The Dual Mechanism of Separase Regulation by Securin
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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Current Biology, Vol. 12, 973–982, June 25, 2002, 2002 Elsevier Science Ltd. All rights reserved. PII S0960-9822(02)00847-3 The Dual Mechanism of Separase Regulation by Securin Nadine C.D. Hornig,1 Philip P. Knowles,2 chromatid pairs on the metaphase spindle and their Neil Q. McDonald,2,3 and Frank Uhlmann1,4 segregation into daughter cells in anaphase, the status 1Chromosome Segregation Laboratory of cohesin and the activity of separase must be tightly 2 Structural Biology Laboratory controlled. The nature and possible regulation of the Cancer Research UK cohesiveness of cohesin is still poorly understood. It is London Research Institute clear, however, that cohesin is the central mediator of Lincoln’s Inn Fields Laboratories sister chromatid cohesion in metaphase and that cleav- 44 Lincoln’s Inn Fields age of its Scc1 subunit by separase destroys its cohe- London WC2A 3PX siveness in order to initiate sister chromatid separation 3 Department of Crystallography in anaphase in, probably, all eukaryotes [3–7]. Birkbeck College Separases are large proteins of 150–230 kDa in differ- London WC1E 7HX ent species (an exception, Drosophila, in which the sep- United Kingdom arase gene seems to have split in two, is described in the Discussion). Separases have been initially identified as Esp1 in budding yeast and Cut1 in fission yeast [8, Summary 9]. A C-terminal region, spanning ca. 50 kDa, is con- served in all species and has been called “separase Background: Sister chromatid separation and segrega- domain”. The second half of this separase domain har- tion at anaphase onset are triggered by cleavage of the bors the conserved cysteine and histidine residues of chromosomal cohesin complex by the protease separ- the protease active site and has been predicted to adopt ase. Separase is regulated by its binding partner securin the fold of CD clan proteases [4, 10]. The large regions in two ways: securin is required to support separase N-terminal of the separase domain do not show obvious activity in anaphase; and, at the same time, securin conservation between species. The contribution of must be destroyed via ubiquitylation before separase these extended N termini to separase function has re- becomes active. The molecular mechanisms underlying mained unclear. In fission yeast, N-terminal sequences this dual regulation of separase by securin are unknown. are required for the function of separase and have been Results: We show that, in budding yeast, securin sup- implicated in its nuclear localization, and more central ports separase localization. Separase enters the nu- sequences have been implicated in the possible cyto- cleus independently of securin, but securin is required plasmic retention of the protein [11]. Also in fission yeast, and sufficient to cause accumulation of separase in the as well as in budding yeast, N-terminal regions are nucleus, where its known cleavage targets reside. Sec- thought to be the sites of interaction with separase’s urin also ensures that separase gains full proteolytic binding partner, securin [12–14]. activity in anaphase. We also show that securin, while The initial characterization of securin, Pds1 in budding present, directly inhibits the proteolytic activity of separ- yeast, uncovered two complementary roles: securin, al- ase. Securin prevents the binding of separase to its though not essential, is required for efficient chromo- substrates. It also hinders the separase N terminus from some segregation in anaphase; and, at the same time, interacting with and possibly inducing an activating con- securin is needed to prevent anaphase in response to formational change at the protease active site 150 kDa spindle and DNA damage [12, 13, 15–18]. It became downstream at the protein’s C terminus. clear that securin is a separase inhibitor that has to be Conclusions: Securin inhibits the proteolytic activity of degraded via ubiquitylation by the anaphase-promoting separase in a 2-fold manner. While inhibiting separase, complex (APC) [19–21]. While present in cells, securin securin is able to promote nuclear accumulation of sep- is bound to separase [13, 14], and, in crude in vitro arase and help separase to become fully activated after systems, this prevents separase from cleaving Scc1 [3, securin’s own destruction at anaphase onset. 22]. Whether securin is itself sufficient to inhibit separ- ase, and by which mechanism securin prevents separ- ase from attacking Scc1, is unknown. In particular, it is Introduction unclear how binding of securin to the separase N termi- nus could influence the activity of the protease domain Replicated sister chromatids in the G2 phase of the cell that is located far downstream at the protein’s C termi- cycle are held together pairwise by the chromosomal nus. We describe here that protease activity requires cohesin complex. In metaphase, cohesin provides the the separase N terminus that binds to the proteolytic cohesion between sister chromatids to withstand the site at the protein’s C terminus. Securin disrupts this pulling force of the mitotic spindle. Sister chromatid interaction, suggesting a mechanism for how securin separation at anaphase onset is triggered when the Scc1 inhibits separase activity. subunit of cohesin is cleaved by the protease separase The inhibition of separase by securin could suggest to destroy the cohesin complex (reviewed in [1, 2]). For that, in cells lacking securin, separase is overly active, the faithful execution of both the alignment of sister but this is not the case. Indeed, budding yeast and human cells lacking securin show compromised separ- 4 Correspondence: [email protected] ase function [13, 23], and, in fission yeast and Drosoph- Current Biology 974 ila, securin is essential for sister chromatid separation Securin binds and inactivates separase before cells and therefore apparently for separase activity [12, 24]. enter S phase, but separase gradually accumulates in The supportive role of securin for separase activity has nuclei throughout G2, reaching maximum levels only been explained in two ways. Securin might ensure that in mitosis. This could mean that cell cycle-dependent separase adopts its proper fold required for proteolytic events other than the presence of securin contribute to activity [23]. Securin has also been implicated in the nuclear accumulation of separase. However, separase subcellular localization of separase [11, 14]. Here, we nuclear accumulation was unchanged when DNA repli- show that securin in budding yeast is both sufficient to cation was blocked using the replication inhibitor hy- drive the nuclear accumulation of separase and enable droxyurea or when all of the mitotic cyclins, Clb1–4, full catalytic activity of separase after its own destruction were inactivated [28] (data not shown). Alternatively, the in anaphase. presence of securin might be sufficient to cause nuclear accumulation of separase, but it might be a relatively slow process or require an excess of securin over separ- Results ase. In this case, even G1 cells should accumulate nu- clear separase if securin is ectopically expressed. To Securin Promotes Separase Nuclear Accumulation test this, we arrested cells lacking securin stably in G1 Independent of the Cell Cycle by pheromone treatment and then induced expression A possible reason why cells lacking securin show re- of securin from the galactose-inducible GAL1 promoter duced separase function in budding yeast might be the (Figure 1C) [20]. As soon as securin appeared in the incorrect localization of separase in the absence of sec- nucleus, separase had redistributed and was also con- urin. This has recently been studied in budding yeast centrated in the nucleus, reminiscent of cells in mitosis. strains overexpressing separase [14]. To address this (Figure 1C). This demonstrates that expression of sec- under more natural conditions, we observed separase urin in G1 is sufficient to cause nuclear accumulation Esp1 by virtue of myc epitopes that were added to the of separase. Because levels of securin after expression genomic copy of the ESP1 gene (Figure 1A). In G1 cells, from the GAL1 promoter were about 10-fold higher than when securin was absent, about 50% of the cells endogenous levels in metaphase, an excess of securin showed a weak separase accumulation in the nucleus. might be sufficient to promote fast nuclear separase At the G1/S transition, separase was enriched in the accumulation. Together, this suggests that separase nucleus of over 80% of cells, and the nuclear accumula- can enter the nucleus independently of securin, but that tion further increased during the G2 and M period. All the presence of securin is required and sufficient to cells in early anaphase, when securin abruptly disap- cause nuclear concentration of separase. peared, showed a strong nuclear concentration of sep- One possible mechanism to explain this observation arase (Figure 1A). In marked contrast, cells deleted of is that separase is in an equilibrium of import and export securin showed a seeming exclusion of separase from from the nucleus. Securin, once present in the nucleus, the nucleus at all cell cycle stages (Figure 1A). This could act to prevent nuclear export of separase. To test shows that securin is required for accumulation of sep- this hypothesis, we searched the amino acid sequence arase in the nucleus. Although securin disappeared in of separase and found a putative bipartite nuclear local- anaphase and was absent in wild-type G1 cells, separ- ization sequence (NLS) RKAQNLALSLLKKKNK at amino ase was still concentrated in the nuclei of some of these acids 798–813 as well as several possible nuclear export cells. In contrast, separase seemed to be excluded from sequences, Lx1-3Lx2-3LxL, for the major nuclear export all nuclei of cells deleted of securin. The reason for receptor exportin Crm1/Xpo1. However, mutation of the this is unclear; it could be because separase leaves the putative NLS did not interfere with nuclear localization nucleus rather slowly after securin has been destroyed.