Hurwitz N, Segal M, Marbach I, Levitzki A
Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Israel.
Proc Natl Acad Sci U S A. 1995 Nov 21;92(24):11009-13. doi: 10.1073/pnas.92.24.11009.
Although both Ras1 and Ras2 activate adenylyl cyclase in yeast, a number of differences can be observed regarding their function in the cAMP pathway. To explore the relative contribution of conserved and variable domains in determining these differences, chimeric RAS1-RAS2 or RAS2-RAS1 genes were constructed by swapping the sequences encoding the variable C-terminal domains. These constructs were expressed in a cdc25ts ras1 ras2 strain. Biochemical data show that the difference in efficacy of adenylyl cyclase activation between the two Ras proteins resides in the highly conserved N-terminal domain. This finding is supported by the observation that Ras2 delta, in which the C-terminal domain of Ras2 has been deleted, is a more potent activator of the yeast adenylyl cyclase than Ras1 delta, in which the C-terminal domain of Ras1 has been deleted. These observations suggest that amino acid residues other than the highly conserved residues of the effector domain within the N terminus may determine the efficiency of functional interaction with adenylyl cyclase. Similar levels of intracellular cAMP were found in Ras1, Ras1-Ras2, Ras1 delta, Ras2, and Ras2-Ras1 strains throughout the growth curve. This was found to result from the higher expression of Ras1 and Ras1-Ras2, which compensate for their lower efficacy in activating adenylyl cyclase. These results suggest that the difference between the Ras1 and the Ras2 phenotype is not due to their different efficacy in activating the cAMP pathway and that the divergent C-terminal domains are responsible for these differences, through interaction with other regulatory elements.
尽管Ras1和Ras2在酵母中均可激活腺苷酸环化酶，但在cAMP信号通路中它们的功能存在一些差异。为了探究保守结构域和可变结构域在决定这些差异中的相对作用，通过交换编码可变C末端结构域的序列构建了嵌合的RAS1-RAS2或RAS2-RAS1基因。这些构建体在cdc25ts ras1 ras2菌株中表达。生化数据表明，两种Ras蛋白在激活腺苷酸环化酶效力上的差异存在于高度保守的N末端结构域。这一发现得到以下观察结果的支持：缺失Ras2 C末端结构域的Ras2 delta比缺失Ras1 C末端结构域的Ras1 delta是酵母腺苷酸环化酶更有效的激活剂。这些观察结果表明，除了N末端效应结构域的高度保守残基之外的氨基酸残基可能决定与腺苷酸环化酶功能相互作用的效率。在整个生长曲线中，Ras1、Ras1-Ras2、Ras1 delta、Ras2和Ras2-Ras1菌株中发现了相似水平的细胞内cAMP。这是由于Ras1和Ras1-Ras2的较高表达，它们弥补了其激活腺苷酸环化酶效力较低的缺陷。这些结果表明，Ras1和Ras2表型之间的差异并非由于它们激活cAMP信号通路的效力不同，并且不同的C末端结构域通过与其他调节元件相互作用导致了这些差异。
