Matviw H, Yu G, Young D
Department of Medical Biochemistry, University of Calgary Health Sciences Centre, Alberta, Canada.
Gene. 1993 Jul 15;129(1):147-52. doi: 10.1016/0378-1119(93)90711-b.
Ira1 is a negative regulator of Ras proteins in Saccharomyces cerevisiae. Deletion of IRA1 leads to constitutive activation of the Ras/cyclic AMP (cAMP) pathway, which results in several phenotypes including sensitivity to heat-shock (HS) treatment. We have identified eight Schizosaccharomyces pombe cDNAs that, when overexpressed, suppress the HS-sensitive phenotype associated with the deletion of IRA1 in S. cerevisiae. To determine where these cDNAs act, we tested their ability to suppress other mutations that activate the Ras/cAMP pathway in S. cerevisiae. Two of the cDNA clones, pPSI1 and pPSI2, failed to suppress the HS-sensitive phenotype induced by the activating RAS2Val19 mutation. Clone pPSI2 encodes Gap1/Sar1, a Sz. pombe homologue of Ira1, which has been previously identified. Three of the six RAS2Val19 suppressors could suppress the deletion of PDE1 and PDE2, the cAMP phosphodiesterase (Pde)-encoding genes, suggesting that they act downstream from adenylyl cyclase (Cyr). The remaining three clones, pPSI3, pPSI6 and pPSI7, encode proteins that may suppress the HS-sensitive phenotype by reducing Ras and/or Cyr activity. One of these, pPSI3, contains a cDNA that encodes the C-terminal region (aa 166-550) of the Sz. pombe Dbp2 protein, a homologue of the human p68 RNA helicase. We have amplified cDNAs encoding the full-length Sz. pombe Dbp2 protein by the polymerase chain reaction method and have cloned them into a S. cerevisiae expression vector. The ira1- cells harboring these plasmids retained their HS-sensitive phenotype. These results suggest that the truncated Dbp2, but not the full-length protein, is capable of interfering with Ras and/or Cyr activity.
Ira1是酿酒酵母中Ras蛋白的负调控因子。Ira1的缺失会导致Ras/环磷酸腺苷（cAMP）途径的组成型激活，从而产生多种表型，包括对热休克（HS）处理敏感。我们鉴定出了八个粟酒裂殖酵母cDNA，当它们过表达时，可抑制酿酒酵母中与Ira1缺失相关的HS敏感表型。为了确定这些cDNA的作用位点，我们测试了它们抑制酿酒酵母中其他激活Ras/cAMP途径的突变的能力。两个cDNA克隆，pPSI1和pPSI2，未能抑制由激活型RAS2Val19突变诱导的HS敏感表型。克隆pPSI2编码Gap1/Sar1，它是Ira1的粟酒裂殖酵母同源物，此前已被鉴定。六个RAS2Val19抑制子中的三个可以抑制PDE1和PDE2（编码cAMP磷酸二酯酶（Pde）的基因）的缺失，这表明它们在腺苷酸环化酶（Cyr）的下游起作用。其余三个克隆，pPSI3、pPSI6和pPSI7，编码的蛋白质可能通过降低Ras和/或Cyr活性来抑制HS敏感表型。其中之一pPSI3包含一个cDNA，它编码粟酒裂殖酵母Dbp2蛋白的C末端区域（氨基酸166 - 550），该蛋白是人p68 RNA解旋酶的同源物。我们通过聚合酶链反应方法扩增了编码全长粟酒裂殖酵母Dbp2蛋白的cDNA，并将它们克隆到酿酒酵母表达载体中。携带这些质粒的ira1 - 细胞保留了它们的HS敏感表型。这些结果表明，截短的Dbp2能够干扰Ras和/或Cyr活性，而全长蛋白则不能。
