Properties of the SDC25 C-domain, a GDP to GTP exchange factor of RAS proteins 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 stimulator on RAS proteins (Créchet, J.B., Poullet, P., Mistou, M. Y., Parmeggiani, 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 SDC25 C-domain activates adenylyl cyclase by rapidly recycling the active RAS2. or RAS1.GTP complex from the respective GDP complex. This is also supported by the observation that the stimulation of adenylyl cyclase 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 adenylyl cyclase was detected. Biochemical evidence was obtained, showing that in the presence of the functional target of RAS, the adenylyl cyclase, the effects of SDC25 C-domain and the catalytic domain of GTPase-activating protein are antagonistic. This in vitro system allowed a quantitative evaluation of the effects of positive and negative effectors of RAS on adenylyl cyclase and the biochemical analysis of conditions inducing a phenotype of permanently activated adenylyl cyclase.
Créchet
J B
JB
S.D.I. 61840 du Centre National de la Recherche Scientifique, Laboratoire de Biochimie, Ecole Polytechnique, Palaiseau, France.
Poullet
P
P
Bernardi
A
A
Fasano
O
O
Parmeggiani
A
A
D000262:Adenylyl Cyclases / Q000378:metabolism*; D004789:Enzyme Activation; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005838:Genotype; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D020668:rap GTP-Binding Proteins; D018631:ras Proteins
D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D019204:GTP-Binding Proteins; C489014:RAS1 protein, S cerevisiae; C489015:RAS2 protein, S cerevisiae; D020668:rap GTP-Binding Proteins; D018631:ras Proteins; D000262:Adenylyl Cyclases