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In vivo analysis of the domains of yeast Rvs167p suggests Rvs167p function is mediated through multiple protein
1Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
Abstract:
Morphological changes during cell division in the yeast Saccharomyces cerevisiae are controlled by cell-cycle regulators. The Pcl-Pho85p kinase complex has been implicated in the regulation of the actin cytoskeleton at least in part through Rvs167p. Rvs167p consists of three domains called BAR, GPA, and SH3. Using a two-hybrid assay, we demonstrated that each region of Rvs167p participates in protein-protein interactions: the BAR domain bound the BAR domain of another Rvs167p protein and that of Rvs161p, the GPA region bound Pcl2p, and the SH3 domain bound Abp1p. We identified Rvs167p as a Las17p/Bee1p-interacting protein in a two-hybrid screen and showed that Las17p/Bee1p bound the SH3 domain of Rvs167p. We tested the extent to which the Rvs167p protein domains rescued phenotypes associated with deletion of RVS167: salt sensitivity, random budding, and endocytosis and sporulation defects. The BAR domain was sufficient for full or partial rescue of all rvs167 mutant phenotypes tested but not required for the sporulation defect for which the SH3 domain was also sufficient. Overexpression of Rvs167p inhibits cell growth. The BAR domain was essential for this inhibition and the SH3 domain had only a minor effect. Rvs167p may link the cell cycle regulator Pcl-Pho85p kinase and the actin cytoskeleton. We propose that Rvs167p is activated by phosphorylation in its GPA region by the Pcl-Pho85p kinase. Upon activation, Rvs167p enters a multiprotein complex, making critical contacts in its BAR domain and redundant or minor contacts with its SH3 domain.
Insights
The Rvs167p protein, crucial for yeast cell division, interacts with cell-cycle regulators and the actin cytoskeleton via its BAR, GPA, and SH3 domains. Its BAR domain rescues most defects, while the SH3 domain aids sporulation.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Morphological changes in Saccharomyces cerevisiae cell division are regulated by cell-cycle proteins.
- The Pcl-Pho85p kinase complex influences the actin cytoskeleton, partly via Rvs167p.
- Rvs167p has three domains: BAR, GPA, and SH3, involved in protein interactions.
Purpose of the Study:
- To investigate the protein-protein interactions of Rvs167p domains.
- To determine the functional roles of Rvs167p domains in yeast phenotypes.
- To elucidate the mechanism by which Rvs167p links cell-cycle regulation to the actin cytoskeleton.
Main Methods:
- Two-hybrid assays were used to identify interacting proteins for Rvs167p domains.
- Functional analysis involved testing domain-specific rescue of rvs167 deletion mutant phenotypes.
- The impact of Rvs167p overexpression on cell growth was assessed.
Main Results:
- The BAR domain of Rvs167p interacted with itself and Rvs161p; the GPA domain bound Pcl2p; the SH3 domain bound Abp1p and Las17p/Bee1p.
- The BAR domain rescued salt sensitivity, random budding, and endocytosis defects, while the SH3 domain was sufficient for sporulation rescue.
- Rvs167p overexpression inhibited growth, primarily mediated by the BAR domain.
Conclusions:
- Rvs167p integrates cell-cycle regulation by Pcl-Pho85p kinase with actin cytoskeleton dynamics.
- Phosphorylation of the GPA region by Pcl-Pho85p kinase likely activates Rvs167p.
- Activated Rvs167p forms a complex, with the BAR domain mediating key interactions and the SH3 domain playing a supporting role.