Overexpression of RPI1, a novel inhibitor of the yeast Ras-cyclic AMP pathway, down-regulates normal but not mutationally activated ras function.
A high-copy-number plasmid genomic library was screened for genes that when overexpressed down-regulate Ras protein activity in Saccharomyces cerevisiae. We report on the structure and characterization of one such gene, RPI1, which potentially encodes a novel 46-kDa negative regulator of the Ras-cyclic AMP pathway. Three lines of evidence suggest that the RPI1 gene product operates upstream to negatively regulate the activity of normal but not mutationally activated Ras proteins: (i) overexpressed RPI1 lowers cyclic AMP levels in wild-type yeast cells but not in yeast cells carrying the RAS2Val-19 mutation, (ii) overexpressed RPI1 suppresses the heat shock sensitivity phenotype induced by overexpression of normal RAS2 but does not suppress the same phenotype induced by RAS2Val-19, and (iii) disruption of RPI1 results in a heat shock sensitivity phenotype which can be suppressed by mutations that lower normal Ras activity. Thus, RPI1 appears to encode an inhibitor of Ras activity that shares a common feature with Ras GTPase-activating proteins in that it fails to down-regulate activated RAS2Val-19 function. We present evidence that the down-regulatory effect of RPI1 requires the presence of one of the two Ras GTPase activators, IRA1 and IRA2.
Kim
J H
JH
Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854.
Powers
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D000595:Amino Acid Sequence; D001483:Base Sequence; D000242:Cyclic AMP / Q000378:metabolism*; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D015698:Genomic Library; D005838:Genotype; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010957:Plasmids; D012097:Repressor Proteins; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013489:Suppression, Genetic; D018631:ras Proteins
D005656:Fungal Proteins; D015345:Oligonucleotide Probes; C069934:RPI1 protein, S cerevisiae; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; C489015:RAS2 protein, S cerevisiae; D018631:ras Proteins