The dual mechanism of separase regulation by securin.

BACKGROUND: Sister chromatid separation and segregation at anaphase onset are triggered by cleavage of the chromosomal cohesin complex by the protease separase. Separase is regulated by its binding partner securin in two ways: securin is required to support separase activity in anaphase; and, at the same time, securin must be destroyed via ubiquitylation before separase becomes active. The molecular mechanisms underlying this dual regulation of separase by securin are unknown. RESULTS: We show that, in budding yeast, securin supports separase localization. Separase enters the nucleus independently of securin, but securin is required and sufficient to cause accumulation of separase in the nucleus, where its known cleavage targets reside. Securin also ensures that separase gains full proteolytic activity in anaphase. We also show that securin, while present, directly inhibits the proteolytic activity of separase. Securin prevents the binding of separase to its substrates. It also hinders the separase N terminus from interacting with and possibly inducing an activating conformational change at the protease active site 150 kDa downstream at the protein's C terminus. CONCLUSIONS: Securin inhibits the proteolytic activity of separase in a 2-fold manner. While inhibiting separase, securin is able to promote nuclear accumulation of separase and help separase to become fully activated after securin's own destruction at anaphase onset.

Preferential cleavage of chromatin-bound cohesin after targeted phosphorylation by Polo-like kinase.

The final irreversible step in the duplication and dissemination of eukaryotic genomes takes place when sister chromatid pairs split and separate in anaphase. This is triggered by the protease separase that cleaves the Scc1 subunit of 'cohesin', the protein complex responsible for holding sister chromatids together in metaphase. Only part of cellular cohesin is bound to chromosomes in metaphase, and it is unclear whether and how separase specifically targets this fraction for cleavage. We established an assay to compare cleavage of chromatin-bound versus soluble budding yeast cohesin. Scc1 in chromosomal cohesin is significantly preferred by separase over Scc1 in soluble cohesin. The difference is most likely due to preferential phosphorylation of chromatin-bound Scc1 by Polo-like kinase. Site-directed mutagenesis of 10 Polo phosphorylation sites in Scc1 slowed cleavage of chromatin-bound cohesin, and hyperphosphorylation of soluble Scc1 by Polo overexpression accelerated its cleavage to levels of chromosomal cohesin. Polo is bound to chromosomes independently of cohesin's presence, providing a possible explanation for chromosome-specific cohesin modification and targeting of separase cleavage.

Studies on substrate recognition by the budding yeast separase.

Sister chromatid cohesion is resolved at anaphase onset when separase, a site-specific protease, cleaves the Scc1 subunit of the chromosomal cohesin complex that is responsible for holding sister chromatids together. This mechanism to initiate anaphase is conserved in eukaryotes from budding yeast to man. Budding yeast separase recognizes and cleaves two conserved peptide motifs within Scc1. In addition, separase cleaves a similar motif in the kinetochore and spindle protein Slk19. Separase may cleave further substrate proteins to orchestrate multiple cellular events that take place during anaphase. To investigate substrate recognition by budding yeast separase we analyzed the sequence requirements at one of the Scc1 cleavage site motifs by systematic mutagenesis. We derived a cleavage site consensus motif (not(FKRWY))(ACFHILMPVWY)(DE)X(AGSV)R/X. This motif is found in 1,139 of 5,889 predicted yeast proteins. We analyzed 28 candidate proteins containing this motif as well as 35 proteins that contain a core (DE)XXR motif. We could so far not confirm new separase substrates, but we have uncovered other forms of mitotic regulation of some of the proteins. We studied whether determinants other than the cleavage site motif mediate separase-substrate interaction. When the separase active site was occupied with a peptide inhibitor covering the cleavage site motif, separase still efficiently interacted with its substrate Scc1. This suggests that separase recognizes both a cleavage site consensus sequence as well as features outside the cleavage site.