Genetic selection in Saccharomyces of mutant mammalian adenylyl cyclases with elevated basal activities

S. A. Haney, J. Xu, S. Y. Lee, C. L. Ma, E. Duzic, James Broach, J. P. Manfredi

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

We show that co-expression of rat Gαs 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 cyr1 is not observed in the absence of Gαs, indicating that the mammalian ACs retain their normal regulatory behavior in yeast. Selection for Gαs-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.

Original languageEnglish (US)
Pages (from-to)1120-1128
Number of pages9
JournalMolecular Genetics and Genomics
Volume265
Issue number6
DOIs
StatePublished - Aug 14 2001

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Saccharomyces
Genetic Selection
Adenylyl Cyclases
Genetic Suppression
HEK293 Cells
Amino Acid Substitution
Growth
Saccharomyces cerevisiae
Yeasts
Mutation
Proteins

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Genetics

Cite this

Haney, S. A. ; Xu, J. ; Lee, S. Y. ; Ma, C. L. ; Duzic, E. ; Broach, James ; Manfredi, J. P. / Genetic selection in Saccharomyces of mutant mammalian adenylyl cyclases with elevated basal activities. In: Molecular Genetics and Genomics. 2001 ; Vol. 265, No. 6. pp. 1120-1128.
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Genetic selection in Saccharomyces of mutant mammalian adenylyl cyclases with elevated basal activities. / Haney, S. A.; Xu, J.; Lee, S. Y.; Ma, C. L.; Duzic, E.; Broach, James; Manfredi, J. P.

In: Molecular Genetics and Genomics, Vol. 265, No. 6, 14.08.2001, p. 1120-1128.

Research output: Contribution to journalArticle

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AU - Xu, J.

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AU - Broach, James

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