Segal M, Marbach I, Engelberg D, Simchen G, Levitzki A
Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Israel.
J Biol Chem. 1992 Nov 15;267(32):22747-51.
In order to characterize the interaction between the Saccharomyces cerevisiae Cdc25 protein and Harvey-ras (p21H-ras), we have constructed a yeast strain disrupted at the RAS1 and RAS2 loci, expressing both p21H-ras and the catalytic domain of the bovine GTPase activating protein (GAP) and containing the cdc25-2 mutation. Such a strain exhibits a temperature-sensitive phenotype. The shift to the nonpermissive temperature is accompanied by the loss of guanyl nucleotide-dependent activity of adenylylcyclase in vitro. The temperature-sensitive phenotype can be rescued by CDC25 itself, as well as by a plasmid containing a truncated SDC25 gene. In addition, wild type CDC25 significantly improves the guanyl nucleotide response observed in the background of the cdc25ts allele at the permissive temperature in a dosage-dependent manner and restores the guanyl nucleotide response at the restrictive temperature. Both CDC25 and a truncated SDC25 also restored p21H-ras-dependent guanyl nucleotide response in a strain isogenic to the one described above but containing a disrupted CDC25 locus instead of the temperature-sensitive allele. These results suggest that the S. cerevisiae Cdc25 protein interacts with p21H-ras expressed in yeast by promoting GDP-GTP exchange. It follows that the yeast system can be used for characterizing the interaction between guanyl nucleotide exchangers of Ras proteins and mammalian p21H-ras.
为了表征酿酒酵母Cdc25蛋白与哈维 - 拉斯（p21H - ras）之间的相互作用，我们构建了一个在RAS1和RAS2基因座处被破坏的酵母菌株，该菌株表达p21H - ras和牛GTP酶激活蛋白（GAP）的催化结构域，并含有cdc25 - 2突变。这样的菌株表现出温度敏感型表型。转移到非允许温度伴随着体外腺苷酸环化酶鸟苷酸依赖性活性的丧失。温度敏感型表型可以被CDC25本身以及含有截短的SDC25基因的质粒挽救。此外，野生型CDC25以剂量依赖性方式显著改善在允许温度下cdc25ts等位基因背景中观察到的鸟苷酸反应，并在限制温度下恢复鸟苷酸反应。CDC25和截短的SDC25还在与上述菌株同基因但含有破坏的CDC25基因座而非温度敏感等位基因的菌株中恢复了p21H - ras依赖性鸟苷酸反应。这些结果表明，酿酒酵母Cdc25蛋白通过促进GDP - GTP交换与酵母中表达的p21H - ras相互作用。因此，酵母系统可用于表征Ras蛋白的鸟苷酸交换剂与哺乳动物p21H - ras之间的相互作用。
