Biochemical Insights on Degradation of Arabidopsis DELLA Proteins Gained From a Cell-Free Assay System
Biochemical Insights on Degradation of Arabidopsis DELLA Proteins Gained From a Cell-Free Assay System
复制标题
从无细胞检测系统获得的拟南芥 DELLA 蛋白降解的生化见解
DOI：
10.1105/tpc.108.065433
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发表时间：
2009-08-01
期刊：
影响因子：
11.6
通讯作者：
Deng, Xing Wang
中科院分区：
文献类型：
--
作者：
Wang, Feng;Zhu, Danmeng;Deng, Xing Wang
植物激素赤霉素（GA）调节植物生长发育的各个方面。GA反应是由在GA信号通路中起抑制作用的DELLA蛋白降解引发的。最近对拟南芥和水稻的研究表明，DELLA蛋白的降解是通过泛素-蛋白酶体系统进行的。在这里，我们开发了一个拟南芥无细胞系统来概括体外DELLA蛋白降解。使用这种无细胞系统，我们证明了泛素的Lys-29是泛素链形成介导DELLA蛋白降解的主要位点。我们还证实了GA受体和多亚基E3连接酶组分在调节DELLA蛋白降解中的特定作用。此外，在我们的无细胞实验中，用PP1/PP2A磷酸酶抑制剂阻断DELLA降解表明，DELLA蛋白的降解需要蛋白丝氨酸/苏氨酸去磷酸化活性。此外，我们的数据显示，拟南芥DELLA蛋白的LZ结构域对其稳定性和活性都至关重要。因此，我们的体外降解系统提供了对DELLA蛋白降解调控的生化见解。这种体外分析系统可以广泛适用于解剖细胞信号通路，其中调节蛋白水解是一个关键的反复主题。
The phytohormone gibberellic acid (GA) regulates diverse aspects of plant growth and development. GA responses are triggered by the degradation of DELLA proteins, which function as repressors in GA signaling pathways. Recent studies in Arabidopsis thaliana and rice (Oryza sativa) have implied that the degradation of DELLA proteins occurred via the ubiquitin-proteasome system. Here, we developed an Arabidopsis cell-free system to recapitulate DELLA protein degradation in vitro. Using this cell-free system, we documented that Lys-29 of ubiquitin is the major site for ubiquitin chain formation to mediate DELLA protein degradation. We also confirmed the specific roles of GA receptors and multisubunit E3 ligase components in regulating DELLA protein degradation. In addition, blocking DELLA degradation with a PP1/PP2A phosphatase inhibitor in our cell-free assay suggested that degradation of DELLA proteins required protein Ser/Thr dephosphorylation activity. Furthermore, our data revealed that the LZ domain of Arabidopsis DELLA proteins is essential for both their stability and activity. Thus, our in vitro degradation system provides biochemical insights into the regulation of DELLA protein degradation. This in vitro assay system could be widely adapted for dissecting cellular signaling pathways in which regulated proteolysis is a key recurrent theme.