COILED-COIL INTERACTION OF N-TERMINAL-36 RESIDUES OF CYCLASE-ASSOCIATED PROTEIN WITH ADENYLYL-CYCLASE IS SUFFICIENT FOR ITS FUNCTION IN SACCHAROMYCES-CEREVISIAE RAS PATHWAY

In the budding yeast Saccharomyces cerevisiae, association with the 70 -kDa cyclase-associated protein (CAP) is required for proper response of adenylyl cyclase to Pas 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 R AS2(Val-19)-dependent heat shock sensitivity to yeast cells. The CAP-b inding site of adenylyl cyclase was mapped to a segment of 119 amino a cid residues near its C terminus. Both of these regions contained tand em repetitions of a heptad motif alpha XX alpha XXX (where alpha repre sents a hydrophobic amino acid and X represents any amino acid), sugge sting a coiled-coil interaction. When mutants of CAP defective in asso ciating with adenylyl cyclase were isolated by screening of a pool of randomly mutagenized CAP, they were found to carry substitution mutati ons in one of the key hydrophobic residues in the heptad repeats. Furt hermore, mutations of the key hydrophobic residues in the heptad repea ts of adenylyl cyclase also resulted in loss of association with CAP. These results indicate the coiled-coil mechanism as a basis of the CAP -adenylyl cyclase interaction.