Significant reduction of BiFC non-specific assembly facilitates in planta assessment of heterotrimeric G-protein interactors.
Significant reduction of BiFC non-specific assembly facilitates in planta assessment of heterotrimeric G-protein interactors.
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DOI：
10.1111/tpj.12639
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发表时间：
2014-11
期刊：
影响因子：
--
通讯作者：
Assmann SM
中科院分区：
文献类型：
--
作者：
Gookin TE;Assmann SM
蛋白质网络和信号级联是细胞内和细胞间信号转导的关键机制。识别蛋白质的相互作用伙伴可以提供有关其生理作用的重要线索。双分子荧光互补（BiFC）测定已成为体内分析蛋白质-蛋白质相互作用及其亚细胞定位的常规工具。虽然BiFC系统自成立以来已经有所改进，但用于植物分析的可用选项仍然受到非常低的信噪比的影响，并且缺乏BiFC混淆背景信号的系统比较。背景信号可以掩盖弱相互作用，提供假阳性，并降低真阳性的置信度。为了克服这些问题，我们对后生动物和植物中使用的已发表的BiFC片段进行了广泛的植物内分析，然后开发了优化的单载体BiFC系统，该系统利用在残基210处分裂的单体Venus（mVenus），并包含整合的mTurquoise 2标记以精确地鉴定转化的细胞以区分真阴性。在这里，我们提供了我们的流线型双ORF表达（pDOE）BiFC系统，并表明我们在BiFC方法的功能，甚至与内部融合的mVenus 210片段的进步。我们通过提供拟南芥MLO 1与钙调素样（CML）蛋白相互作用的直接可视化，以及通过显示异源三聚体G蛋白亚基Gα（GPA 1）和Gβ（AGB 1）在植物细胞中相互作用来说明该系统的功效。我们进一步证明，GPA 1和AGB 1在植物中与PLDα1相互作用，而PLDα1“DRY”基序的突变消除了这两种相互作用。
Protein networks and signaling cascades are key mechanisms for intra- and intercellular signal transduction. Identifying the interacting partners of a protein can provide vital clues regarding its physiological role. The bimolecular fluorescence complementation (BiFC) assay has become a routine tool for in vivo analysis of protein–protein interactions and their subcellular location. Although the BiFC system has improved since its inception, the available options for in planta analysis are still subject to very low signal-to-noise ratios, and a systematic comparison of BiFC confounding background signals has been lacking. Background signals can obscure weak interactions, provide false positives, and decrease confidence in true positives. To overcome these problems, we performed an extensive in planta analysis of published BiFC fragments used in metazoa and plants, and then developed an optimized single vector BiFC system which utilizes monomeric Venus (mVenus) split at residue 210, and contains an integrated mTurquoise2 marker to precisely identify transformed cells in order to distinguish true negatives. Here we provide our streamlined double ORF expression (pDOE) BiFC system, and show that our advance in BiFC methodology functions even with an internally fused mVenus210 fragment. We illustrate the efficacy of the system by providing direct visualization of Arabidopsis MLO1 interacting with a calmodulin-like (CML) protein, and by showing that heterotrimeric G-protein subunits Gα (GPA1) and Gβ (AGB1) interact in plant cells. We further demonstrate that GPA1 and AGB1 each physically interact with PLDα1 in planta, and that mutation of the so-called PLDα1 ‘DRY’ motif abolishes both of these interactions.
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DOI：
10.1007/978-1-61779-160-4_12
发表时间：
2011
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
--
作者：
Hynes, Thomas R;Yost, Evan A;Yost, Stacy M;Berlot, Catherine H
通讯作者：
Berlot, Catherine H
影响因子：
2.7
作者：
Kodama, Yutaka;Hu, Chang-Deng
通讯作者：
Hu, Chang-Deng
DOI：
10.1073/pnas.0509763102
发表时间：
2006-01-03
影响因子：
11.1
作者：
Gibson, SK;Gilman, AG
通讯作者：
Gilman, AG
影响因子：
4.8
作者：
Mal, Tapas K.;Takahata, Shinya;Ikura, Mitsuhiko
通讯作者：
Ikura, Mitsuhiko
DOI：
10.1016/s1388-1981(00)00182-7
发表时间：
2001-02-26
影响因子：
4.8
作者：
Lein, W;Saalbach, G
通讯作者：
Saalbach, G