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.     

Protein phosphatase 2A and separase form a complex regulated by separase autocleavage

The onset of anaphase is triggered by the activation of a site-specific protease called separase. Separase cleaves the chromosomal cohesins holding the duplicated sister chromatids together, allowing sisters to simultaneously separate and segregate to opposite ends of the cell before division. Activated separase cleaves not only cohesin, but also itself; however, the biological significance of separase self-cleavage has remained elusive. Before anaphase, separase is inhibited by at least two mechanisms. The first involves the binding of securin, whereas the second requires the phosphorylation-dependent binding of cyclin-dependent kinase 1 (Cdk1)/cyclin B1. Because securin and Cdk1/cyclin B1 interact with separase in a mutually exclusive manner, the degradation of both these inhibitors plays an important role in activating separase at anaphase. Here we identify a new separase interacting partner, a specific subtype of the heterotrimeric protein phosphatase 2A (PP2A). PP2A associates with separase through the B' (B56) regulatory subunit and does so independently of securin and cyclin B1 binding. The association of PP2A with separase requires a 55-amino acid domain closely juxtaposed to separase autocleavage sites. Strikingly, mutation of these cleavage sites increases PP2A binding, suggesting that separase cleavage disrupts the interaction of PP2A with separase. Furthermore, expression of a non-cleavable separase, but not a non-cleavable mutant that cannot bind PP2A, causes a premature loss of centromeric cohesion. Together these observations provide a new mechanistic insight into a physiological function for separase self-cleavage.     

Orchestrating anaphase and mitotic exit: separase cleavage and localization of Slk19

Anaphase in budding yeast is triggered by cleavage of the central subunit, Scc1, of the chromosomal cohesin complex by the protease separase. Here we show that separase also cleaves the kinetochore-associated protein Slk19 at anaphase onset. Separase activity is also required for the proper localization of a stable Slk19 cleavage product to the spindle midzone in anaphase. The cleavage and localization of Slk19 are necessary to stabilize the anaphase spindle, and we show that a stable spindle is a prerequisite for timely exit from mitosis. This demonstrates the cleavage of targets other than cohesin by separase in the orchestration of high-fidelity anaphase.     

Separase biosensor reveals that cohesin cleavage timing depends on phosphatase PP2A(Cdc55) regulation

In anaphase, sister chromatids separate abruptly and are then segregated by the mitotic spindle. The protease separase triggers sister separation by cleaving the Scc1/Mcd1 subunit of the cohesin ring that holds sisters together. Polo-kinase phosphorylation of Scc1 promotes its cleavage, but the underlying regulatory circuits are unclear. We developed a separase biosensor in Saccharomyces cerevisiae that provides a quantitative indicator of cohesin cleavage in single cells. Separase is abruptly activated and cleaves most cohesin within 1?min, after which anaphase begins. Cohesin near centromeres and telomeres is cleaved at the same rate and time. Protein phosphatase PP2A(Cdc55) inhibits cohesin cleavage by counteracting polo-kinase phosphorylation of Scc1. In early anaphase, the previously described separase inhibition of PP2A(Cdc55) promotes cohesin cleavage. Thus, separase acts directly on Scc1 and also indirectly, through inhibition of PP2A(Cdc55), to stimulate cohesin cleavage, providing a feedforward loop that may contribute to a robust and timely anaphase.     

Separase Cleaves the N-Tail of the CENP-A Related Protein CPAR-1 at the Meiosis I Metaphase-Anaphase Transition in C. elegans

Centromeres are defined epigenetically in the majority of eukaryotes by the presence of chromatin containing the centromeric histone H3 variant CENP-A. Most species have a single gene encoding a centromeric histone variant whereas C. elegans has two: HCP-3 (also known as CeCENP-A) and CPAR-1. Prior RNAi replacement experiments showed that HCP-3 is the functionally dominant isoform, consistent with CPAR-1 not being detectable in embryos. GFP::CPAR-1 is loaded onto meiotic chromosomes in diakinesis and is enriched on bivalents until meiosis I. Here we show that GFP::CPAR-1 signal loss from chromosomes precisely coincides with homolog segregation during anaphase I. This loss of GFP::CPAR-1 signal reflects proteolytic cleavage between GFP and the histone fold of CPAR-1, as CPAR-1::GFP, in which GFP is fused to the C-terminus of CPAR-1, does not exhibit any loss of GFP signal. A focused candidate screen implicated separase, the protease that initiates anaphase by cleaving the kleisin subunit of cohesin, in this cleavage reaction. Examination of the N-terminal tail sequence of CPAR-1 revealed a putative separase cleavage site and mutation of the signature residues in this site eliminated the cleavage reaction, as visualized by retention of GFP::CPAR-1 signal on separating homologous chromosomes at the metaphase-anaphase transition of meiosis I. Neither cleaved nor uncleavable CPAR-1 were centromere-localized in mitosis and instead localized throughout chromatin, indicating that centromere activity has not been retained in CPAR-1. Although the functions of CPAR-1 and of its separase-dependent cleavage remain to be elucidated, this effort reveals a new substrate of separase and provides an in vivo biosensor to monitor separase activity at the onset of meiosis I anaphase.     

Separase sensor reveals dual roles for separase coordinating cohesin cleavage and cdk1 inhibition

Complete dissociation of sister chromatid cohesion and subsequent induction of poleward movement of disjoined sisters are two essential events underlying chromosome segregation; however, how cells coordinate these two processes is not well understood. Here, we developed a fluorescence-based sensor for the protease separase that mediates cohesin cleavage. We found that separase undergoes an abrupt activation shortly before anaphase onset in the vicinity of chromosomes. This activation profile of separase depends on the abilities of two of its binding proteins, securin and cyclin B1, to inhibit its protease activity and target it to chromosomes. Subsequent to its proteolytic activation, separase then binds to and inhibits a subset of cyclin B1-cdk1, which antagonizes cdk1-mediated phosphorylation on chromosomes and facilitates poleward movement of sisters in anaphase. Therefore, by consecutively acting as a protease and a cdk1 inhibitor, separase coordinates two key processes to achieve simultaneous and abrupt separation of sister chromatids.     

A non-proteolytic function of separase links the onset of anaphase to mitotic exit

Separase is a protease that triggers chromosome segregation at anaphase onset by cleaving cohesin, the chromosomal protein complex responsible for sister chromatid cohesion. After anaphase, cells exit from mitosis; that is, they complete downregulation of cyclin-dependent kinase activity, undergo cytokinesis and enter G1 of the next cell cycle. Here we show that separase activation at the onset of anaphase is sufficient to promote release from the nucleolus and activation of the budding yeast phosphatase, Cdc14, a key step in mitotic exit. The ability of separase to activate Cdc14 is independent of its protease function but may involve promoting phosphorylation of the Cdc14 inhibitor Net1. This novel separase function is coregulated with its proteolytic activity by the separase inhibitor securin. This helps to explain the coupling of anaphase and mitotic exit--after securin degradation at anaphase onset, separase cleaves cohesin to trigger chromosome segregation and concurrently uses a non-proteolytic mechanism to initiate mitotic exit.     

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.     

PP2A delays APC/C‐dependent degradation of separase‐associated but not free securin

The universal triggering event of eukaryotic chromosome segregation is cleavage of centromeric cohesin by separase. Prior to anaphase, most separase is kept inactive by association with securin. Protein phosphatase 2A (PP2A) constitutes another binding partner of human separase, but the functional relevance of this interaction has remained enigmatic. We demonstrate that PP2A stabilizes separase‐associated securin by dephosphorylation, while phosphorylation of free securin enhances its polyubiquitylation by the ubiquitin ligase APC/C and proteasomal degradation. Changing PP2A substrate phosphorylation sites to alanines slows degradation of free securin, delays separase activation, lengthens early anaphase, and results in anaphase bridges and DNA damage. In contrast, separase‐associated securin is destabilized by introduction of phosphorylation‐mimetic aspartates or extinction of separase‐associated PP2A activity. G2‐ or prometaphase‐arrested cells suffer from unscheduled activation of separase when endogenous securin is replaced by aspartate‐mutant securin. Thus, PP2A‐dependent stabilization of separase‐associated securin prevents precocious activation of separase during checkpoint‐mediated arrests with basal APC/C activity and increases the abruptness and fidelity of sister chromatid separation in anaphase.     

Phosphorylation of the cohesin subunit Scc1 by Polo/Cdc5 kinase regulates sister chromatid separation in yeast

At the onset of anaphase, a caspase-related protease (separase) destroys the link between sister chromatids by cleaving the cohesin subunit Scc1. During most of the cell cycle, separase is kept inactive by binding to an inhibitory protein called securin. Separase activation requires proteolysis of securin, which is mediated by an ubiquitin protein ligase called the anaphase-promoting complex. Cells regulate anaphase entry by delaying securin ubiquitination until all chromosomes have attached to the mitotic spindle. Though no longer regulated by this mitotic surveillance mechanism, sister separation remains tightly cell cycle regulated in yeast mutants lacking securin. We show here that the Polo/Cdc5 kinase phosphorylates serine residues adjacent to Scc1 cleavage sites and strongly enhances their cleavage. Phosphorylation of separase recognition sites may be highly conserved and regulates sister chromatid separation independently of securin.     

Shugoshin prevents dissociation of cohesin from centromeres during mitosis in vertebrate cells

Cohesion between sister chromatids is essential for their bi-orientation on mitotic spindles. It is mediated by a multisubunit complex called cohesin. In yeast, proteolytic cleavage of cohesin's alpha kleisin subunit at the onset of anaphase removes cohesin from both centromeres and chromosome arms and thus triggers sister chromatid separation. In animal cells, most cohesin is removed from chromosome arms during prophase via a separase-independent pathway involving phosphorylation of its Scc3-SA1/2 subunits. Cohesin at centromeres is refractory to this process and persists until metaphase, whereupon its alpha kleisin subunit is cleaved by separase, which is thought to trigger anaphase. What protects centromeric cohesin from the prophase pathway? Potential candidates are proteins, known as shugoshins, that are homologous to Drosophila MEI-S332 and yeast Sgo1 proteins, which prevent removal of meiotic cohesin complexes from centromeres at the first meiotic division. A vertebrate shugoshin-like protein associates with centromeres during prophase and disappears at the onset of anaphase. Its depletion by RNA interference causes HeLa cells to arrest in mitosis. Most chromosomes bi-orient on a metaphase plate, but precocious loss of centromeric cohesin from chromosomes is accompanied by loss of all sister chromatid cohesion, the departure of individual chromatids from the metaphase plate, and a permanent cell cycle arrest, presumably due to activation of the spindle checkpoint. Remarkably, expression of a version of Scc3-SA2 whose mitotic phosphorylation sites have been mutated to alanine alleviates the precocious loss of sister chromatid cohesion and the mitotic arrest of cells lacking shugoshin. These data suggest that shugoshin prevents phosphorylation of cohesin's Scc3-SA2 subunit at centromeres during mitosis. This ensures that cohesin persists at centromeres until activation of separase causes cleavage of its alpha kleisin subunit. Centromeric cohesion is one of the hallmarks of mitotic chromosomes. Our results imply that it is not an intrinsically stable property, because it can easily be destroyed by mitotic kinases, which are kept in check by shugoshin.     

Secured cutting: controlling separase at the metaphase to anaphase transition

The final irreversible step in the duplication and distribution of genomes to daughter cells takes place at the metaphase to anaphase transition. At this point aligned sister chromatid pairs split and separate. During metaphase, cohesion between sister chromatids is maintained by the chromosomal multi-subunit cohesin complex. Here, I review recent findings as to how anaphase is initiated by proteolytic cleavage of the Scc1 subunit of cohesin. Scc1 is cleaved by a site-specific protease that is conserved in all eukaryotes, and is now called ‘separase’. As a result of this cleavage, the cohesin complex is destroyed, allowing the spindle to pull sister chromatids into opposite halves of the cell. Because of the final and irreversible nature of Scc1 cleavage, this reaction is tightly controlled. Several independent mechanisms seem to impose regulation on Scc1 cleavage, acting on both the activity of separase and the susceptibility of the substrate.     

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.     

Centrosomal Aki1 and cohesin function in separase-regulated centriole disengagement

Sister chromatid separation at anaphase is triggered by cleavage of the cohesin subunit Scc1, which is mediated by separase. Centriole disengagement also requires separase. This dual role of separase permits concurrent control of these events for accurate metaphase to anaphase transition. Although the molecular mechanism underlying sister chromatid cohesion has been clarified, that of centriole cohesion is poorly understood. In this study, we show that Akt kinase–interacting protein 1 (Aki1) localizes to centrosomes and regulates centriole cohesion. Aki1 depletion causes formation of multipolar spindles accompanied by centriole splitting, which is separase dependent. We also show that cohesin subunits localize to centrosomes and that centrosomal Scc1 is cleaved by separase coincidentally with chromatin Scc1, suggesting a role of Scc1 as a connector of centrioles as well as sister chromatids. Interestingly, Scc1 depletion strongly induces centriole splitting. Furthermore, Aki1 interacts with cohesin in centrosomes, and this interaction is required for centriole cohesion. We demonstrate that centrosome-associated Aki1 and cohesin play pivotal roles in preventing premature cleavage in centriole cohesion.     

Analyzing protease specificity and detecting in vivo proteolytic events using tandem mass spectrometry

Although trypsin remains the most commonly used protease in MS, other proteases may be employed for increasing peptide coverage or generating overlapping peptides. Knowledge of the accurate specificity rules of these proteases is helpful for database search tools to detect peptides, and becomes crucial when label‐free MS is used to discover in vivo proteolytic cleavages. Since in vivo cleavages are inferred by subtracting digestion‐induced cleavages from all observed cleavages, it is important to ensure that the specificity rule used to identify digestion‐induced cleavages are broad enough to capture even minor cleavages produced in digestion, to avoid erroneously identifying them as in vivo cleavages. In this study, we describe MS‐Proteolysis, a software tool for identifying putative sites of in vivo proteolytic cleavage using label‐free MS. The tool is used in conjunction with digestion by trypsin and three other proteases, whose specificity rules are revised and extended before inferring proteolytic cleavages. Finally, we show that comparative analysis of multiple proteases can be used to detect putative in vivo proteolytic sites on a proteome‐wide scale.     

Separase is required for chromosome segregation during meiosis I in Caenorhabditis elegans.

BACKGROUND: Chromosome segregation during mitosis and meiosis is triggered by dissolution of sister chromatid cohesion, which is mediated by the cohesin complex. Mitotic sister chromatid disjunction requires that cohesion be lost along the entire length of chromosomes, whereas homolog segregation at meiosis I only requires loss of cohesion along chromosome arms. During animal cell mitosis, cohesin is lost in two steps. A nonproteolytic mechanism removes cohesin along chromosome arms during prophase, while the proteolytic cleavage of cohesin's Scc1 subunit by separase removes centromeric cohesin at anaphase. In Saccharomyces cerevisiae and Caenorhabditis elegans, meiotic sister chromatid cohesion is mediated by Rec8, a meiosis-specific variant of cohesin's Scc1 subunit. Homolog segregation in S. cerevisiae is triggered by separase-mediated cleavage of Rec8 along chromosome arms. In principle, chiasmata could be resolved proteolytically by separase or nonproteolytically using a mechanism similar to the mitotic "prophase pathway." RESULTS: Inactivation of separase in C. elegans has little or no effect on homolog alignment on the meiosis I spindle but prevents their timely disjunction. It also interferes with chromatid separation during subsequent embryonic mitotic divisions but does not directly affect cytokinesis. Surprisingly, separase inactivation also causes osmosensitive embryos, possibly due to a defect in the extraembryonic structures, referred to as the "eggshell." CONCLUSIONS: Separase is essential for homologous chromosome disjunction during meiosis I. Proteolytic cleavage, presumably of Rec8, might be a common trigger for the first meiotic division in eukaryotic cells. Cleavage of proteins other than REC-8 might be necessary to render the eggshell impermeable to solutes.     

Regulation of human separase by securin binding and autocleavage

BACKGROUND: Sister chromatid separation is initiated by separase, a protease that cleaves cohesin and thereby dissolves sister chromatid cohesion. Separase is activated by the degradation of its inhibitor securin and by the removal of inhibitory phosphates. In human cells, separase activation also coincides with the cleavage of separase, but it is not known if this reaction activates separase, which protease cleaves separase, and how separase cleavage is regulated.RESULTS: Inhibition of separase expression in human cells by RNA interference causes the formation of polyploid cells with large lobed nuclei. In mitosis, many of these cells contain abnormal chromosome plates with unseparated sister chromatids. Inhibitor binding experiments in vitro reveal that securin prevents the access of substrate analogs to the active site of separase. Upon securin degradation, the active site of full-length separase becomes accessible, allowing rapid autocatalytic cleavage of separase at one of three sites. The resulting N- and C-terminal fragments remain associated and can be reinhibited by securin. A noncleavable separase mutant retains its ability to cleave cohesin in vitro.CONCLUSIONS: Our results suggest that separase is required for sister chromatid separation during mitosis in human cells. Our data further indicate that securin inhibits separase by blocking the access of substrates to the active site of separase. Securin proteolysis allows autocatalytic processing of separase into a cleaved form, but separase cleavage is not essential for separase activation.     

Essential CDK1-inhibitory role for separase during meiosis I in vertebrate oocytes

Separase not only triggers anaphase of meiosis I by proteolytic cleavage of cohesin on chromosome arms, but in vitro vertebrate separase also acts as a direct inhibitor of cyclin-dependent kinase 1 (Cdk1) on liberation from the inhibitory protein, securin. Blocking separase-Cdk1 complex formation by microinjection of anti-separase antibodies prevents polar-body extrusion in vertebrate oocytes. Importantly, proper meiotic maturation is rescued by chemical inhibition of Cdk1 or expression of Cdk1-binding separase fragments lacking cohesin-cleaving activity.     

Nuclear Exclusion of Separase Prevents Cohesin Cleavage in Interphase Cells

During mitosis, equal transmission of the duplicated chromosomes demands a strict regulation of separase, which cleaves cohesin and triggers sister chromatid separation in anaphase. Vertebrate separase is inhibited by securin and the inhibitory phosphorylation of separase. However, knockout experiments indicate that securin is dispensable and the inhibitory phosphorylation was observed only in M phase cells. This begs the question how cohesin cleavage by separase is prevented in the absence these two mechanisms. Here we show that separase is excluded from cohesin by the nuclear envelope, which forms in telophase and disassembles in mitosis. The exclusion is achieved passively by its large physical mass and may be backed up by the CRM1-dependent nuclear export. A functional NES motif is identified in separase. We demonstrated that the nuclear envelope is sufficient to prevent active separase from cleaving nuclear cohesin. We propose that the nuclear exclusion is important to prevent cohesin cleavage during interphase in the absence of securin and the phosphorylation inhibition.     

Protease-Dead Separase Is Dominant Negative in the C. elegans Embryo

Separase is a protease that promotes chromosome segregation at anaphase by cleaving cohesin. Several non-proteolytic functions of separase have been identified in other organisms. We created a transgenic C. elegans line that expresses protease-dead separase in embryos to further characterize separase function. We find that expression of protease-dead separase is dominant-negative in C. elegans embryos, not previously reported in other systems. The C. elegans embryo is an ideal system to study developmental processes in a genetically tractable system. However, a major limitation is the lack of an inducible gene expression system for the embryo. We have developed two methods that allow for the propagation of lines carrying dominant-negative transgenes and have applied them to characterize expression of protease-dead separase in embryos. Using these methods, we show that protease-dead separase causes embryo lethality, and that protease-dead separase cannot rescue separase mutants. These data suggest that protease-dead separase interferes with endogenous separase function, possibly by binding substrates and protecting them from cleavage.