Regulation of Separase in meiosis: Separase is activated at the metaphase I-II transition in Xenopus oocytes during meiosis.

Separase is a cysteine protease conserved in all eukaryotes and functions to remove sister chromatid cohesion in anaphase by cleaving the SCC1 subunit of the cohesin complex. Separase activity is regulated by its inhibitor securin and by an inhibitory phosphorylation in vertebrates. However, these regulations have never been directly investigated in the meiotic cell cycle of vertebrates. In this study, we cloned the full-length gene encoding Xenopus separase from an oocyte cDNA library. Purified xSeparase can cleave the human alpha-kleisin subunit of cohesin in vitro but cannot bind to hSecurin when these two proteins are coexpressed in 293T cells. Similar to its human counterpart, xSeparase cleaves itself upon activation but at a single site. The cleavage site is conserved with one of the three self-cleavage sites in hSeparase. Using self-cleavage as a reporter for its activation, we demonstrated that xSeparase is transiently activated between the two meioses and may be involved in homolog separation, as is observed in other organisms. Taking advantage of the inability of xSecurin to interact with hSeparase, we demonstrated that CSF extract can reinhibit both full-length and auto-cleaved hSeparase, indicating that inhibition of separase by phosphorylation does occur under physiological conditions. In addition, we found that endogenous xSecurin accumulated in response to progesterone-induced oocyte maturation and was degraded at both anaphase I and II in an APC/C-dependent manner.