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Molecular mechanism of separase dependent PP2ACdc55 downregulation

Final Report Summary - CHROMOSOME STABILITY (Molecular mechanism of separase dependent PP2ACdc55 downregulation)

The initial objective of this project was to elucidate the mechanism by which Separase promotes PP2ACdc55 inactivation at anaphase onset in budding yeast. For this aim we took two different approaches:

- Analysis of the role of known regulators of PP2A.
PP2A is regulated through its methylation-demethylation by Ppm1 and Ppe1, and two peptidyl-prolyl isomerases Rrd1 and Rrd2. We analysed the phenotype of the different deletions of these regulators as well as changes in their localisation, abundance and post-translational status in the presence or absence of Separase activity.
(i) Deletion of Rrd2 but not of Rrd1 shows an early activation of Cdc14 in cells arrested in metaphase, suggesting that Rrd2 could have a role in the regulation of PP2A at anaphase onset.
(ii) Neither Ppe1 nor Rrd2 show changes in their abundance or mobility upon anaphase onset.
(iii) Localisation of Rrd2 is pan-cellular throughout the cell cycle, not showing changes dependent on Separase activation.
(iv) PP2ACdc55 complex formation was not analysed under these conditions and remains a feasible explanation for the early release of Cdc14 in rrd2 mutants.

- Study of the role of FEAR components in PP2ACdc55 inactivation.
Of all the FEAR components, we had special interest in Slk19, as it regulates Separase localisation and is itself a substrate for Separase. We could confirm that:
(i) Interaction of Separase and Slk19 is enhanced during anaphase.
(ii) Securin hinders this interaction during metaphase.
These results prompted us to test whether forcing the interaction of Separase and Slk19 could promote Cdc14 release in metaphase-arrested cells. We expressed a fusion protein of Slk19 and the N-terminus non-catalytic domain of Separase; this chimera did not advance Cdc14 release, suggesting that although the proteolytic activity of Separase is not required, the C-terminal domain might be important for its mitotic exit function.

While the above lines of investigation shed some insight onto the role of Separase in PP2ACdc55 regulation, we analysed the contribution of Cdc14 to the irreversibility of mitotic exit. It is believed that the timely proteolysis of major regulators (ie. Wee1 and Cyclin B) is the source of this irreversibility. Nonetheless, in living cells protein degradation is counteracted by de novo protein synthesis, and both processes together define the steady state level of a protein in the cell.

We used a strain in which we could regulate degradation of Clb2 and we observed:
(i) Clb2 degradation is sufficient to trigger mitotic exit events.
(ii) If Clb2 degradation is terminated, those events remain reversible.
(iii) Mitotic exit events become irreversible if Cdc14 activates a double negative feedback loop, involving the Cdk inhibitor Sic1.
(iv) Deletion of SIC1 allows reversible mitotic exit under conditions that would otherwise be irreversible.
(v) Irreversible mitotic exit can be achieved even in the absence of Clb2 degradation if the feedback loop is engaged by using a chemical Cdk inhibitor.
These results were integrated into a mathematical model developed by Bela Novak (Oxford University) and published in May 2009.

During the last year, we extended our study of the regulation of mitotic exit phosphatases to the fission yeast Schizosaccharomyces pombe. We generated temperature-sensitive (ts) alleles of the main phosphatases in fission yeast, namely PP1 and PP2A by mutagenic PCR and characterised their phenotypes as well as tried to identify their physiological substrates; initial observations showed that loss of PP1 function leads to a metaphase arrest that is largely dependent on the integrity of the mitotic checkpoint. In contrast PP2A mutants arrest after mitosis with a defect in cell separation. A set of potential substrates has been tagged and their mobility during a time course has been examined.