Yi Tau-Mu, Kitano Hiroaki, Simon Melvin I
Systems Biology Group, Exploratory Research for Advanced Technology Kitano Symbiotic Systems Project, Japan Science and Technology Corporation, Shibuya, Tokyo, Japan.
Proc Natl Acad Sci U S A. 2003 Sep 16;100(19):10764-9. doi: 10.1073/pnas.1834247100. Epub 2003 Sep 5.
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)-Galpha and Gbetagamma-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 alpha-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.
酵母交配反应是目前了解最为透彻的异源三聚体G蛋白信号通路之一。然而，对该系统的大多数描述都是定性的。我们基于直接的体内测量对酵母中的异源三聚体G蛋白循环进行了定量表征。我们利用荧光共振能量转移来监测青色荧光蛋白（CFP）标记的Gα和黄色荧光蛋白（YFP）标记的Gβγ的结合状态，并且发现受体介导的G蛋白激活导致荧光共振能量转移丧失。我们获得了具有改变的信息素反应的野生型和突变细胞的定量时间进程和剂量反应数据。这些结果描绘了诸如Sst2p和α-因子受体的C末端尾巴等调节因子如何调节G蛋白信号传导的动力学和敏感性的定量图景。我们探究了动力学的关键特征，包括早期反应期间活性G蛋白的快速上升和随后的下降，以及G蛋白激活剂量反应曲线与细胞周期停滞和转录诱导的下游剂量反应曲线之间的关系。将数据拟合到一个数学模型得出了酵母中异源三聚体G蛋白激活和失活的体内速率估计值。
