AN ACTIN MONOMER BINDING-ACTIVITY LOCALIZES TO THE CARBOXYL-TERMINAL HALF OF THE SACCHAROMYCES-CEREVISIAE CYCLASE-ASSOCIATED PROTEIN

The Saccharomyces cerevisiae adenylyl cyclase complex contains at leas t two subunits, a 200-kDa catalytic subunit and a 70-kDa cyclase-assoc iated protein, CAP (also called Srv2p). Genetic studies suggested two roles for CAP, one as a positive regulator of cAMP levels in yeast and a second role as a cytoskeletal regulator. We present evidence showin g that CAP sequesters monomeric actin (K-d in the range of 0.5-5 mu M) , decreasing actin incorporation into actin filaments. Anti-CAP monocl onal antibodies co-immunoprecipitate a protein with a molecular size o f about 46 kDa. When CAP was purified from yeast using an anti-CAP mon oclonal antibody column, the 46-kDa protein co-purified with a stoichi ometry of about 1:1 with CAP. Western blots identified the 46-kDa prot ein as yeast actin. CAP also bound to muscle actin in vitro in immunop recipitation assays and falling ball viscometry assays. Experiments wi th pyrene-labeled actin demonstrated that CAP sequesters actin monomer s. The actin monomer binding activity is localized to the carboxyl-ter minal half of CAP. Together, these data suggest that yeast CAP regulat es the yeast cytoskeleton by sequestering actin monomers.