A quantitative characterization of the yeast heterotrimeric G protein cycle
A quantitative characterization of the yeast heterotrimeric G protein cycle
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DOI：
10.1073/pnas.1834247100
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发表时间：
2003-09
影响因子：
11.1
通讯作者：
T. Yi;H. Kitano;M. Simon
中科院分区：
文献类型：
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作者：
T. Yi;H. Kitano;M. Simon
酵母交配反应是最被理解的异三聚体G蛋白信号通路之一。然而，对这一系统的大多数描述都是定性的。我们基于体内的直接测量，定量地描述了酵母中的异源三聚体G蛋白周期。我们利用荧光共振能量转移来监测青色荧光蛋白-G-α和G-βγ-黄色荧光蛋白的结合状态，我们发现受体介导的G蛋白激活会导致荧光共振能量转移的丧失。获得了具有改变信息素反应的野生型和突变型细胞的定量时间过程和剂量-反应数据。这些结果定量地描绘了调控因子Sst2p和α因子受体的C末端尾巴如何调节G蛋白信号的动力学和敏感性。我们探索了动力学的关键特征，包括活性G蛋白在早期反应中的快速上升和随后下降，以及G蛋白激活剂量-反应曲线与下游细胞周期停滞和转录诱导的剂量-反应曲线之间的关系。将数据与数学模型进行拟合，得到了酵母中异源三聚体G蛋白激活和失活的体内速率的估计。
The yeast mating response is one of the best understood heterotrimeric G protein signaling pathways. Yet, most descriptions of this system have been qualitative. We have quantitatively characterized the heterotrimeric G protein cycle in yeast based on direct in vivo measurements. We used fluorescence resonance energy transfer to monitor the association state of cyan fluorescent protein (CFP)-Gα and Gβγ-yellow fluorescent protein (YFP), and we found that receptor-mediated G protein activation produced a loss of fluorescence resonance energy transfer. Quantitative time course and dose–response data were obtained for both wild-type and mutant cells possessing an altered pheromone response. These results paint a quantitative portrait of how regulators such as Sst2p and the C-terminal tail of α-factor receptor modulate the kinetics and sensitivity of G protein signaling. We have explored critical features of the dynamics including the rapid rise and subsequent decline of active G proteins during the early response, and the relationship between the G protein activation dose–response curve and the downstream dose–response curves for cell-cycle arrest and transcriptional induction. Fitting the data to a mathematical model produced estimates of the in vivo rates of heterotrimeric G protein activation and deactivation in yeast.