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Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80
Published on: May 3, 2015
Cdk1 and SUMO regulate Swe1 stability.
Kobi J Simpson-Lavy1, Michael Brandeis
1The Department of Genetics, The Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel.
Budding yeast Swe1 protein levels are regulated by SUMOylation at K594, a modification critical for its timely degradation and proper cell cycle control. Impaired SUMOylation delays degradation, impacting cell cycle progression.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Swe1/Wee1 kinase is a key regulator of the cell cycle, inhibiting Cdk1-Clb2 and acting as a mitotic switch.
- Swe1 protein levels are primarily controlled by ubiquitin-mediated degradation, influenced by interactions with mitotic kinases.
- Previous work demonstrated Swe1's ability to sense cell cycle progression via Cdk1-Clb2, Cdc5, and Hsl1 levels.
Purpose of the Study:
- To investigate a novel mechanism regulating Swe1 protein levels.
- To elucidate the role of post-translational modifications in Swe1 stability and function.
- To understand how Swe1 degradation is controlled during the cell cycle.
Main Methods:
- Site-directed mutagenesis to create the swe1(K594R) mutant.
- Analysis of Swe1 protein levels and activity in wild-type and mutant yeast strains.
- SUMOylation assays using Siz1 SUMO ligase.
- Cellular localization studies using microscopy.
- Sensitivity assays to osmotic stress.
Main Results:
- Saccharomyces cerevisiae Swe1 is modified by Smt3/SUMOylation at residue K594 in a Cdk1-dependent manner.
- The swe1(K594R) mutant, unable to be SUMOylated, exhibits significantly delayed degradation compared to wild-type Swe1.
- Cells expressing the swe1(K594R) mutant show elevated Swe1 protein levels and activity, indicated by increased Cdk1-Y19 phosphorylation.
- The K594R mutant is mislocalized and not targeted to the bud neck, the site of wild-type Swe1 degradation.
- Deletion of the Siz1 SUMO ligase gene (siz1Δ) results in elevated Swe1 protein levels and activity.
- The swe1(K594R) mutant displays sensitivity to osmotic stress, correlating with its impaired Swe1 degradation regulation.
Conclusions:
- SUMOylation of Swe1 at K594 by Siz1 is a novel regulatory mechanism controlling its stability and degradation.
- This SUMOylation event is crucial for targeting Swe1 to the bud neck for degradation, ensuring proper cell cycle progression.
- Disruption of Swe1 SUMOylation leads to increased Swe1 activity and sensitivity to cellular stress, highlighting the importance of regulated degradation.

