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Spatiotemporal Analysis of Cytokinetic Events in Fission Yeast
Published on: February 20, 2017
Fission yeast Cut2 required for anaphase has two destruction boxes
H Funabiki1, H Yamano, K Nagao
1Department of Biophysics, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto 606, Japan.
The EMBO Journal
|October 6, 1997
Summary
The Cut2 protein in fission yeast is crucial for separating sister chromatids. Its destruction, mediated by the Anaphase-Promoting Complex, relies on specific N-terminal sequences acting as destruction boxes.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- The Cut2 protein in *Schizosaccharomyces pombe* is essential for sister chromatid separation during cell division.
- Cut2 protein degradation occurs during anaphase, coinciding with mitotic cyclin destruction.
- This proteolysis is dependent on the Anaphase-Promoting Complex (APC)-cyclosome, a ubiquitin ligase complex.
Purpose of the Study:
- To investigate the role of N-terminal destruction boxes in Cut2 protein proteolysis.
- To determine if these sequences are sufficient for ubiquitination and degradation.
- To understand the mechanism regulating Cut2 protein levels during mitosis.
Main Methods:
- In vitro polyubiquitination and proteolysis assays using Xenopus and HeLa cell extracts.
- Site-directed mutagenesis of putative destruction box sequences in the Cut2 N-terminus.
- Analysis of cell cycle progression in yeast strains expressing wild-type and mutated Cut2 proteins.
Main Results:
- Two specific N-terminal sequences (33RAPLGSTKQ and 52RTVLGGKST) were identified as functional destruction boxes.
- Mutation of these destruction boxes inhibited Cut2 proteolysis and polyubiquitination in vitro.
- Doubly mutated Cut2 inhibited anaphase, while singly mutated versions could suppress *cut2* mutations.
- Overexpression of the N-terminal region containing destruction boxes led to cell cycle arrest in metaphase.
Conclusions:
- The N-terminal destruction boxes of Cut2 are critical for its regulated proteolysis by the APC/cyclosome.
- These sequences mediate polyubiquitination and are essential for proper sister chromatid separation.
- Cut2 degradation is a key regulatory step in the fission yeast cell cycle.
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