Multicolor bimolecular fluorescence complementation reveals simultaneous formation of alternative CBL/CIPK complexes in planta
Multicolor bimolecular fluorescence complementation reveals simultaneous formation of alternative CBL/CIPK complexes in planta
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DOI：
10.1111/j.1365-313x.2008.03612.x
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发表时间：
2008-11-01
期刊：
影响因子：
7.2
通讯作者：
Kudla, Joerg
中科院分区：
文献类型：
--
作者：
Waadt, Rainer;Schmidt, Lena K.;Kudla, Joerg
生物系统中细胞内信号传导和发育模式的特异性依赖于特定细胞区室中不同蛋白质之间的选择性相互作用。这种蛋白质-蛋白质相互作用的鉴定对于解开复杂的信号传导和调控网络是必不可少的。最近，双分子荧光互补（BiFC）已成为一个强大的技术，有效地检测蛋白质相互作用在其天然亚细胞定位。在这里，我们报告了植物BiFC方法的重大技术进步。我们描述了一系列通用的BiFC矢量集，与以前生成的矢量完全兼容。新载体能够产生C-末端和N-末端融合蛋白，并携带优化的荧光蛋白基因，大大提高了BiFC的灵敏度。使用这些载体，我们描述了一种用于同时可视化在同一细胞中的多个蛋白质相互作用的双BiFC（mcBiFC）方法。应用于蛋白质相互作用网络中的钙介导的信号转导揭示了并行相互作用的蛋白激酶CIPK 24与钙传感器CBL 1和CBL 10在质膜和液泡膜，分别。我们还通过mcBiFC观察到在质膜上同时形成CBL 1/CIPK 1和CBL 9/CIPK 1蛋白复合物。因此，mcBiFC为探索植物中复杂的调控网络提供了一个有用的新工具。
The specificity of intracellular signaling and developmental patterning in biological systems relies on selective interactions between different proteins in specific cellular compartments. The identification of such protein-protein interactions is essential for unraveling complex signaling and regulatory networks. Recently, bimolecular fluorescence complementation (BiFC) has emerged as a powerful technique for the efficient detection of protein interactions in their native subcellular localization. Here we report significant technical advances in the methodology of plant BiFC. We describe a series of versatile BiFC vector sets that are fully compatible with previously generated vectors. The new vectors enable the generation of both C-terminal and N-terminal fusion proteins and carry optimized fluorescent protein genes that considerably improve the sensitivity of BiFC. Using these vectors, we describe a multicolor BiFC (mcBiFC) approach for the simultaneous visualization of multiple protein interactions in the same cell. Application to a protein interaction network acting in calcium-mediated signal transduction revealed the concurrent interaction of the protein kinase CIPK24 with the calcium sensors CBL1 and CBL10 at the plasma membrane and tonoplast, respectively. We have also visualized by mcBiFC the simultaneous formation of CBL1/CIPK1 and CBL9/CIPK1 protein complexes at the plasma membrane. Thus, mcBiFC provides a useful new tool for exploring complex regulatory networks in plants.