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 CYR1 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. cerevisiae. The interactions between CAP, adenylyl cyclase and RAS were explored in a strain of yeast that lacked CAP and contained an epitope tagged adenylyl cyclase. Adenylyl cyclase activity in this strain was not immunoprecipitated with anti-CAP antibodies, but was immunoprecipitated with anti-epitope antibodies. Two anti-CAP polyclonal antisera and five anti-CAP monoclonal antibodies were used in these studies. Like CAP-bound adenylyl cyclase, the CAP-free adenylyl cyclase was fully activated by yeast RAS2. Transformation of cap strains with plasmids expressing portions of CAP allowed the adenylyl cyclase binding sites on CAP to be mapped by immunoprecipitation experiments. In other experiments, deletion mutations of adenylyl cyclase were used to map the CAP binding site on adenylyl cyclase. The adenylyl cyclase binding site localized to the amino one third of CAP (amino acids 1-168), and the CAP binding site localized to the carboxyl terminus of adenylyl cyclase (amino acids 1768-2026).
Mintzer
K A
KA
Department of Biological Sciences, Columbia University, New York, NY 10027.
Field
J
J
D048868:Adaptor Proteins, Signal Transducing; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D001665:Binding Sites; D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D015342:DNA Probes; D029721:Drosophila Proteins; D000939:Epitopes; D005656:Fungal Proteins / Q000276:immunology / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism; D051379:Mice; D008840:Microfilament Proteins; D008969:Molecular Sequence Data; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D018631:ras Proteins
D048868:Adaptor Proteins, Signal Transducing; D000911:Antibodies, Monoclonal; C410502:Capt protein, Drosophila; D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D015342:DNA Probes; D029721:Drosophila Proteins; D000939:Epitopes; D005656:Fungal Proteins; D008840:Microfilament Proteins; C073751:SRV2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D019204:GTP-Binding Proteins; C489015:RAS2 protein, S cerevisiae; D018631:ras Proteins; D000262:Adenylyl Cyclases