The Eukaryotic Linear Motif resource for
Functional Sites in Proteins
Functional site class:
Separase cleavage motif
Functional site description:
Separase plays an important role in sister chromatid separation at the metaphase-to-anaphase transition in cell division. In particular, they execute separation by cleaving a subunit of the chromosomal protein complex cohesin, responsible for sister chromatid cohesion. Separase recognizes its substrates based on a short linear motif. The amino acids of the substrate around the separase cleavage site are named N- to C-terminal: P6, P5, P4, P3, P2, P1, P-1. The scissile bond between P1 and P-1 is cleaved by separase and the positions P6 to P1 are important for substrate specificity and recognition.
ELMs with same func. site: CLV_Separin_Fungi  CLV_Separin_Metazoa 
ELM Description:
The amino acids of the substrate around the separase cleavage site are designated N- to C-terminal: P6, P5, P4, P3, P2, P1, P-1. The scissile bond between P1 and P-1 is cleaved by separase and the positions P6 to P1 are important for substrate specificity and recognition. P1 is always arginine (R), a positively charged, polar amino acid. Even the conservative substitution for lysine (K) drastically reduces cleavage efficiency (Sullivan,2004). At P2, small and hydrophobic amino acids are preferred, usually restricted to glycine (G). Hydrophobic amino acids like isoleucine (I) and valine (V) were found most frequently at P3. This position seems to be a conserved hydrophobic residue. Glutamate is the most preferred amino acid at P4. Substitution for aspartate (D), which is negatively charged, is also allowed. This suggests, that the negative charge is essential at P4, forming a core motif with P1 (ExxR). At the next position, P5, amino acids with hydrophobic character are important. Acidic amino acids like glutamate (E) or phosphorylated serine (S) are preferred at P6, but in nature and multiple alignments almost exclusively serine was found at this position. Surprisingly, small and medium sized hydrophobic amino acids like glycine (G) or leucine (L) are also tolerated at P6 with just minor decrease in cleavage efficiency (Sullivan,2004).
Pattern Probability: 0.0000040
Present in taxon: Fungi
Interaction Domain:
Peptidase_C50 (PF03568) Peptidase family C50 (Stochiometry: 1 : 1)
o See 4 Instances for CLV_Separin_Fungi
o Abstract
Separases, caspase-like cysteine endopeptidases, are involved in the mitotic and meiotic processes in all Eukaryotes from yeast to mammals. The protease is best known for its role in the irreversible separation of sister chromatids at the end of metaphase, thereby initializing anaphase (Uhlmann,1999; Uhlmann,2001). Sister chromatids are connected to each other instantaneously after their replication. A multiprotein complex called cohesin, composed of four conserved subunits Smc1, Smc3, Scc1/Rad21 and one version of Scc3 (SA1 or SA2), is responsible for the sister chromatid connection. Separase cleaves the Scc1 subunit of the cohesin complex, thereby releasing sister chromatids and promoting their poleward movement. Unlike in budding yeast, this event takes place in two steps in Vertebrates (Waizenegger,2000). First, the bulk of cohesin dissociates separase-independently from the chromosome arms in prophase. Just a small amount of cohesin remains associated with the centromeres up to metaphase and dissociates in a second, separase-mediated step. The meiotic equivalent of Scc1 is the cohesin subunit Rec8, featuring centromeric cleavage protection in anaphase I by shugoshin proteins (Buonomo,2000). This prevents random chromatid segregation in anaphase II. In meiosis II, shugoshins disappear from the cells and Rec8 can be cleaved (Katis,2010; PMC2248297).
It is of high importance that the separation mechanism is tightly regulated. Premature cleavage causes missegregation of chromatids in cell division, which in turn leads to severe diseases like cancer or trisomy in Metazoans (Shepard,2007). Moreover, sister chromatid linkage is important to ensure spatial proximity for homologous recombination, counteracts the pulling of microtubules towards the spindle poles when centromeres align on the equatorial plate at transition to metaphase and prevents the need to reidentify pairs of replication products before anaphase to ensure equipartition on daughter cells. In interphase, separase is located in the cytoplasm and thereby physically isolated from cohesin. Before anaphase, separase is inhibited by securin (Stemmann,2001; Waizenegger,2002; Viadiu,2005). Ubiquitination, which marks securin for proteasomal degradation, is catalyzed by APCCdc20 (anaphase promoting complex, activated by Cdc20 at the metaphase-to-anaphase transition), which in turn is activated by Cdks (cyclin-dependent kinases; mitosis-promoting factor) (Uhlmann,2001). On the other hand, in some organisms like budding yeast or human, securin is surprisingly not essential for timing or accuracy of sister chromatid separation (PMC2248297), suggesting that further mechanisms must regulate separase activity.
In vertebrate cells, there is another inhibitory mechanism of separase, which is securin-independent. Phosphorylation of separase at one major site (Ser1126; human) by Cdk1 facilitates binding of Cdk1 via its activator cyclin B1, thereby inactivating separase (Gorr,2005; Holland,2006). Interestingly, securin and Cdk1 bind separase in a mutually exclusive manner. The redundancy in separase inhibition is a safeguard against partial defects in the regulatory machinery that might occur (PMC2248297). Unusually separase also regulates Cdk1 activity via its inhibition by Cdk1. Like securin, Cdk1 inactivation is mediated by APCCdc20, triggered via ubiquitination of its activator cyclin B1. But degradation of cyclin B1 alone is not sufficient to decrease Cdk1 activity well enough and hence to promote fast chromatid poleward movement after their separation in anaphase (Shindo,2012). In the separase-Cdk1 complex, Cdk1 is also inactive, thereby assisting APCCdc20 in promoting mitotic exit. It has been shown, that securin has a higher affinity for separase than Cdk1 (Shindo,2012). Therefore, most separase is bound by securin in metaphase and Cdk1 inhibition becomes more important after degradation of securin at onset of anaphase. However, it has been found that there is a different mechanism in budding yeast to decrease Cdk1 activity, also mediated by separase. The mitotic phosphatase Cdc14 activates Cdh1 (another activator of APC) via dephosphorylation. As described before, APC further down-regulates Cdk1 activity. In addition, Cdc14 dephosphorylates Cdk1 substrates, thereby counteracting Cdk1 activity. Separase initiates this pathway by mediating Net1 phosphorylation, the inhibitor of Cdc14, which in turn down-regulates PP2ACdc55 (Protein phosphatase type 2A in conjunction with its regulatory subunit Cdc55) (Queralt,2008). It has also been shown that PP2A and separase co-immunoprecipitate, but the mechanism by which separase finally interacts with PP2ACdc55 is still unknown (Holland,2007; Queralt,2008; Queralt,2008). In this way, budding yeast separase not only triggers anaphase, but also has additional roles in cell-cycle progression.
Cleaved separase forms have been discovered in Metazoans. In human cells, it has been shown that with degradation of securin, separase becomes proteolytically active and cleaves itself autocatalytically at one of three possible, closely clustered cleavage sites. It has been shown by immunoprecipitation that the cleaved N-terminal and C-terminal fragments remain noncovalently bound to each other (Waizenegger,2002). In other organisms like Xenopus or Drosophila, there is just one separase auto cleavage site, conserved with one of the three cleavage sites in human separase (Fan,2006; Herzig,2002). Both cleaved and noncleaved forms exhibit the same activity in cleaving Scc1. Hence sister chromatid separation, which coincides with separase cleavage, is not affected by separase cleavage (Waizenegger,2002). The biological significance of separase autocleavage is still not absolutely cleared. It is conceivable that cleavage serves for rapid degradation of the C-terminal cleavage product – containing the active site – by the N-end rule pathway to recover cohesin for the subsequent cell cycle (Waizenegger,2002).
Kinetochore protein Slk19, a protein implicated in spindle stabilization in Saccharomyces cerevisiae, is also cleaved by separase at anaphase onset (Zeng,1999; Sullivan,2001). Slk19 cleavage coincides with Scc1 cleavage at the metaphase-to-anaphase transition (Sullivan,2004). Recently, kendrin (or pericentrin) has been discovered as a novel separase substrate in Vertebrata (Matsuo,2012). Kendrin is a compound of the pericentriolar material (PCM). PCM is a protein matrix surrounding the centrioles, microtubule-based cylindrical structures. Together they form the centrosome, which is known to direct the formation of bipolar spindles during mitosis (Loncarek,2008; Delaval,2010; Matsuo,2012). Centriol overduplication is associated with chromosome instability and cancer progression (Nigg,2002). Therefore, it is of high importance to strictly regulate the centrosome duplication. It has been shown that separase cleaves kendrin at the centrosome in late mitosis, which enables centriole disengagement and thereby centriole duplication at the beginning of the following S phase (Matsuo,2012).
Very little is known about function and regulation of separases in plants (Moschou,2012). But it has already been demonstrated that the meiotic cohesin subunit Rec8 homolog in Arabidopsis thaliana, called SYN1/DIF1, is cleaved by AESP (Arabidopsis homolog of separase) (Liu,2006).
o 5 selected references:

o 9 GO-Terms:

o 4 Instances for CLV_Separin_Fungi
(click table headers for sorting; Notes column: =Number of Switches, =Number of Interactions)
Acc., Gene-, NameStartEndSubsequenceLogic#Ev.OrganismNotes
P30776 rad21
226 232 DNQSQISIEVGRDAPAAAAT TP 3 Schizosaccharomyces pombe 972h-
Q12188 REC8
448 454 SSSSTRSHEYGRKSFRNNKN TP 3 Saccharomyces cerevisiae S288c
Q12158 MCD1
175 181 AAPWDTSLEVGRRFSPDEDF TP 5 Saccharomyces cerevisiae S288c
Q12158 MCD1
263 269 NDDDDNSVEQGRRLGESIMS TP 3 Saccharomyces cerevisiae S288c
Please cite: ELM 2016-data update and new functionality of the eukaryotic linear motif resource. (PMID:26615199)

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