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Proteolytic activation of Rim1p, a positive regulator of yeast sporulation and invasive growth
1Department of Microbiology and Institute of Cancer Research, Columbia University, New York, New York 10032, USA.
Abstract:
In the yeast Saccharomyces cerevisiae, rim1, 8, 9, or 13 mutations cause four phenotypes: poor growth at low temperature, altered colony morphology, inefficient sporulation due to reduced expression of the meiotic activator IME1, and, as shown here, defective invasive growth. In this report, we have determined the relationship between RIM1 and the other genes, RIM8, 9, and 13, in this group. We have analyzed production of epitope-tagged Rim1p derivatives with HA epitopes at the N-terminus or in the middle of the protein. These Rim1p derivatives exist primarily as a small form (90 kD for Rim1-HA2p) in wild-type cells and as a large form (98 kD for Rim1-HA2p) in rim8, 9, and 13 mutants. We have also analyzed production of beta-galactosidase in strains that express a RIM1-lacZ fusion gene. beta-galactosidase exists primarily as a approximately 130 kD form in wild-type cells and as a approximately 190 kD form in rim9 mutants. These results indicate that Rim1p undergoes C-terminal proteolytic cleavage, and that rim8, 9, and 13 mutations block cleavage. Expression of a Rim1p C-terminal deletion derivative suppresses rim8, 9, and 13 mutations. Thus the phenotypes of rim8, 9, and 13 mutants arise from the defect in Rim1p C-terminal cleavage. Cleavage of Rim1p, like that of its Aspergillus nidulans homologue PacC, is stimulated under alkaline growth conditions. Therefore, Rim1p, PacC and their respective processing pathways may represent a conserved signal transduction pathway.
Insights
Mutations in RIM genes disrupt Rim1p processing in yeast, affecting growth and sporulation. This cleavage defect, similar to PacC in Aspergillus, suggests a conserved signal transduction pathway.
Area of Science:
- Molecular Biology
- Yeast Genetics
- Signal Transduction
Background:
- RIM1, RIM8, RIM9, and RIM13 mutations in Saccharomyces cerevisiae lead to pleiotropic phenotypes including poor growth, altered colony morphology, inefficient sporulation, and defective invasive growth.
- These phenotypes are linked to reduced expression of the meiotic activator IME1.
Purpose of the Study:
- To elucidate the relationship between RIM1 and the genes RIM8, RIM9, and RIM13.
- To investigate the molecular mechanism underlying the phenotypes associated with mutations in these RIM genes.
Main Methods:
- Analysis of epitope-tagged Rim1p derivatives (N-terminal and middle HA tags) in wild-type and mutant yeast strains.
- Analysis of beta-galactosidase production in yeast strains expressing a RIM1-lacZ fusion gene.
- Assessment of the effect of Rim1p C-terminal deletion on mutant phenotypes.
Main Results:
- Rim1p derivatives exist as a smaller form in wild-type cells and a larger form in rim8, rim9, and rim13 mutants, indicating Rim1p undergoes C-terminal proteolytic cleavage.
- RIM8, RIM9, and RIM13 mutations block this C-terminal cleavage of Rim1p.
- Expression of a Rim1p C-terminal deletion derivative suppresses the mutations in rim8, rim9, and rim13, confirming the cleavage defect is the cause of the observed phenotypes.
Conclusions:
- The phenotypes of rim8, rim9, and rim13 mutants are a direct consequence of a defect in Rim1p C-terminal proteolytic cleavage.
- Rim1p cleavage is stimulated by alkaline growth conditions, analogous to the processing of its Aspergillus nidulans homologue, PacC.
- The Rim1p and PacC processing pathways likely represent a conserved signal transduction mechanism across different fungal species.