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An adenylate cyclase from Saccharomyces cerevisiae that is stimulated by RAS proteins with effector mutations
M S Marshall1, J B Gibbs, E M Scolnick
1Department of Virus and Cell Biology, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania 19486.
Abstract:
Conservative amino acid substitutions were introduced into the proposed effector regions of both mammalian Ha-ras (residues 32 to 40) and Saccharomyces cerevisiae RAS2 (residues 39 to 47) proteins. The RAS2[Ser 42] protein had reduced biological function in the yeast S. cerevisiae. A S. cerevisiae strain with a second-site suppressor mutation, SSR2-1, was isolated which could grow on nonfermentable carbon sources when the endogenous RAS2 protein was replaced by the RAS2[Ser 42] protein. The SSR2-1 mutation was mapped to the structural gene for adenylate cyclase (CYR1), and the gene containing SSR2-1 was cloned and sequenced. SSR2-1 corresponded to a point mutation that would create an amino acid substitution of a tyrosine residue for an aspartate residue at position 1547. The SSR2-1 gene encodes an adenylate cyclase that is dependent on ras proteins for activity, but is stimulated by Ha-ras and RAS2 mutant proteins that are unable to stimulate wild-type adenylate cyclase.
Insights
Conservative amino acid substitutions in ras proteins were studied. A mutation in adenylate cyclase (CYR1) restored function to a defective RAS2 protein, revealing a novel interaction mechanism.
Area of Science:
- Molecular Biology
- Yeast Genetics
- Protein Function
Background:
- Ras proteins are crucial signaling molecules in both mammals and yeast.
- Understanding Ras protein function requires studying interactions with downstream effectors like adenylate cyclase.
- Mutational analysis is key to elucidating protein function and regulatory pathways.
Purpose of the Study:
- To investigate the functional consequences of conservative amino acid substitutions in ras protein effector regions.
- To identify genetic suppressors that restore biological function to mutant ras proteins.
- To characterize the molecular basis of ras protein interaction with adenylate cyclase.
Main Methods:
- Site-directed mutagenesis of Ha-ras and Saccharomyces cerevisiae RAS2 proteins.
- Isolation and genetic mapping of second-site suppressor mutations in yeast.
- Cloning and sequencing of the mutated adenylate cyclase gene (CYR1).
Main Results:
- A RAS2[Ser 42] mutant protein exhibited reduced biological function in yeast.
- A second-site suppressor mutation, SSR2-1, was identified in the CYR1 gene.
- The SSR2-1 mutation resulted in an aspartate to tyrosine substitution at position 1547 in adenylate cyclase.
- The SSR2-1 encoded adenylate cyclase showed altered dependency and stimulation by ras proteins.
Conclusions:
- Conservative substitutions in ras effector regions can impact protein function.
- Second-site suppressors can reveal novel interactions and regulatory mechanisms.
- The identified mutation in adenylate cyclase alters its interaction with ras proteins, suggesting complex regulation.