Thaminy Safia, Miller John, Stagljar Igor
Institut of Veterinary Biochemistry and Molecular Biology, University of Zurich-Irchel, Zurich, Switzerland.
Methods Mol Biol. 2004;261:297-312. doi: 10.1385/1-59259-762-9:297.
Protein-protein interactions are essential in almost all biological processes, extending from the formation of cellular macromolecular structures and enzymatic complexes to the regulation of signal transduction pathways. It is assumed that approximately one-third of all proteins in eukaryotic cells are membrane associated. Because of their hydrophobic nature, the analysis of membrane-protein interactions is difficult to be studied in a conventional two-hybrid assay. We described here a new genetic method for in vivo detection of membrane-protein interactions in the budding yeast Saccharomyces cerevisiae. The system uses the split-ubiquitin approach based on the detection of the in vivo processing of a reconstituted split ubiquitin. On interaction of X and Y proteins, ubiquitin reconstitution occurs and leads to the proteolytic cleavage and subsequent release of a transcription factor that triggers the activation of a reporter system enabling easy detection. In this manner, and in contrast to the conventional yeast-two hybrid system in which interactions occur in the nucleus, the membrane-based yeast two-hybrid system represents an in vivo system that detects interactions between membrane proteins in their natural environment.
蛋白质-蛋白质相互作用在几乎所有生物过程中都至关重要，涵盖从细胞大分子结构和酶复合物的形成到信号转导通路的调控。据推测，真核细胞中约三分之一的蛋白质与膜相关。由于其疏水性，膜蛋白相互作用的分析难以在传统的双杂交实验中进行研究。我们在此描述了一种用于在芽殖酵母酿酒酵母中体内检测膜蛋白相互作用的新遗传方法。该系统基于对重组分裂泛素体内加工的检测，采用分裂泛素方法。当X和Y蛋白相互作用时，泛素会发生重组，导致蛋白水解切割并随后释放转录因子，从而触发报告系统的激活，便于检测。通过这种方式，与在细胞核中发生相互作用的传统酵母双杂交系统不同，基于膜的酵母双杂交系统是一种体内系统，可在其自然环境中检测膜蛋白之间的相互作用。
