Snider Jamie, Kittanakom Saranya, Curak Jasna, Stagljar Igor
Department of Biochemistry, University of Toronto, Canada.
J Vis Exp. 2010 Feb 1(36):1698. doi: 10.3791/1698.
The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (C(ub)) and the N-terminal half of the ubiquitin moiety (N(ub)). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to C(ub) which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the N(ub) moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.
整合膜蛋白在生物学和临床上具有重要的基础意义，这促使人们开发一种基于酵母的系统，用于高通量鉴定全长跨膜蛋白的蛋白质-蛋白质相互作用（PPI）。为此，我们实验室开发了基于分裂泛素的膜酵母双杂交（MYTH）系统。该技术利用酿酒酵母作为宿主生物体，能够灵敏地检测瞬时和稳定的蛋白质相互作用。MYTH利用了泛素可被分离成两个稳定部分的特性：酵母泛素的C末端一半（C(ub)）和泛素部分的N末端一半（N(ub)）。在MYTH中，这一原理被用作蛋白质-蛋白质相互作用的“传感器”。简而言之，整合膜诱饵蛋白与连接到人工转录因子的C(ub)融合。猎物蛋白以单个或文库形式与N(ub)部分融合。诱饵和猎物之间的蛋白质相互作用导致泛素部分的重组，形成全长的“假泛素”分子。该分子进而被胞质去泛素化酶识别，导致转录因子的切割，随后诱导报告基因表达。该系统具有高度的适应性，特别适合高通量筛选。它已成功用于研究酵母和其他生物体的整合膜蛋白之间的相互作用。
