Analysis of the function of the 70-kilodalton cyclase-associated protein (CAP) by using mutants of yeast adenylyl cyclase defective in CAP binding.
In Saccharomyces cerevisiae, adenylyl cyclase forms a complex with the 70-kDa cyclase-associated protein (CAP). By in vitro mutagenesis, we assigned a CAP-binding site of adenylyl cyclase to a small segment near its C terminus and created mutants which lost the ability to bind CAP. CAP binding was assessed first by observing the ability of the overproduced C-terminal 150 residues of adenylyl cyclase to sequester CAP, thereby suppressing the heat shock sensitivity of yeast cells bearing the activated RAS2 gene (RAS2Val-19), and then by immunoprecipitability of adenylyl cyclase activity with anti-CAP antibody and by direct measurement of the amount of CAP bound. Yeast cells whose chromosomal adenylyl cyclase genes were replaced by the CAP-nonbinding mutants possessed adenylyl cyclase activity fully responsive to RAS2 protein in vitro. However, they did not exhibit sensitivity to heat shock in the RAS2Val-19 background. When glucose-induced accumulation of cyclic AMP (cAMP) was measured in these mutants carrying RAS2Val-19, a rapid transient rise indistinguishable from that of wild-type cells was observed and a high peak level and following persistent elevation of the cAMP concentration characteristic of RAS2Val-19 were abolished. In contrast, in the wild-type RAS2 background, similar cyclase gene replacement did not affect the glucose-induced cAMP response. These results suggest that the association with CAP, although not involved in the in vivo response to the wild-type RAS2 protein, is somehow required for the exaggerated response of adenylyl cyclase to activated RAS2.
Department of Physiology, Kobe University School of Medicine, Japan.
J
J
Wang
N
N
Suzuki
Y
Y
Nishida
T
T
Kataoka
D048868:Adaptor Proteins, Signal Transducing; D000262:Adenylyl Cyclases / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D003598:Cytoskeletal Proteins; D029721:Drosophila Proteins; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D005947:Glucose / Q000378:metabolism; D006358:Hot Temperature; D015151:Immunoblotting; D008840:Microfilament Proteins; D016296:Mutagenesis; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D018631:ras Proteins
D048868:Adaptor Proteins, Signal Transducing; C410502:Capt protein, Drosophila; D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D029721:Drosophila Proteins; D005656:Fungal Proteins; D008840:Microfilament Proteins; C073751:SRV2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D000242:Cyclic AMP; C489015:RAS2 protein, S cerevisiae; D018631:ras Proteins; D000262:Adenylyl Cyclases; D005947:Glucose