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 the sisterchromatid cohesion in anaphase by cleaving the SCC1 subunit of the cohesin complex. Theregulation of separase activity as the degradation of its inhibitor, securin, and the downregulationof the inhibitory phosphorylation has never been directly investigated in themeiotic cell cycle of vertebrates. In this study, we cloned the full-length gene encodingXenopus separase from an oocyte cDNA library. Purified xSeparase can cleave the human ?-kleisin subunit of cohesin in vitro but cannot bind with hSecurin when these two proteins areco-expressed in 293T cells. Similar to its human counterpart, xSeparase cleaves itself uponactivation but at a single site. The cleavage site is conserved with one of the three selfcleavagesites in hSeparase. Using self-cleavage as a reporter for its activation, wedemonstrated that xSeparase was transiently activated between the two meioses and may beinvolved in the homologous chromosome separation, as observed in other organisms. Takingthe advantage of the inability of xSecurin to interact with hSeparase, we demonstrated that theCSF extract re-inhibit both full-length and auto-cleaved hSeparase, indicating thatphosphorylation inhibition of separase does occur under the physiological condition. Inaddition, we found that the endogenous xSecurin was accumulated in response toprogesterone-induced oocyte maturation, and was degraded at both the anaphase I and II in anAPC/C-dependent manner.