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 least two subunits, a 200-kDa catalytic subunit and a 70-kDa cyclase-associated 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 showing that CAP sequesters monomeric actin (Kd in the range of 0.5-5 microM), decreasing actin incorporation into actin filaments. Anti-CAP monoclonal antibodies co-immunoprecipitate a protein with a molecular size of about 46 kDa. When CAP was purified from yeast using an anti-CAP monoclonal antibody column, the 46-kDa protein co-purified with a stoichiometry of about 1:1 with CAP. Western blots identified the 46-kDa protein as yeast actin. CAP also bound to muscle actin in vitro in immunoprecipitation assays and falling ball viscometry assays. Experiments with pyrene-labeled actin demonstrated that CAP sequesters actin monomers. The actin monomer binding activity is localized to the carboxyl-terminal half of CAP. Together, these data suggest that yeast CAP regulates the yeast cytoskeleton by sequestering actin monomers.
Freeman
N L
NL
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia 19104.
Chen
Z
Z
Horenstein
J
J
Weber
A
A
Field
J
J
D000199:Actins / Q000302:isolation & purification / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing; D000262:Adenylyl Cyclases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D018797:Cell Cycle Proteins; D002846:Chromatography, Affinity; D003598:Cytoskeletal Proteins; D029721:Drosophila Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008840:Microfilament Proteins; D009132:Muscles / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013045:Species Specificity; D013462:Sulfur Radioisotopes; D014783:Viscosity
D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; D000911:Antibodies, Monoclonal; C410502:Capt protein, Drosophila; D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D029721:Drosophila Proteins; D005656:Fungal Proteins; D046911:Macromolecular Substances; D008840:Microfilament Proteins; C073751:SRV2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D013462:Sulfur Radioisotopes; D000262:Adenylyl Cyclases