Tandem Fluorescent Protein Timers for Noninvasive Relative Protein Lifetime Measurement in Plants
Tandem Fluorescent Protein Timers for Noninvasive Relative Protein Lifetime Measurement in Plants
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DOI：
10.1104/pp.19.00051
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发表时间：
2019-06-01
期刊：
影响因子：
7.4
通讯作者：
Wirtz, Markus
中科院分区：
文献类型：
--
作者：
Zhang, Hongtao;Linster, Eric;Wirtz, Markus
靶向蛋白降解是植物对环境的感知和响应以及发育信号传递所必需的重要而普遍的调控机制。尽管这一过程具有重要意义，但相对较少的研究以定量的方式评估植物蛋白周转。串联荧光蛋白计时器（tFTs）为评估单个或多个细胞的不同亚细胞区室的体内蛋白质周转提供了一种强有力的方法。tFT是两种不同荧光蛋白的融合，具有不同的荧光团成熟动力学，这使得可以从两种荧光蛋白的荧光强度的比率来估计蛋白质的年龄。在这里，我们使用短寿命的生长素信号蛋白和模型n端规则（n - recognition in）通路报告者来证明tft在研究拟南芥（Arabidopsis thaliana）和烟叶（Nicotiana benthamiana）活植物细胞中蛋白质转换的效用。我们提出瞬时表达tFTs作为相对蛋白质寿命的有效筛选，用于测试突变和不同遗传背景对蛋白质稳定性的影响。这项工作证明了利用稳定表达的tft来研究外源或内源刺激下高时间分辨率的天然蛋白动力学的潜力。
Targeted protein degradation is an important and pervasive regulatory mechanism in plants, required for perception and response to the environment as well as developmental signaling. Despite the significance of this process, relatively few studies have assessed plant protein turnover in a quantitative fashion. Tandem fluorescent protein timers (tFTs) offer a powerful approach for the assessment of in vivo protein turnover in distinct subcellular compartments of single or multiple cells. A tFT is a fusion of two different fluorescent proteins with distinct fluorophore maturation kinetics, which enable protein age to be estimated from the ratio of fluorescence intensities of the two fluorescent proteins. Here, we used short-lived auxin signaling proteins and model N-end rule (N-recognin) pathway reporters to demonstrate the utility of tFTs for studying protein turnover in living plant cells of Arabidopsis (Arabidopsis thaliana) and Nicotiana benthamiana. We present transient expression of tFTs as an efficient screen for relative protein lifetime, useful for testing the effects of mutations and different genetic backgrounds on protein stability. This work demonstrates the potential for using stably expressed tFTs to study native protein dynamics with high temporal resolution in response to exogenous or endogenous stimuli.