于事。这是一个实质性的问题，因为在典型的真核细胞中，构成膜的或与膜相关的蛋白质约占所有蛋白质的三分之一。我们之前已经证明，改进的分裂泛素系统可以作为一种遗传实验来在体内检测两种特征的酵母跨膜蛋白Ost1p和Wbp1p之间的相互作用。这个所谓的分裂泛素膜酵母双杂交(YTH)系统使用分裂泛素方法，其中两半泛素的重建是通过蛋白质-蛋白质相互作用来调节的。在这里，我们将分裂泛素膜YTH系统转化为一种普遍适用的体内筛选方法，以确定特定哺乳动物跨膜蛋白的相互作用伙伴。我们用哺乳动物的ErbB3受体作为诱饵，证明了这种方法的有效性，并鉴定了三种以前未知的ErbB3相互作用蛋白。此外，我们还通过免疫共沉淀证实了ErbB3和膜相关RGS4蛋白之间的一个新发现的相互作用。我们期望分裂泛素膜YTH技术在从许多模式生物中鉴定完整的膜蛋白的潜在相互作用伙伴方面有价值。
Analysis of membrane protein interactions is difficult because of the hydrophobic nature of these proteins, which often renders conventional biochemical and genetic assays fruitless. This is a substantial problem because proteins that are integral or associated with membranes represent approximately one-third of all proteins in a typical eukaryotic cell. We have shown previously that the modified split-ubiquitin system can be used as a genetic assay for the in vivo detection of interactions between the two characterized yeast transmembrane proteins, Ost1p and Wbp1p. This so-called split-ubiquitin membrane yeast two-hybrid (YTH) system uses the split-ubiquitin approach in which reconstitution of two ubiquitin halves is mediated by a protein-protein interaction. Here we converted the split-ubiquitin membrane YTH system into a generally applicable in vivo screening approach to identify interacting partners of a particular mammalian transmembrane protein. We have demonstrated the effectiveness of this approach by using the mammalian ErbB3 receptor as bait and have identified three previously unknown ErbB3-interacting proteins. In addition, we have confirmed one of the newly found interactions between ErbB3 and the membrane-associated RGS4 protein by coimmunoprecipitating the two proteins from human cells. We expect the split-ubiquitin membrane YTH technology to be valuable for the identification of potential interacting partners of integral membrane proteins from many model organisms.