Toward a protein-protein interaction map of the budding yeast: A comprehensive system to examine two-hybrid interactions in all possible combinations between the yeast proteins
Toward a protein-protein interaction map of the budding yeast: A comprehensive system to examine two-hybrid interactions in all possible combinations between the yeast proteins
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DOI：
10.1073/pnas.97.3.1143
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发表时间：
2000-02-01
影响因子：
11.1
通讯作者：
Sakaki, Y
中科院分区：
文献类型：
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作者：
Ito, T;Tashiro, K;Sakaki, Y
蛋白质-蛋白质相互作用在细胞的结构和功能组织的各个方面起着关键作用，它们的完整描述对于彻底理解细胞是必不可少的。作为实现这一目标的方法，在这里，我们报告了一个全面的系统来检查酿酒酵母蛋白质之间所有可能组合中的双杂交相互作用。我们将所有酵母ORF单独克隆为MATa菌株中的DNA结合结构域融合体（“诱饵”）和MAT α菌株中的激活结构域融合体（“猎物”），随后将它们分成池，每个含有96个克隆。这些诱饵和猎物克隆池彼此系统地交配，转化体进行严格的选择，激活三个报告基因，然后进行序列标记。我们对约4 × 10（6）种不同组合的初步研究，占待测组合总数的约10%，发现了183种独立的双杂交相互作用，其中一半以上是全新的。值得注意的是，所获得的二进制数据使我们能够提取更复杂的相互作用网络，包括可以解释目前未解决的囊泡运输不同步骤之间的连接机制的网络。因此，这里描述的方法将为整合各种细胞功能提供许多线索，并作为完成蛋白质-蛋白质相互作用图谱的主要驱动力。
Protein-protein interactions play pivotal roles in various aspects of the structural and functional organization of the cell, and their complete description is indispensable to thorough understanding of the cell. As an approach toward this goal, here we report a comprehensive system to examine two-hybrid interactions in all of the possible combinations between proteins of Saccharomyces cerevisiae, We cloned all of the yeast ORFs individually as a DNA-binding domain fusion ("bait") in a MATa strain and as an activation domain fusion ("prey") in a MAT alpha strain, and subsequently divided them into pools, each containing 96 clones. These bait and prey clone pools were systematically mated with each other, and the transformants were subjected to strict selection for the activation of three reporter genes followed by sequence tagging. Our initial examination of approximate to 4 x 10(6) different combinations, constituting approximate to 10% of the total to be tested, has revealed 183 independent two-hybrid interactions, more than half of which are entirely novel. Notably, the obtained binary data allow us to extract more complex interaction networks, including the one that may explain a currently unsolved mechanism for the connection between distinct steps of vesicular transport. The approach described here thus will provide many leads for integration of various cellular functions and serve as a major driving force in the completion of the protein-protein interaction map.