PROPERTIES OF THE SDC25 C-DOMAIN, A GDP TO GTP EXCHANGE FACTOR OF RASPROTEINS AND IN-VITRO MODULATION OF ADENYLYL-CYCLASE

The SDC25 C-domain, the product encoded by the 3'-terminal part of the Saccharomyces cerevisiae SDC25 gene, acts as a GDP dissociation stimu lator on RAS proteins (Crechet, J. B., Poullet, P., Mistou, M. Y., Par meggiani, A., Camonis, J., Boy-Marcotte, E., Damak, F., and Jacquet, M . (1990b) Science 248, 866-868). To define further its role in the RAS -adenylyl cyclase pathway, an in vitro system was used, which utilized cell membranes from yeast strains with appropriate genotypes carrying alterations in the positive regulators of adenylyl cyclase activity. The SDC25 C-domain was able to stimulate the adenylyl cyclase activity of membranes from RAS2 cdc25 strains. Our results indicate that the S DC25 C-domain activates adenylyl cyclase by rapidly recycling the acti ve RAS2. or RAS1.GTP complex from the respective GDP complex. This is also supported by the observation that the stimulation of adenylyl cyc lase activity by RAS2T152I, a mutant characterized by a constitutively fast GDP to GTP exchange, was insensitive to the action of the SDC25 C-domain. No direct influence of this GDP dissociation stimulator on a denylyl cyclase was detected. Biochemical evidence was obtained, showi ng that in the presence of the functional target of RAS, the adenylyl cyclase, the effects of SDC25 C-domain and the catalytic domain of GTP ase-activating protein are antagonistic. This in vitro system allowed a quantitative evaluation of the effects of positive and negative effe ctors of RAS on adenylyl cyclase and the biochemical analysis of condi tions inducing a phenotype of permanently activated adenylyl cyclase.