Separin an endopeptidase necessary for separating chromatids

The budding yeast securin has what appears to be a single stable partner, a 180 kDa protein called Espl (Ciosk et al 1998). In fission yeast, Cut2 had previously been found to be associated with Cut 1, an Espl homologue (Funabiki et al 1996b).

Vertebrate securins are likewise associated with an Esp1 homologue (Zou et al 1999). Esp1/Cut1-like proteins, now known as separins, are found in most if not all eukaryotes. They are usually large proteins, with molecular weights from 180 to 200 kDa, containing a conserved C-terminal 'separin' domain. In budding yeast (Ciosk et al 1998, McGrew et al 1992), fission yeast (Funabiki et al 1996b) and .Aspergillus (May et al 1992), separins are essential for sister chromatid separation. Despite failing to separate sister chromatids, separin mutants proceed with most if not all other aspects of the cell cycle. It has been proposed that separins are dedicated 'sister-separating' proteins whose activity is held in check by their association with securins. According to this hypothesis, the APC mediates sister chromatid separation by liberating separin from its inhibitory embrace by securin (Fig. 5) (Ciosk etal 1998).

A clue to the mechanism by which separin splits sister chromatids was the observation that in budding yeast (contrary to most other eukaryotic cells) most Scc1 remains bound to chromosomes until the metaphase to anaphase transition (Michaelis et al 1997). Scc1's dissociation from chromosomes at the onset of anaphase depends on separin (Ciosk et al 1998) and is accompanied by Scc1's proteolytic cleavage, both in vivo and in vitro (Uhlmann et al 1999). Separin induces Scc1 cleavage at two related sites, each with an arginine in the P1 position. Mutation of either arginine to aspartic acid abolishes cleavage at that site but is not lethal to the cell. However, simultaneous mutation of both sites is lethal and prevents both sister chromatid separation and Scc1's dissociation from chromosomes (Uhlmann et al 1999). Similar potential cleavage sites are found in Rad21, the fission yeast Scc1 homologue, and their simultaneous (but not single) mutation also blocks chromosome segregation (Tomonaga et al 2000). Cleavage of cohesin's Sec1 subunit might therefore be a conserved feature of sister chromatid separation, at least in fungi (Fig. 5).

With the recent addition of several other separins to the databases, the conserved amino acid residues within the separin domain have been clarified. They include a universally conserved histidine and cysteine residue, which is a hallmark of cysteine endopeptidases (Barrett et al 1998). The sequences flanking these two residues are characteristic of cysteine endopeptidases of the CD subclass, which includes caspases, legumains, and two bacterial proteases, gingipain and clostripain (Chen et al 1998). Thus separin might indeed be the protease that cleaves Scc1. Whether cohesin's Scc1 subunit is separin's sole target is presently unclear but certainly possible, for the only other yeast protein to contain good matches to the Scc1/Rad21 consensus is Rec8, a related protein that replaces Scc1 in the cohesin complex during meiosis (Klein et al 1999). It will be crucial to address whether cleavage of Scc1 alone is sufficient to trigger anaphase in yeast and whether sister separation in animal and plant cells also depends on cleavage of cohesion proteins.

Separin's proposed C-terminal catalytic domain depends (at least for in vivo activity) on a long N-terminal domain, which is bound by its inhibitory securin chaperone (Kumada et al 1998). Securin must do more than just inhibit separin, because sister separation fails to occur in cut2 (Funabiki et al 1996b) and pimples (Stratmann & Lehner 1996) mutants and is inefficient in pdsl securin mutants (Ciosk et al 1998). Securin might either target separin to its future sites of action in the cell or help separin adopt a potentially active conformation, which is only unleashed on the cell when securins are destroyed by the APC.

0 0

Post a comment