Mol Genet Genomics 2001 Aug;265(6):1120-8
Genetic selection in Saccharomyces of mutant mammalian adenylyl
cyclases with elevated basal activities.
Haney SA, Xu J, Lee SY, Ma CL, Duzic E, Broach JR, Manfredi JP.
Cadus Pharmaceutical Corporation, Tarrytown, NY 10591, USA.
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. |
