Detection of in vivo protein interactions between Snf1-related kinase subunits with intron-tagged epitope-labelling in plants cells
Detection of in vivo protein interactions between Snf1-related kinase subunits with intron-tagged epitope-labelling in plants cells
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DOI：
10.1093/nar/29.17.3685
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发表时间：
2001-09-01
影响因子：
14.9
通讯作者：
Koncz, C
中科院分区：
文献类型：
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作者：
Ferrando, A;Koncz-Kálmán, Z;Koncz, C
酵母蔗糖非发酵激酶（Snf1）和哺乳动物amp活化蛋白激酶（AMPK）的植物同源物代表了一类新兴的代谢和应激信号的重要调节因子。植物Snf1相关激酶（SnRKs）的催化α -亚基在酵母双杂交系统中与不同的蛋白相互作用，这些蛋白与Snf1和AMPKs的β -亚基和γ -亚基共享保守结构域。然而，由于缺乏一种强大的技术来检测植物细胞中的蛋白质相互作用，这些蛋白质是否确实存在于体内的SnRK复合体中尚不清楚。在这里，我们描述了一种双重标记技术，使用内含子标记的血凝素（HA）和c-Myc表位序列，这为在农杆菌转化的拟南芥细胞中表达的相互作用蛋白的共免疫纯化提供了一种简单的工具。这种普遍适用的植物蛋白相互作用实验证明，作为保守的Snf1/AMPK β -亚基的植物同源物，AKIN β 2在体内与拟南芥SnRK蛋白激酶AKIN10和AKIN11的催化α -亚基形成不同的复合物。
Plant orthologs of the yeast sucrose non-fermenting (Snf1) kinase and mammalian AMP-activated protein kinase (AMPK) represent an emerging class of important regulators of metabolic and stress signalling. The catalytic alpha -subunits of plant Snf1-related kinases (SnRKs) interact in the yeast two-hybrid system with different proteins that share conserved domains with the beta- and gamma -subunits of Snf1 and AMPKs. However, due to the lack of a robust technique allowing the detection of protein interactions in plant cells, it is unknown whether these proteins indeed occur in SnRK complexes in vivo. Here we describe a double-labelling technique, using intron-tagged hemagglutinin (HA) and c-Myc epitope sequences, which provides a simple tool for co-immunopurification of interacting proteins expressed in Agrobacterium-transformed Arabidopsis cells. This generally applicable plant protein interaction assay was used to demonstrate that AKIN beta2, a plant ortholog of conserved Snf1/AMPK beta -subunits, forms different complexes with the catalytic alpha -subunits of Arabidopsis SnRK protein kinases AKIN10 and AKIN11 in vivo.