Securin can have a separase cleavage site by substitution mutations in the domain required for stabilization and inhibition of separase

Securin-separase complex is required for sister chromatid separation. Securin degrades in an APC/cyclosome dependent manner. Separase is activated on the destruction of securin and cleaves cohesin. Fission yeast securin/Cut2 required for proper separase localization has the motifs for destruction and separase-binding at the N- and C-termini, respectively. We report here the third essential domain, which becomes toxic when the 76-amino acid fragment (81-156) in the middle is overproduced. The fragment inhibits separase, while separase is recruited normally and securin is destroyed. It may interfere with separase activation after destruction of securin. If the 127DIE129 stretch is substituted for AIA, the fragment toxicity and the full-length function are abolished. Interestingly, Cut2 is cleaved in a separase dependent manner if the cleavage consensus is introduced following the DIE sequence. This finding is consistent with the proposed model that the DIE region may mimic the cleavage site of separase and inhibit the activation of separase. Evidence for physical interaction between the fragment and separase is provided. A temperature sensitive mutation cut1-K73 isolated by its specific resistance to the fragment toxicity resides in the superhelical region of separase, suggesting that the catalytic site and the helical region in separase may cooperate for activation.     

Phosphorylation of the mitotic regulator Pds1/securin by Cdc28 is required for efficient nuclear localization of Esp1/separase

Sister chromatid separation at the metaphase-to-anaphase transition is induced by the proteolytic cleavage of one of the cohesin complex subunits. This process is mediated by a conserved protease called separase. Separase is associated with its inhibitor, securin, until the time of anaphase initiation, when securin is degraded in an anaphase-promoting complex/cyclosome (APC/C)-dependent manner. In budding yeast securin/Pds1 not only inhibits separase/Esp1, but also promotes its nuclear localization. The molecular mechanism and regulation of this nuclear targeting are presently unknown. Here we show that Pds1 is a substrate of the cyclin-dependent kinase Cdc28. Phosphorylation of Pds1 by Cdc28 is important for efficient binding of Pds1 to Esp1 and for promoting the nuclear localization of Esp1. Our results uncover a previously unknown mechanism for regulating the Pds1-Esp1 interaction and shed light on a novel role for Cdc28 in promoting the metaphase-to-anaphase transition in budding yeast.     

Spo13 regulates cohesin cleavage

A key aspect of meiotic chromosome segregation is that cohesin, the protein complex that holds sister chromatids together, dissociates from chromosome arms during meiosis I and from centromeric regions during meiosis II. The budding yeast protein Spo13 plays a key role in preventing centromeric cohesin from being lost during meiosis I. We have determined the molecular basis for the metaphase arrest obtained when SPO13 is overexpressed during the mitotic cell cycle. Overexpression of SPO13 inhibits anaphase onset by at least two mechanisms. First, Spo13 causes a transient delay in degradation of the anaphase inhibitor Pds1. Second, Spo13 inhibits cleavage of the cohesin subunit Scc1/Mcd1 or its meiosis-specific homolog, Rec8, by the separase Esp1. The finding that Spo13 did not prevent cleavage of another Esp1 substrate, Slk19, suggests that overexpression of SPO13 is sufficient to prevent cohesin cleavage by protecting specific substrates from separase activity.     

Drosophila separase is required for sister chromatid separation and binds to PIM and THR

Drosophila PIM and THR are required for sister chromatid separation in mitosis and associate in vivo. Neither of these two proteins shares significant sequence similarity with known proteins. However, PIM has functional similarities with securin proteins. Like securin, PIM is degraded at the metaphase-to-anaphase transition and this degradation is required for sister chromatid separation. Securin binds and inhibits separase, a conserved cysteine endoprotease. Proteolysis of securin at the metaphase-to-anaphase transition activates separase, which degrades a conserved cohesin subunit, thereby allowing sister chromatid separation. To address whether PIM regulates separase activity or functions with THR in a distinct pathway, we have characterized a Drosophila separase homolog (SSE). SSE is an unusual member of the separase family. SSE is only about one-third the size of other separases and has a diverged endoprotease domain. However, our genetic analyses show that SSE is essential and required for sister chromatid separation during mitosis. Moreover, we show that SSE associates with both PIM and THR. Although our work shows that separase is required for sister chromatid separation in higher eukaryotes, in addition, it also indicates that the regulatory proteins have diverged to a surprising degree, particularly in Drosophila.     

LAB-1 antagonizes the Aurora B kinase in C. elegans

The Shugoshin/Aurora circuitry that controls the timely release of cohesins from sister chromatids in meiosis and mitosis is widely conserved among eukaryotes, although little is known about its function in organisms whose chromosomes lack a localized centromere. Here we show that Caenorhabditis elegans chromosomes rely on an alternative mechanism to protect meiotic cohesin that is shugoshin-independent and instead involves the activity of a new chromosome-associated protein named LAB-1 (Long Arm of the Bivalent). LAB-1 preserves meiotic sister chromatid cohesion by restricting the localization of the C. elegans Aurora B kinase, AIR-2, to the interface between homologs via the activity of the PP1/Glc7 phosphatase GSP-2. The localization of LAB-1 to chromosomes of dividing embryos and the suppression of mitotic-specific defects in air-2 mutant embryos with reduced LAB-1 activity support a global role of LAB-1 in antagonizing AIR-2 in both meiosis and mitosis. Although the localization of a GFP fusion and the analysis of mutants and RNAi-mediated knockdowns downplay a role for the C. elegans shugoshin protein in cohesin protection, shugoshin nevertheless helps to ensure the high fidelity of chromosome segregation at metaphase I. We propose that, in C. elegans, a LAB-1-mediated mechanism evolved to offset the challenges of providing protection against separase activity throughout a larger chromosome area.     

Cell cycle mechanisms of sister chromatid separation; roles of Cut1/separin and Cut2/securin

The correct transmission of chromosomes from mother to daughter cells is fundamental for genetic inheritance. Separation and segregation of sister chromatids in growing cells occurs in the cell cycle stage called 'anaphase'. The basic process of sister chromatid separation is similar in all eukaryotes: many gene products required are conserved. In this review, the roles of two proteins essential for the onset of anaphase in fission yeast, Cut2/securin and Cut1/separin, are discussed with regard to cell cycle regulation, and compared with the postulated roles of homologous proteins in other organisms. Securin, like mitotic cyclins, is the target of the anaphase promoting complex (APC)/cyclosome and is polyubiquitinated before destruction in a manner dependent upon the destruction sequence. The anaphase never occurs properly in the absence of securin destruction. In human cells, securin is an oncogene. Separin is a large protein (MW approximately 180 kDa), the C-terminus of which is conserved, and is thought to be inhibited by association with securin at the nonconserved N-terminus. In the budding yeast, Esp1/separin is thought to be a component of proteolysis against Scc1, an essential subunit of cohesin which is thought to link duplicated sister chromatids up to the anaphase. Whether fission yeast Cut1/separin is also implicated in proteolysis of cohesin is discussed.     

Western blot screening for monoclonal antibodies against human separase

Separase is a cysteine protease that participates in separation of sister chromatids during mitosis. Human separase is a 230-kDa enzyme that is inhibited by binding to its protein inhibitor securin, specific phosphorylation, and subcellular localization. To further characterize human separase, we raised monoclonal antibodies specific against a C-terminal fragment of the protein. A critical step in monoclonal antibody production procedure is the primary screening of hybridoma supernatants. Here we report primary screening protocol utilizing Western blot analysis. The described screening protocol is carried out using fusion of a human separase fragment with two different purification tags, maltose-binding protein (MBP) and glutathione S-transferase (GST). Immunization by MBP-fusion was followed by primary screening with both MBP- and GST-separase fusions combined in the same preparation separated in SDS-PAGE. This highly sensitive screening approach reduced the number of positive signals by eliminating antibodies specific for the purification tag used in the immunization procedure. The described separase-specific antibodies were suitable for detection of endogenous separase in crude extracts, immunoprecipitation, and immunofluorescent cell staining experiments. The presented procedure is fast, reproducible and could be adopted as a primary screening scheme for a variety of protein antigens.     

Meikin‐associated polo‐like kinase specifies Bub1 distribution in meiosis I

In meiosis I, sister chromatids are captured by microtubules emanating from the same pole (mono‐orientation), and centromeric cohesion is protected throughout anaphase. Shugoshin, which is localized to centromeres depending on the phosphorylation of histone H2A by Bub1 kinase, plays a central role in protecting meiotic cohesin Rec8 from separase cleavage. Another key meiotic kinetochore factor, meikin, may regulate cohesion protection, although the underlying molecular mechanisms remain elusive. Here, we show that fission yeast Moa1 (meikin), which associates stably with CENP‐C during meiosis I, recruits Plo1 (polo‐like kinase) to the kinetochores and phosphorylates Spc7 (KNL1) to accumulate Bub1. Consequently, in contrast to the transient kinetochore localization of mitotic Bub1, meiotic Bub1 persists at kinetochores until anaphase I. The meiotic Bub1 pool ensures robust Sgo1 (shugoshin) localization and cohesion protection at centromeres by cooperating with heterochromatin protein Swi6, which binds and stabilizes Sgo1. Furthermore, molecular genetic analyses show a hierarchical regulation of centromeric cohesion protection by meikin and shugoshin that is important for establishing meiosis‐specific chromosome segregation. We provide evidence that the meiosis‐specific Bub1 regulation is conserved in mouse.     

Proteolytic cleavage of the THR subunit during anaphase limits Drosophila separase function

Sister-chromatid separation in mitosis requires proteolytic cleavage of a cohesin subunit. Separase, the corresponding protease, is activated at the metaphase-to-anaphase transition. Activation involves proteolysis of an inhibitory subunit, securin, following ubiquitination mediated by the anaphase-promoting complex/cyclosome. In Drosophila, the securin PIM associates not only with separase (SSE), but also with an additional protein, THR. Here we show that THR is cleaved after the metaphase-to-anaphase transition. THR cleavage only occurs in functional SSE complexes and in a region that matches the separase cleavage-site consensus. Mutations in this region abolish mitotic THR cleavage. These results indicate that THR is cleaved by SSE. Expression of noncleavable THR variants results in cold-sensitive maternal-effect lethality. This lethality can be suppressed by a reduction of catalytically active SSE levels, indicating that THR cleavage inactivates SSE complexes. THR cleavage is particularly important during the process of cellularization, which follows completion of the last syncytial mitosis of early embryogenesis, suggesting that Drosophila separase has other targets in addition to cohesin subunits.     

Spo13 protects meiotic cohesin at centromeres in meiosis I

In the absence of Spo13, budding yeast cells complete a single meiotic division during which sister chromatids often separate. We investigated the function of Spo13 by following chromosomes tagged with green fluorescent protein. The occurrence of a single division in spo13Delta homozygous diploids depends on the spindle checkpoint. Eliminating the checkpoint accelerates meiosis I in spo13Delta cells and allows them to undergo two divisions in which sister chromatids often separate in meiosis I and segregate randomly in meiosis II. Overexpression of Spo13 and the meiosis-specific cohesin Rec8 in mitotic cells prevents separation of sister chromatids despite destruction of Pds1 and activation of Esp1. This phenotype depends on the combined overexpression of both proteins and mimics one aspect of meiosis I chromosome behavior. Overexpressing the mitotic cohesin, Scc1/Mcd1, does not substitute for Rec8, suggesting that the combined actions of Spo13 and Rec8 are important for preventing sister centromere separation in meiosis I.     

An interactive gene network for securin-separase, condensin, cohesin, Dis1/Mtc1 and histones constructed by mass transformation

The small genome of fission yeast Schizosaccharomyces pombe contains 4824 predicted genes and gene disruption suggests that approximately 850 are essential for viability. To obtain information on interactions among genes required for chromosome segregation, an approach called Strategy B was taken using mass transformation of the 1015 temperature-sensitive (ts) mutants that were made by random mutagenesis and transformed by plasmids carrying the genes for securin, separase, condensin, cohesin, kinetochore microtubule-binding proteins Dis1/Mtc1 or histones. Mutant strains whose phenotypes were either suppressed or inhibited by plasmids were selected. Each plasmid interacted positively or negatively with the average 14 strains. Identification of the mutant gene products by cloning revealed many hitherto unknown interactions. The interactive networks of segregation therefore may consist of genes with a variety of functions. For example, separase/Cut1 interacts with Cdc48/p97/VCP, which stabilizes securin and separase. Surprisingly, S. pombe cdc48 mutants displayed the mitotic phenotype highly similar to separase/cut1 mutants. This approach also provides a novel way of mutant isolation, resulting in two histone H2B strains and a cohesion mutant with a new phenotype.     

Cohesin release is required for sister chromatid resolution,but not for condensin-mediated compaction,at the onset of mitosis

The establishment of metaphase chromosomes is an essential prerequisite of sister chromatid separation in anaphase. It involves the coordinated action of cohesin and condensin, protein complexes that mediate cohesion and condensation, respectively. In metazoans, most cohesin dissociates from chromatin at prophase, coincident with association of condensin. Whether loosening of cohesion at the onset of mitosis facilitates the compaction process, resolution of the sister chromatids, or both, remains unknown. We have found that the prophase release of cohesin is completely blocked when two mitotic kinases, aurora B and polo-like kinase (Plx1), are simultaneously depleted from Xenopus egg extracts. Condensin loading onto chromatin is not affected under this condition, and rod-shaped chromosomes are produced that show an apparently normal level of compaction. However, the resolution of sister chromatids within these chromosomes is severely compromised. This is not because of inhibition of topoisomerase II activity that is also required for the resolution process. We propose that aurora B and Plx1 cooperate to destabilize the sister chromatid linkage through distinct mechanisms that may involve phosphorylation of histone H3 and cohesin, respectively. More importantly, our results strongly suggest that cohesin release at the onset of mitosis is essential for sister chromatid resolution but not for condensin-mediated compaction.     

Shugoshin-2 is essential for the completion of meiosis but not for mitotic cell division in mice

Shugoshin-2 (SGOL2) is one of the two mammalian orthologs of the Shugoshin/Mei-S322 family of proteins that regulate sister chromatid cohesion by protecting the integrity of the multiprotein cohesin complexes. This protective system is essential for faithful chromosome segregation during mitosis and meiosis, which is the physical basis of Mendelian inheritance. Regardless of its evolutionary conservation from yeast to mammals, little is known about the in vivo relevance and specific role that SGOL2 plays in mammals. Here we show that disruption of the gene encoding mouse SGOL2 does not cause any alteration in sister chromatid cohesion in embryonic cultured fibroblasts and adult somatic tissues. Moreover, mutant mice develop normally and survive to adulthood without any apparent alteration. However, both male and female Sgol2-deficient mice are infertile. We demonstrate that SGOL2 is necessary for protecting centromeric cohesion during mammalian meiosis I. In vivo, the loss of SGOL2 promotes a premature release of the meiosis-specific REC8 cohesin complexes from anaphase I centromeres. This molecular alteration is manifested cytologically by the complete loss of centromere cohesion at metaphase II leading to single chromatids and physiologically with the formation of aneuploid gametes that give rise to infertility.     

Cut1/separase C-terminus affects spindle pole body positioning in interphase of fission yeast: pointed nuclear formation

BACKGROUND: The separase-securin complex is required for anaphase. Separase activated by securin destruction cleaves the cohesin subunit Scc1/Rad21 enriched in kinetochores. Fission yeast Cut1/separase resides in interphase cytoplasm and mobilizes to the spindle and the spindle pole bodies (SPBs) in mitosis, while Cut2/securin remains in the nucleus from interphase to metaphase, and temporarily locates at the short spindle. RESULTS: We here report a novel SPB-led dynamic nuclear movement in fission yeast, when the Cut1 C-terminal fragment is over-expressed. The tip of the pointed nucleus contained both SPB and centromeric DNA, and rapidly moved along the bundled cytoplasmic microtubules. The same pointed nucleus was produced when the human separase C-fragment was over-expressed. The pointed nuclear formation did not require the protease site of separase, but required the conserved C-terminus and a microtubule- and kinetochore-binding protein Mtc1/Alp14, a homologue of frog XMAP215 and budding yeast Stu2. The movement-inducing C-fragment should be cytoplasmic, as the pointed nucleus was abolished when the fragment contained the NLS (nuclear localization signal). CONCLUSIONS: Overproduced separase C-fragment abolishes correct SPB-positioning in interphase. Resulting pointed nuclear formation (alternatively called 'pigtail movement') requires cytoplasmic microtubules and Mtc1/Alp14.     

Genetic interactions of separase regulatory subunits reveal the diverged Drosophila Cenp-C homolog

Faithful transmission of genetic information during mitotic divisions depends on bipolar attachment of sister kinetochores to the mitotic spindle and on complete resolution of sister-chromatid cohesion immediately before the metaphase-to-anaphase transition. Separase is thought to be responsible for sister-chromatid separation, but its regulation is not completely understood. Therefore, we have screened for genetic loci that modify the aberrant phenotypes caused by overexpression of the regulatory separase complex subunits Pimples/securin and Three rows in Drosophila. An interacting gene was found to encode a constitutive centromere protein. Characterization of its centromere localization domain revealed the presence of a diverged CENPC motif. While direct evidence for an involvement of this Drosophila Cenp-C homolog in separase activation at centromeres could not be obtained, in vivo imaging clearly demonstrated that it is required for normal attachment of kinetochores to the spindle.     

Sororin is enriched at the central region of synapsed meiotic chromosomes

During meiotic prophase, cohesin complexes mediate cohesion between sister chromatids and promote pairing and synapsis of homologous chromosomes. Precisely how the activity of cohesin is controlled to promote these events is not fully understood. In metazoans, cohesion establishment between sister chromatids during mitotic divisions is accompanied by recruitment of the cohesion-stabilizing protein Sororin. During somatic cell division cycles, Sororin is recruited in response to DNA replication-dependent modification of the cohesin complex by ESCO acetyltransferases. How Sororin is recruited and acts in meiosis is less clear. Here, we have surveyed the chromosomal localization of Sororin and its relationship to the meiotic cohesins and other chromatin modifiers with the objective of determining how Sororin contributes to meiotic chromosome dynamics. We show that Sororin localizes to the cores of meiotic chromosomes in a manner that is dependent on synapsis and the synaptonemal complex protein SYCP1. In contrast, cohesin, with which Sororin interacts in mitotic cells, shows axial enrichment on meiotic chromosomes even in the absence of synapsis between homologs. Using high-resolution microscopy, we show that Sororin is localized to the central region of the synaptonemal complex. These results indicate that Sororin regulation during meiosis is distinct from its regulation in mitotic cells and may suggest that it interacts with a distinctly different partner to ensure proper chromosome dynamics in meiosis.     

Releasing cohesin from chromosome arms in early mitosis: opposing actions of Wapl–Pds5 and Sgo1

The cohesin complex establishes sister chromatid cohesion during S phase. In metazoan cells, most if not all cohesin dissociates from chromatin during mitotic prophase, leading to the formation of metaphase chromosomes with two cytologically discernible chromatids. This process, known as sister chromatid resolution, is believed to be a prerequisite for synchronous separation of sister chromatids in subsequent anaphase. To dissect this process at a mechanistic level, we set up an in vitro system. Sister chromatid resolution is severely impaired upon depletion of Wapl from Xenopus egg extracts. Exogenously added human Wapl can rescue these defects and, remarkably, it can do so in a very short time window of early mitosis. A similar set of observations is made for Pds5, a factor implicated previously in the stabilization of interphase cohesion. Characteristic amino acid motifs (the FGF motifs) in Wapl coordinate its physical and functional interactions with Pds5 and cohesin subunits. We propose that Wapl and Pds5 directly modulate conformational changes of cohesin to make it competent for dissociation from chromatin during prophase. Evidence is also presented that Sgo1 plays a hitherto underappreciated role in stabilizing cohesin along chromosome arms, which is antagonized by the mitotic kinases polo-like kinsase (Plk1) and aurora B.     

A brute force postgenome approach to identify temperature-sensitive mutations that negatively interact with separase and securin plasmids

BACKGROUND: The fission yeast Schizosaccharomyces pombe separase/Cut1 and securin/Cut2 are required for anaphase-specific activation of proteolysis that leads to proper sister chromatid separation. We intended to identify ts (temperature sensitive) strains whose growth was inhibited by multicopy plasmid pCUT1 or pCUT2 at the permissive temperature. RESULTS: After a one-by-one transformation of 1015 randomly isolated ts strains, 18 transformants that retarded in colony formation at the permissive or semipermissive temperature were isolated. Six of them, in the absence of pCUT1 or pCUT2, produced mitotic phenotypes with condensed chromosomes at the restrictive temperature. Gene cloning established that these mutants were defective in either the subunits (Cut9, Cut23, Cut20 or Apc10) of APC (anaphase promoting complex)/cyclosome or Cut8, a regulator for 26S proteasome localization. The inhibitory effect of separase against APC/cyclosome mutations was abolished when the catalytic site mutation C1730A was introduced and overproduced, indicating that inhibition needs an active separase. Securin/Cut2 overproduction also caused a negative effect on these mutants. Surprisingly, the phenotypes of cut9 and cut23 in the presence of pCUT1 or pCUT2 were not the mitotic arrest, and they were strikingly different depending on pCUT1 or pCUT2. CONCLUSIONS: This study shows the functional link between separase/Cut1 and APC/cyclosome in a separase activity-dependent manner. The negative effect of active separase overproduction on APC/cyclosome mutations is possibly due to the direct inhibition of APC/cyclosome. In addition, the manner of the inhibition by high copy securin and separase plasmids were quite different each other and did not result in the mitotic block.