Identification and genetic analysis of Schizosaccharomyces pombe cDNAs that suppress deletion of IRA1 in Saccharomyces cerevisiae.
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.
Matviw
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Department of Medical Biochemistry, University of Calgary Health Sciences Centre, Alberta, Canada.
Yu
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Young
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D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics*; D020690:GTPase-Activating Proteins; D016153:Genes, Suppressor; D011905:Genes, ras; D020365:RNA Helicases; D012316:RNA Nucleotidyltransferases / Q000235:genetics*; D012097:Repressor Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D012568:Schizosaccharomyces / Q000235:genetics*
D004271:DNA, Fungal; D005656:Fungal Proteins; D020690:GTPase-Activating Proteins; C065725:IRA1 protein, S cerevisiae; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D012316:RNA Nucleotidyltransferases; D020365:RNA Helicases