Identification of Evening Complex Associated Proteins in Arabidopsis by Affinity Purification and Mass Spectrometry.
Identification of Evening Complex Associated Proteins in Arabidopsis by Affinity Purification and Mass Spectrometry.
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DOI：
10.1074/mcp.m115.054064
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发表时间：
2016-01
期刊：
影响因子：
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通讯作者：
Nusinow DA
中科院分区：
文献类型：
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作者：
Huang H;Alvarez S;Bindbeutel R;Shen Z;Naldrett MJ;Evans BS;Briggs SP;Hicks LM;Kay SA;Nusinow DA
许多物种具有内源性生物钟，以使内部生理与振荡的外部环境同步。在植物中，生物钟在每日和季节的时间尺度上协调生长，代谢和发育。昼夜节律网络中的许多蛋白质形成振荡复合物，其在时间上调节无数过程，包括信号转导、转录、蛋白质降解和翻译后修饰。在拟南芥中，一个由EARLY FLOWER 4（ELF 4）、EARLY FLOWER 3（ELF 3）和LUX ARRHYTHMO（LUX）组成的三元复合体，称为夜晚复合体，通过转录调控来调节基因表达和生长的每日节律。然而，人们对将昼夜节律系统与其他途径连接起来的物理相互作用知之甚少。我们使用亲和纯化和质谱（AP-MS）方法鉴定与A. thaliana.确定了昼夜节律网络内以及与光信号传导途径的新联系，包括夜间复合物，CAB表达时间1（TOC 1），咖啡时间（TIC），所有光敏色素和TANDEM锌结/PLUS 3（TZP）之间的联系。将遗传突变与亲和纯化相结合，测试光敏色素B（phyB）、EARLY FLOWER 4和EARLY FLOWER 3作为连接夜晚复合物与时钟和光信号传导途径的节点的作用。这些实验建立了通路之间的层次关联，并表明直接和间接的相互作用。具体而言，结果表明，EARLY FLOWERSHIP 3和光敏色素B作为连接时钟和红光信号通路的枢纽。最后，我们描述了一个相关的核激酶的进化枝，这些核激酶调节A. thaliana.质谱和遗传学的结合是一种快速、直接鉴定复杂信号通路内和之间的新组分和连接的有力方法。
Many species possess an endogenous circadian clock to synchronize internal physiology with an oscillating external environment. In plants, the circadian clock coordinates growth, metabolism and development over daily and seasonal time scales. Many proteins in the circadian network form oscillating complexes that temporally regulate myriad processes, including signal transduction, transcription, protein degradation and post-translational modification. In Arabidopsis thaliana, a tripartite complex composed of EARLY FLOWERING 4 (ELF4), EARLY FLOWERING 3 (ELF3), and LUX ARRHYTHMO (LUX), named the evening complex, modulates daily rhythms in gene expression and growth through transcriptional regulation. However, little is known about the physical interactions that connect the circadian system to other pathways. We used affinity purification and mass spectrometry (AP-MS) methods to identify proteins that associate with the evening complex in A. thaliana. New connections within the circadian network as well as to light signaling pathways were identified, including linkages between the evening complex, TIMING OF CAB EXPRESSION1 (TOC1), TIME FOR COFFEE (TIC), all phytochromes and TANDEM ZINC KNUCKLE/PLUS3 (TZP). Coupling genetic mutation with affinity purifications tested the roles of phytochrome B (phyB), EARLY FLOWERING 4, and EARLY FLOWERING 3 as nodes connecting the evening complex to clock and light signaling pathways. These experiments establish a hierarchical association between pathways and indicate direct and indirect interactions. Specifically, the results suggested that EARLY FLOWERING 3 and phytochrome B act as hubs connecting the clock and red light signaling pathways. Finally, we characterized a clade of associated nuclear kinases that regulate circadian rhythms, growth, and flowering in A. thaliana. Coupling mass spectrometry and genetics is a powerful method to rapidly and directly identify novel components and connections within and between complex signaling pathways.