Posttranslational modification of Ras protein has been shown to be critical for interaction with its effector molecules, including Saccharomyces cerevisiae adenylyl cyclase. However, the mechanism of its action was unknown. In this study, we used a reconstituted system with purified adenylyl cyclase and Ras proteins carrying various degrees of the modification to show that the posttranslational modification, especially the farnesylation step, is responsible for 5-to 10-fold increase in Ras-dependent activation of adenylyl cyclase activity even though it has no significant effect on their binding affinity. The stimulatory effect of farnesylation is found to depend on the association of adenylyl cyclase with 70-kDa adenylyl cyclase-associated protein (CAP), which was known to be required for proper in vivo response of adenylyl cyclase to Ras protein, by comparing the levels of Ras-dependent activation of purified adenylyl cyclase with and without bound CAP. The region of CAP required for this effect is mapped to its N-terminal segment of 168 amino acid residues, which coincides with the region required for the in vivo effect. Furthermore, the stimulatory effect is successfully reconstituted by in vitro association of CAP with the purified adenylyl cyclase molecule lacking the bound CAP. These results indicate that the association of adenylyl cyclase with CAP is responsible for the stimulatory effect of posttranslational modification of Ras on its activity and that this may be the mechanism underlying its requirement for the proper in vivo cyclic AMP response.The budding yeast Saccharomyces cerevisiae has two RAS genes, RAS1 and RAS2, whose protein products are structurally, functionally, and biochemically similar to mammalian Ras proto-oncoproteins (for reviews, see references 1 and 13). The yeast Ras proteins are essential regulatory elements of adenylyl cyclase (2, 38), which catalyzes the production of cyclic AMP (cAMP), a second messenger vital for yeast cell growth. The Ras-adenylyl cyclase pathway has been implicated in transduction of a glucose-triggered signal to an intracellular environment where a protein phosphorylation cascade is induced by cAMP. Yeast cells bearing the activated RAS2 gene, RAS2

In the budding yeast Saccharomyces cerevisiae, association with the 70-kDa cyclase-associated protein (CAP) is required for proper response of adenylyl cyclase to Ras proteins. We show here that a small segment comprising the N-terminal 36 amino acid residues of CAP is sufficient for association with adenylyl cyclase as well as for its function in the Ras-adenylyl cyclase pathway as assayed by the ability to confer RAS2Val-19 -dependent heat shock sensitivity to yeast cells. The CAPbinding site of adenylyl cyclase was mapped to a segment of 119 amino acid residues near its C terminus. Both of these regions contained tandem repetitions of a heptad motif ␣XX␣XXX (where ␣ represents a hydrophobic amino acid and X represents any amino acid), suggesting a coiled-coil interaction. When mutants of CAP defective in associating with adenylyl cyclase were isolated by screening of a pool of randomly mutagenized CAP, they were found to carry substitution mutations in one of the key hydrophobic residues in the heptad repeats. Furthermore, mutations of the key hydrophobic residues in the heptad repeats of adenylyl cyclase also resulted in loss of association with CAP. These results indicate the coiled-coil mechanism as a basis of the CAPadenylyl cyclase interaction.

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