Separase-Mediated Cleavage of Cohesins Initiates Chromosome Segregation
As mentioned in the previous section, each sister chromatid of a metaphase chromosome is attached to microtubules via its kinetochore (see Figure 19-20). At metaphase, the mitotic spindle is in a state of tension, with forces pulling the two kinetochores toward the opposite spindle poles, but sister chromatids do not separate because they are held together at their centromeres by cohesins. In all organisms analyzed to date, loss of cohesins from chromosomes triggers anaphase chromosome movement. The mechanism that brings about this loss of cohesins from chromosomes is conserved as well. A protease known as separase cleaves the cohesin subunit Scc1 (Rad21), breaking the protein circles linking sister chromatids (Figure 19-25). Once this link is broken, anaphase begins as poleward force exerted on the kinetochores moves the split sister chromatids toward opposite spindle poles.
FIGURE 19-25 Regulation of cohesin cleavage. Separase, a protease that can cleave the Scc1 subunit of cohesin complexes, is inhibited before anaphase by the binding of securin. Mitotic CDKs also inhibit separase by phosphorylating it. When all the kinetochores have attached to spindle microtubules and the spindle apparatus is properly assembled and oriented, the Cdc20 specificity factor associated with APC/C directs it to ubiquitinylate securin and mitotic cyclins. Following securin degradation and a decrease in mitotic CDK activity, the released and dephosphorylated separase cleaves the Scc1 subunit, breaking the cohesin circles and allowing sister chromatids to be pulled apart by the spindle apparatus that is pulling them toward opposite spindle poles.
Cohesin cleavage was discovered in budding yeast. Analysis of the Scc1 subunit by Western blot analysis showed that from G1 until metaphase, the protein migrated in polyacrylamide electrophoresis gels according to its predicted molecular weight, but during anaphase, the protein ran considerably faster in the gels, indicating that it had somehow become smaller. Subsequent studies showed that the faster-migrating form of Scc1 was indeed a cleavage product. Insight into the identity of the protein that was responsible for the cleavage of cohesin came from the analysis of previously identified yeast mutants that failed to segregate chromosomes during anaphase. A mutant form of the gene encoding Esp1—what we now know to be separase—failed to produce the cleavage fragment. Subsequent analyses revealed not only that separase is a protease, but that cleavage of cohesin is essential for chromosome segregation. Cells expressing a form of Scc1 with its cleavage sites mutated fail to segregate their chromosomes. Given the irreversible nature of Scc1 cleavage, it is absolutely essential that separase activity be tightly controlled. In what follows, we discuss its regulation.