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Two types of RAS mutants that dominantly interfere with activators of RAS
1Cold Spring Harbor Laboratory, New York 11724.
Abstract:
In the fission yeast Schizosaccharomyces pombe, ras1 regulates both sexual development (conjugation and sporulation) and cellular morphology. Two types of dominant interfering mutants were isolated in a genetic screen for ras1 mutants that blocked sexual development. The first type of mutation, at Ser-22, analogous to the H-rasAsn-17 mutant (L. A. Feig and G. M. Cooper, Mol. Cell. Biol. 8:3235-3243, 1988), blocked only conjugation, whereas a second type of mutation, at Asp-62, interfered with conjugation, sporulation, and cellular morphology. Analogous mutations at position 64 of Saccharomyces cerevisiae RAS2 or position 57 of human H-ras also resulted in dominant interfering mutants that interfered specifically and more profoundly than mutants of the first type with RAS-associated pathways in both S. pombe or S. cerevisiae. Genetic evidence indicating that both types of interfering mutants function upstream of RAS is provided. Biochemical evidence showing that the mutants are altered in their interaction with the CDC25 class of exchange factors is presented. We show that both H-rasAsn-17 and H-rasTyr-57, compared with wild-type H-ras, are defective in their guanine nucleotide-dependent release from human cdc25 and that this defect is more severe for the H-rasTyr-57 mutant. Such a defect would allow the interfering mutants to remain bound to, thereby sequestering RAS exchange factors. The more severe interference phenotype of this novel interfering mutant suggests that it functions by titrating out other positive regulators of RAS besides those encoded by ste6 and CDC25.
Insights
Researchers identified novel interfering mutants in fission yeast ras1 that disrupt sexual development and cell shape. These mutants, altered at specific amino acids, interfere with RAS pathways by sequestering essential exchange factors.
Area of Science:
- Molecular and Cellular Biology
- Yeast Genetics
- Signal Transduction
Background:
- The ras1 gene in fission yeast (Schizosaccharomyces pombe) is crucial for regulating sexual development and cellular morphology.
- Dominant interfering mutants are valuable tools for dissecting complex genetic pathways.
- RAS proteins are key regulators of cellular processes, interacting with guanine nucleotide exchange factors (GEFs).
Purpose of the Study:
- To isolate and characterize dominant interfering mutants of ras1 in Schizosaccharomyces pombe that affect sexual development.
- To investigate the mechanism by which these mutants interfere with RAS signaling pathways.
- To compare the functional interference of different ras1 mutants and homologous RAS proteins from other species.
Main Methods:
- Genetic screening to identify dominant interfering ras1 mutants in S. pombe.
- Analysis of mutant phenotypes related to conjugation, sporulation, and cellular morphology.
- Biochemical assays to examine the interaction of mutant RAS proteins with CDC25 class exchange factors.
- Comparative analysis of homologous RAS proteins from Saccharomyces cerevisiae and human H-ras.
Main Results:
- Two classes of dominant interfering ras1 mutants were identified, with mutations at Ser-22 and Asp-62.
- The Asp-62 mutant exhibited a more severe phenotype, affecting conjugation, sporulation, and morphology, unlike the Ser-22 mutant which only affected conjugation.
- Mutations analogous to Asp-62 in S. cerevisiae RAS2 and human H-ras also showed potent interference with RAS pathways.
- Biochemical data revealed that these interfering mutants are defective in releasing from CDC25 exchange factors, leading to their sequestration.
Conclusions:
- Dominant interfering ras1 mutants, particularly the Asp-62 type, provide insights into RAS pathway regulation in S. pombe.
- The mechanism of interference involves sequestration of RAS exchange factors due to impaired guanine nucleotide exchange.
- The novel interfering mutant suggests titration of additional positive regulators beyond Ste6 and CDC25.