Adenylyl cyclase in yeast: antibodies and mutations identify a regulatory domain.
The adenylyl cyclase system of the yeast Saccharomyces cerevisiae contains the CYR1 polypeptide, responsible for catalyzing formation of cAMP from ATP, and two RAS polypeptides, responsible for stimulation of cAMP synthesis by guanine nucleotides. We have obtained rabbit antibodies that recognize the CYR1 protein. Antibodies were raised against synthetic oligopeptides and against a recombinant beta-galactosidase/CYR1 fusion protein. These antibodies have allowed the identification of the CYR1 gene product as a 205 kDa protein. Treatment with trypsin (2 micrograms/ml) reduced the size of the CYR1 protein from 205 to 155 kDa and produced an activated enzyme which no longer responded to guanine nucleotides. This result is consistent with a model in which adenylyl cyclase activity is regulated by an inhibitory domain near the amino-terminus of the CYR1 protein. This model is further supported by the finding that adenylyl cyclase activity is also markedly elevated and unresponsive to guanine nucleotides in mutant yeast strains that express only the carboxy-terminal half of the CYR1 protein. Treatment with high trypsin concentrations (greater than 10 micrograms/ml) caused release of adenylyl cyclase activity from the membrane. Comparison of immunoreactive CYR1 fragments released by trypsin and membrane bound genetically altered proteins suggests that the CYR1 protein is attached to the membrane via a separate trypsin sensitive anchoring protein rather than via a membrane anchoring domain.
Heideman
W
W
Department of Pharmacology, University of California, San Francisco 94143-0450.
Casperson
G F
GF
Bourne
H R
HR
D000262:Adenylyl Cyclases / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D004789:Enzyme Activation; D005800:Genes, Fungal; D009154:Mutation; D010446:Peptide Fragments / Q000235:genetics* / Q000276:immunology; D010449:Peptide Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D010446:Peptide Fragments; D000262:Adenylyl Cyclases