An adenylate cyclase from Saccharomyces cerevisiae that is stimulated by RAS proteins with effector mutations

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.