Genetic selection in Saccharomyces of mutant mammalian adenylyl cyclases with elevated basal activities
| Autor: | S. Y. Lee, John Manfredi, Steven A. Haney, Jun Xu, E. Duzic, C. L. Ma, James R. Broach |
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| Rok vydání: | 2001 |
| Předmět: |
Models
Molecular Gs alpha subunit Protein Conformation Mutant Saccharomyces cerevisiae Molecular Sequence Data Endogeny Transfection Saccharomyces Cell Line Adenylyl cyclase chemistry.chemical_compound Genes Reporter Genetics Animals Humans Amino Acid Sequence Molecular Biology Mammals biology Sequence Homology Amino Acid HEK 293 cells Genetic Complementation Test General Medicine biology.organism_classification beta-Galactosidase Molecular biology Heterotrimeric GTP-Binding Proteins Yeast Recombinant Proteins Rats Isoenzymes chemistry Guanosine Triphosphate Adenylyl Cyclases |
| Zdroj: | Molecular genetics and genomics : MGG. 265(6) |
| ISSN: | 1617-4615 |
| Popis: | We show that co-expression of rat Galphas together with type I, II, IV, or VI mammalian adenylyl cyclase (AC) can suppress the growth defect of cyr1 strains of Saccharomyces cerevisiae, which lack a functional endogenous AC. Complemention of cvr1 is not observed in the absence of Galphas, indicating that the mammalian ACs retain their normal regulatory behavior in yeast. Selection for Galphas-independent growth of (cyr1 strains expressing type IV AC yielded several ACIV mutants with enhanced basal activity, each of which had a single amino acid substitution in the conserved C1a or C2a region of the protein. Expression of two of the mutant ACs in HEK293 cells resulted in increased levels of cAMP and elevated adenylyl cyclase activity. Further selection for reverting mutations in one of these constitutively active AC mutants yielded three independent intragenic suppressor mutations. The distribution of the activating and suppressor mutations throughout both C1a and C2a is consistent with a model in which the enhanced basal activity results from an increase in the affinity between C1a and C2a. These results demonstrate the utility of Saccharomyces as a tool for the identification of informative mutant forms of mammalian ACs. |
| Databáze: | OpenAIRE |
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