Analysis of Membrane Protein Complexes Using the Split-Ubiquitin Membrane Yeast Two-Hybrid (MYTH) System
Analysis of Membrane Protein Complexes Using the Split-Ubiquitin Membrane Yeast Two-Hybrid (MYTH) System
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DOI：
10.1007/978-1-59745-540-4_14
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发表时间：
2009-01-01
期刊：
影响因子：
--
通讯作者：
Stagljar, Igor
中科院分区：
文献类型：
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作者：
Kittanakom, Saranya;Chuk, Matthew;Stagljar, Igor
最近的研究已经开始阐明细胞质和膜蛋白相互作用的全球网络。由此产生的相互作用组Snap促进了许多生物学研究，包括那些关于细胞信号、蛋白质运输和蛋白质调控的研究。由于膜蛋白的疏水性，如酪氨酸激酶、G蛋白偶联受体、膜结合磷酸酶和转运蛋白，众所周知，很难研究它们与信号分子、细胞骨架或任何其他相互作用伙伴的关系。虽然传统的酵母双杂交技术是一种简单而稳健的技术，可以有效地鉴定特定的蛋白质之间的相互作用，但它在膜蛋白方面的应用受到了限制。然而，基于分裂泛素膜的酵母双杂交试验(MYTH)被描述为一种允许识别膜蛋白相互作用的工具。在神话系统中，泛素被分成两半，每一半都与一种蛋白质融合，其中至少有一种是膜结合的。当这两种蛋白质相互作用时，泛素的两半被重组，并释放与膜蛋白融合的转录因子。然后转录因子进入细胞核，激活报告基因的转录。目前，人们利用cDNA库或gDNA文库进行大规模的神话筛选，以确定和定位各种膜蛋白的结合伙伴。因此，神话系统被证明是一个强大的工具来阐明特定的蛋白质-蛋白质相互作用，对绘制膜蛋白相互作用组图有很大贡献。
Recent research has begun to elucidate the global network of cytosolic and membrane protein interactions. The resulting interactome snap facilitates numerous biological studies, including those for cell signalling, protein trafficking and protein regulation. Due to the hydrophobic nature of membrane proteins such as tyrosine kinases, G-protein coupled receptors, membrane bound phosphatases and transporters it is notoriously difficult to study their relationship to signaling molecules, the cytoskeleton, or any other interacting partners. Although conventional yeast-two hybrid is a simple and robust technique that is effective in the identification of specific protein-protein interactions, it is limited in its use for membrane proteins. However, the split-ubiquitin membrane based yeast two-hybrid assay (MYTH) has been described as a tool that allows for the identification of membrane protein interactions. In the MYTH system, ubiquitin has been split into two halves, each of which is fused to a protein, at least one of which is membrane bound. Upon interaction of these two proteins, the two halves of ubiquitin are reconstituted and a transcription factor that is fused to the membrane protein is released. The transcription factor then enters the nucleus and activates transcription of reporter genes. Currently, large-scale MYTH screens using cDNA or gDNA libraries are performed to identify and map the binding partners of various membrane proteins. Thus, the MYTH system is proving to be a powerful tool for the elucidation of specific protein-protein interactions, contributing greatly to the mapping of the membrane protein interactome.