Suter Bernhard, Fetchko Michael J, Imhof Ralph, Graham Christopher I, Stoffel-Studer Ingrid, Zbinden Caroline, Raghavan Maanasa, Lopez Lianet, Beneti Lucija, Hort Jacqueline, Fillingham Jeffrey, Greenblatt Jack F, Giaever Guri, Nislow Corey, Stagljar Igor
Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 3E1, Canada.
Genome Res. 2007 Dec;17(12):1774-82. doi: 10.1101/gr.6667007. Epub 2007 Nov 7.
Comprehensive approaches to detect protein-protein interactions (PPIs) have been most successful in the yeast model system. Here we present "Cross-and-Capture," a novel assay for rapid, sensitive assessment of PPIs via pulldown of differently tagged yeast strain arrays. About 500 yeast genes that function in DNA replication, repair, and recombination and nuclear proteins of unknown function were chromosomally tagged with six histidine residues or triple VSV epitopes. We demonstrate that the assay can interrogate a wide range of previously known protein complexes with increased resolution and sensitivity. Furthermore, we use "Cross-and-Capture" to identify two novel protein complexes: Rtt101p-Mms1p and Sae2p-Mre11p. The Rtt101p-Mms1p interaction was subsequently characterized by genetic and functional analyses. Our studies establish the "Cross-and-Capture" assay as a novel, versatile tool that provides a valuable complement for the next generation of yeast proteomic studies.
在酵母模型系统中，检测蛋白质-蛋白质相互作用（PPI）的综合方法最为成功。在此，我们介绍“交叉捕获”，这是一种通过下拉不同标记的酵母菌株阵列来快速、灵敏地评估PPI的新方法。约500个在DNA复制、修复、重组中起作用的酵母基因以及功能未知的核蛋白被染色体标记上六个组氨酸残基或三重VSV表位。我们证明该方法能够以更高的分辨率和灵敏度检测多种先前已知的蛋白质复合物。此外，我们使用“交叉捕获”来鉴定两种新的蛋白质复合物：Rtt101p-Mms1p和Sae2p-Mre11p。随后通过遗传和功能分析对Rtt101p-Mms1p相互作用进行了表征。我们的研究将“交叉捕获”方法确立为一种新颖、通用的工具，为下一代酵母蛋白质组学研究提供了有价值的补充。
