Ssa1p chaperone interacts with the guanine nucleotide exchange factor of ras Cdc25p and controls the cAMP pathway in Saccharomyces cerevisiae.

We have found that the guanine nucleotide exchange factor for ras, Cdc25p, interacts with Ssa1p in Saccharomyces cerevisiae. This interaction was observed with GST-fused Cdc25p polypeptides and confirmed by coimmunoprecipitation with the endogenous Cdc25p. Hsp82 appeared also to be co-immunoprecipitated with Cdc25p, albeit to a lower level than Hsp70. In a strain deleted for SSA1 and SSA2, we observed a reduced cellular content of Cdc25p. Consistent with a reduced activity of the cAMP-dependent PKA pathway, the rate of accumulation of both trehalose and glycogen was stimulated in the ssa-deleted strain. Expression of SSA1 reversed these effects, whereas co-expression of SSA1 and PDE2 restored high accumulation. The expression of genes repressed by cAMP, GAC1 and TPS1, fused to beta-galactosidase, was also stimulated by deletion of SSA genes. The effect of ssa deletion on glycogen accumulation was lost in a strain deleted for CDC25 rescued by the RAS2ile152 allele. Altogether, these results lead to the conclusion that Ssa1p positively controls the cAMP pathway through Cdc25p. We propose that this connection plays a critical role in the adaptation of cells to stress conditions.

Dimerization of Cdc25p, the guanine-nucleotide exchange factor for Ras from Saccharomyces cerevisiae, and its interaction with Sdc25p.

The oligomerization state of Cdc25p, the guanine nucleotide exchange factor for ras from yeast, was analyzed using different complementary approaches. The two-hybrid system showed that the C-terminal part of Cdc25p (Cdc25-Ct) can interact with itself but also with Sdc25p-Ct, the corresponding part of Sdc25p, the other guanine exchange factor from yeast. The homotropic interaction of Cdc25p-Ct has been confirmed in yeast using immunoprecipitation experiments with epitope-tagged and beta-galactosidase-fused polypeptides. No other component was required for this interaction, since dimerization was shown to occur with material synthesized in vitro. The size of Cdc25-Ct produced in Escherichia coli has been directly measured on gel filtration columns and corresponds to a dimer. The dimerization domain is localized in the same part of the molecule as the catalytic domain and the portion responsible for membrane localization. The biological relevance of dimerization is still an open question, however by allowing heterodimerization with Sdc25p it could permit a more complex combinatorial regulation of ras in yeast.

Membrane-anchoring domains of Cdc25p, a Saccharomyces cerevisiae ras exchange factor.

The CDC25 gene product from Saccharomyces cerevisiae, the prototype of the family of ras guanine nucleotide exchange factors, is expressed as a 180-kDa polypeptide, tightly bound to a membrane fraction. The ability to complement a cdc25 defect is located in the 3' part of the gene (codons 877-1589). Sequence analysis reveals only a short hydrophobic domain (residues 1459-1471) and no consensus sequence for post-translational acylation. The SH3 domain present in the N-terminal part of Cdc25p is not involved nor required for membrane localization, since the N-terminal part of Cdc25p did not fractionate with a membrane pellet. In contrast, the C-terminal part was attached to a 18000 g pellet after subcellular fractionation and immunoblotting. This subcellular localization was conserved in a ras1 ras2 double disruption mutant and in a ira2 disruption mutant. Immunofluorescence analysis showed a patchy staining, mainly at the periphery of the cells. These patches were quite distinct from actin patches by double immunolabeling. By analysing a set of truncated derivatives, the elements required for a particulate localization were restricted to residues 1441-1552.

In the budding yeast Kluyveromyces marxianus, adenylate cyclase is regulated by Ras protein(s) in vitro.

The presence of adenylate cyclase activity was first demonstrated in membrane fractions from the budding yeast Kluyveromyces marxianus. The enzyme showed a Mn(2+)- and Mg(2+)-dependent activity, with optimal pH at around 6 as observed in other yeast species. As in Saccharomyces cerevisiae, where adenylate cyclase is regulated by RAS1 and RAS2, we detected a guanyl nucleotide-dependent activity. Interestingly Y13-259 monoclonal antibody, raised against mammalian p21Ha-ras, inhibited Mg2+ plus GTP-gamma-S-dependent cAMP production, suggesting that the GTP binding proteins involved in adenylate cyclase regulation could be Ras proteins. The same antibody recognized on Western blot and immunoprecipitated a 40 kDa polypeptide from K. marxianus crude membranes. This polypeptide was not detected by an anti-RAS2 polyclonal antibody raised against S. cerevisiae RAS2 protein, suggesting that Ras proteins from the two species could be structurally different.

In vitro study of the effect of adenosine on frog adrenocortical cells.

Previous reports have shown that adenosine in rat inhibits both spontaneous and ACTH-induced release of corticosteroids through activation of adenosine A1 receptors. In the present study, we have investigated the possible effect of adenosine in the secretion of corticosteroids in amphibians using a perfusion technique for frog adrenocortical slices. Infusion of adenosine, at concentrations ranging from 10(-7) to 10(-4) M, had no effect on the basal output of corticosterone and aldosterone by frog interrenal cells. Similarly, adenosine did not affect the response of frog adrenocortical slices to ACTH, vasoactive intestinal peptide, or angiotensin II. The stable adenosine A1 receptor agonist N6-phenylisopropyl adenosine (PIA) was also totally devoid of effect on the spontaneous or ACTH-induced release of corticosteroids. These results show that in amphibians, adenosine does not modulate adrenal steroidogenesis.