New GATEWAY vectors for High Throughput Analyses of Protein-Protein Interactions by Bimolecular Fluorescence Complementation
New GATEWAY vectors for High Throughput Analyses of Protein-Protein Interactions by Bimolecular Fluorescence Complementation
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DOI：
10.1093/mp/ssp040
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发表时间：
2009-09-01
期刊：
影响因子：
27.5
通讯作者：
Haensch, Robert
中科院分区：
文献类型：
--
作者：
Gehl, Christian;Waadt, Rainer;Haensch, Robert
复杂的蛋白质相互作用网络构成了植物代谢和信号系统。双分子荧光互补（BiFC）技术是研究植物体内蛋白质复合物形成和蛋白质相互作用定位的一种合适的技术。然而，产生大型质粒集合，以促进复杂的相互作用网络的探索往往受到传统克隆技术的需要的限制。在这里，我们报告了一个GATEWAY载体系统的实施，使大规模的组合和候选蛋白质的研究在BiFC研究。我们描述了一组12个GATEWAY兼容的BiFC载体，它们分别有效地允许候选蛋白质对与S（CFP）3A或Venus的每个可能的N-或C-末端亚片段的组合，并使BiFC（mcBiFC）的性能成为可能。我们使用了植物钼代谢的蛋白质，其中超过20种潜在相互作用的蛋白质被假定形成细胞钼网络，作为建立新载体功能的案例研究。使用这些载体，我们报告的拟南芥蛋白质Cnx 6和Cnx 7的BiFC检测的相互作用，以及同时形成的Cnx 6/Cnx 6和Cnx 6/Cnx 7复合物的mcBiFC揭示的拟南芥蝶呤合酶复合物的形成。因此，这些基于GATEWAY的BiFC载体系统应该显著促进植物细胞中复杂调控网络的大规模研究。
Complex protein interaction networks constitute plant metabolic and signaling systems. Bimolecular fluorescence complementation (BiFC) is a suitable technique to investigate the formation of protein complexes and the localization of protein-protein interactions in planta. However, the generation of large plasmid collections to facilitate the exploration of complex interaction networks is often limited by the need for conventional cloning techniques. Here, we report the implementation of a GATEWAY vector system enabling large-scale combination and investigation of candidate proteins in BiFC studies. We describe a set of 12 GATEWAY-compatible BiFC vectors that efficiently permit the combination of candidate protein pairs with every possible N- or C-terminal sub-fragment of S(CFP)3A or Venus, respectively, and enable the performance of multicolor BiFC (mcBiFC). We used proteins of the plant molybdenum metabolism, in that more than 20 potentially interacting proteins are assumed to form the cellular molybdenum network, as a case study to establish the functionality of the new vectors. Using these vectors, we report the formation of the molybdopterin synthase complex by interaction of Arabidopsis proteins Cnx6 and Cnx7 detected by BiFC as well as the simultaneous formation of Cnx6/Cnx6 and Cnx6/Cnx7 complexes revealed by mcBiFC. Consequently, these GATEWAY-based BiFC vector systems should significantly facilitate the large-scale investigation of complex regulatory networks in plant cells.