EFFECT OF ASSOCIATION WITH ADENYLYL CYCLASE-ASSOCIATED PROTEIN ON THEINTERACTION OF YEAST ADENYLYL-CYCLASE WITH RAS PROTEIN

Posttranslational modification of Pas protein has been shown to be cri tical for interaction with its effector molecules, including Saccharom yces cerevisiae adenylyl cyclase. However, the mechanism of its action was unknown. In this study, we used a reconstituted system with purif ied adenylyl cyclase and Pas proteins carrying various degrees of the modification to show that the posttranslational modification, especial ly the farnesylation step, is responsible for 5- to 10-fold increase i n Pas-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 adeny lyl cyclase with 70-kDa adenylyl cyclase-associated protein (CAP), whi ch was known to be required for proper in vivo response of adenylyl cy clase to Pas protein, by comparing the levels of Pas-dependent activat ion of purified adenylyl cyclase with and without bound CAP. The regio n of CAP required for this effect is mapped to its N-terminal segment of 168 amino acid residues, which coincides with the region required f or the in vivo effect. Furthermore, the stimulatory effect is successf ully reconstituted by in vitro association of CAP with the purified ad enylyl cyclase molecule lacking the bound CAP. These results indicate that the association of adenylyl cyclase with CAP is responsible for t he stimulatory effect of posttranslational modification of Pas on its activity and that this may be the mechanism underlying its requirement for the proper in vivo cyclic AMP response.