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Mutations in RAS2, CYR1, and SCH9 extend the chronological life span in Saccharomyces cerevisiae by activating stress-resistance transcription factors and mitochondrial superoxide dismutase (Sod2). Here we show that mutations in CYR1 and SCH9 also extend the replicative life span of individual yeast mother cells. However, the triple deletion of stress-resistance genes MSN2/MSN4 and RIM15, which causes a major decrease in chronological life span, extends replicative life span. Similarly, the overexpression of superoxide dismutases, which extends chronological survival, shortens the replicative life span and prevents budding in 30–40% of virgin mother cells. These results suggest that stress-resistance transcription factors Msn2/Msn4 negatively regulate budding and the replicative life span in part by increasing SOD2 expression. The role of superoxide dismutases and of other stress-resistance proteins in extending the chronological life span of yeast, worms, and flies indicates that the negative effect of Sod2, Msn2/Msn4/Rim15 on the replicative life span of S. cerevisiae is independent of aging.

We have studied the interaction of the yeast RAS2 protein with guanine nucleotides using energetic parameters for the dissociation of RAS·nucleotide complexes. The results indicated that a Gly → Ser substitution at position 82 led to an altered interaction with GppNHp and, to a lesser extent, also with GDP. It was also possible to conclude that structural perturbation of Gly82 can stimulate nucleotide release by decreasing the energetic barrier for nucleotide dissociation. This, together with the observation that residues 80 and 81 are involved in the response of RAS to nucleotide exchange factors without affecting GDP binding per se, suggests a potential mechanism for exchange factor-stimulated GDP release.

The Saccharomyces cerevisiae Ras2p has been suggested to be a target in the feedback regulation of Ras-cAMP pathway. This work proves that the Ras2p localization is regulated by PKA activity, and that PKA down-regulates Ras2p activity and the protein association between Cdc25p and Ras2-GTP, which is due to a reduced Ras2-GEF Cdc25p activity. These results suggest that Ras2p localization and Ras2-GEF activity of Cdc25p play roles in the feedback regulation of Ras2p in the Ras-cAMP pathway. Structured summary of protein interactions RAF1-RBD physically interacts with Cdc25p and Ras2p by pull down ( View interaction ) RAF1-RBD physically interacts with Ras2p by pull down ( View interaction )

Ras-GEF Cdc25p has been found to be hyperphosphorylated upon glucose addition. This work provides evidence indicating that PKA activity positively regulates the degree of Cdc25p phosphorylation, and that the intracellular association of Cdc25p and Ras2p is independent of PKA activity. In vitro experiments revealed that the Ras2-GEF activity of Cdc25p is inhibited by Cdc25p phosphorylation. These data suggest a negative feedback mechanism by which intracellular cAMP synthesis is inhibited by PKA through Cdc25p phosphorylation.Structured summaryMINT-8053016: CDC25p (uniprotkb:P04821) physically interacts (MI:0915) with ras2p (uniprotkb:P01120) by anti tag co-immunoprecipitation (MI:0007)MINT-8053030: ras2p (uniprotkb:P01120) physically interacts (MI:0915) with CDC25p (uniprotkb:P04821) by anti bait co-immunoprecipitation (MI:0006)

We have used a two-hybrid approach to test various forms of Saccharomyces cerevisiae Ras2p for their ability to interact with the human guanine nucleotide exchange factor HGRF55. We have previously shown that a strong two-hybrid interaction is found between the HGRF55p and the dominant negative Ras2p(G22A) form of ras [Camus et al. (1995) Oncogene 11, 951–959]. We show here that the substitution N123I which weakens the guanine nucleotide binding also promotes ras-GEF interaction. We demonstrate that the R80D substitution alone completely abolishes the interaction of Ras2p(G22A) with GEF, whereas substitutions at positions 81, 82 and 73 have only small effects. Since residue 73 is involved in the response of ras to GEF, we propose that it plays a role in the conformational change induced by the GEF rather than in its binding. Those results emphasize the role of the α2 helix of the switch II region in the recognition of the GEF family. © 1997 Federation of European Biochemical Societies.

Recently the polyphosphoinositides (PI) turnover has been related to the control of growth and cell cycle also in Saccharomvces cerevisiae , and the RAS2 and RAS1 gene products have been shown to be involved in the stimulation of PI turnover in G0/G1 arrested yeast cells. Here we show that addition of glucose to previously glucose-starved cells, stimulates the PI turnover with fast kinetics also in yeast cells that were not arrested in the G0/G1 phase of the cell cycle. In addition PI turnover is equally stimulated in temperature sensitive cdc25-1 and cdc25-5 strains at restrictive temperature, as well as in ras1, ras2 strain, suggesting that PI turnover stimulation is not dependent on the CDC25-RAS mediated signal transduction pathway.

Ras proteins bind either GDP or GTP with high affinity. However, only the GTP-bound form of the yeast Ras2 protein is able to stimulate adenylyl cyclase. To identify amino acid residues that play a role in the conversion from the GDP-bound to the GTP-bound state of Ras proteins, we have searched for single amino acid substitutions that selectively affected the binding of one of the two nucleotides. We have found that the replacement of glycine-82 of the Ras2 protein by serine resulted in an increased rate of dissociation of Gpp(NH)p, a nonhydrolysable analog of GTP, while the GDP dissociation rate was not significantly modified. Glycine-82 resides in a region that is highly conserved between the yeast and human proteins. However, this residue is structurally distant from residues that participate in the binding of the nucleotide, as determined from the crystal structure of the human H-ras gene product. Therefore, the ability of the nucleotide binding site to discriminate between GDP and GTP is dependent not only on residues that are spatially close to the nucleotide, but also on distant amino acids. This is in agreement with the role of glycine-82 as a pivot point during the transition from the GDP- to the GTP-bound form of the Ras proteins.