The Function of Yeast Adenylyl Cyclase-Associated Proteins in Regulation of Cell Growth and Cytoskeletal Structure

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In budding yeast, adenylyl cyclase is regulated by Ras proteins. We found adenylyl cyclase forms a complex with two cyclase-associated proteins ; 70-kDa CAP and 50-kDa p50, and analyzed their modes of interaction with cyclase and their cellular functions.1. CAP is a bifunctional protein ; its N-terminal region is required for association with cyclase and for proper response of cyclase to Ras, while its C-terminal region is presumably involved in cytoskeletal regulation. By mutational analyzes of CAP,we mapped its adenylyl cyclase-binding site to N-terminal 36-amino acid residues. The 36-residues were sufficient for invoking proper cAMP response in yeast lacking CAP.Also, we mapped the CAP-binding site of cyclase to 118 residues near its C terminus. Both of these binding sites contained tandem repetition of LXXLXXX,suggesting the coiled-coil mechanism for their interaction. When the effect of amino acid substitutions of the leucine residues in these sequences was examined, the mutations … More abolished the CAP-cyclase interaction, demonstrating the importance of the coiled-coil mechanism.2. We found that posttranslational modification (especially farnesylation) of Ras is required for activation of adenylyl cyclase, whereas it has no effect on the binding affinity of cyclase for Ras. When cyclase was devoid of its associating CAP,the stimulatory effect of Ras modification on cyclase activation was lost. In this system, overexpression of CAP resumed efficient CAP binding as well as the stimulatory effect of the modification on cyclase. Cyclase mutants lacking the CAP-binding site were insensitive to the stimulatry effect, which was not resumed by CAP overexpression. These results indicate that association with CAP mediates the stimulation of cyclase activation by Ras posttranslational modification.3. By the pyrenyl actin method, we showed that the CAP C-terminal region has G-actin-binding and -sequestering activities. p50 was found not an inherent component of the cyclase complex, and, therefore, was not analyzed any further. Less