INTERACTIONS BETWEEN ADENYLYL, CYCLASE, CAP AND RAS FROM SACCHAROMYCES-CEREVISIAE

The adenylyl cyclase complex from Saccharomyces cerevisiae contains at least two subunits, a catalytic subunit of M(r) 200,000, encoded by C YR1 and a cyclase associated subunit, of apparent M(r) 70,000, encoded by CAP. The complex is a major effector of RAS proteins in S. cerevis iae. The interactions between CAP, adenylyl cyclase and RAS were explo red in a strain of yeast that lacked CAP and contained an epitope tagg ed adenylyl cyclase. Adenylyl cyclase activity in this strain was not immunoprecipitated with anti-CAP antibodies, but was immunoprecipitate d with anti-epitope antibodies. Two anti-CAP polyclonal antisera and f ive anti-CAP monoclonal antibodies were used in these studies. Like CA P-bound adenylyl cyclase, the CAP-free adenylyl cyclase was fully acti vated by yeast RAS2. Transformation of cap strains with plasmids expre ssing portions of CAP allowed the adenylyl cyclase binding sites on CA P to be mapped by immunoprecipitation experiments. In other experiment s, deletion mutations of adenylyl cyclase were used to map the CAP bin ding site on adenylyl cyclase. The adenylyl cyclase binding site local ized to the amino one third of CAP (amino acids 1-168), and the CAP bi nding site localized to the carboxyl terminus of adenylyl cyclase (ami no acids 1768-2026).