Separation of eukaryotic sister chromatids during the cell cycle is timed by the spindle assembly checkpoint (SAC) and ultimately triggered when separase cleaves cohesion-mediating cohesin^1,2,3. Silencing of the SAC during metaphase activates the ubiquitin ligase APC/C (anaphase-promoting complex, also known as the cyclosome) and results in the proteasomal destruction of the separase inhibitor securin¹. In the absence of securin, mammalian chromosomes still segregate on schedule, but it is unclear how separase is regulated under these conditions^4,5. Here we show that human shugoshin 2 (SGO2), an essential protector of meiotic cohesin with unknown functions in the soma^6,7, is turned into a separase inhibitor upon association with SAC-activated MAD2. SGO2–MAD2 can functionally replace securin and sequesters most separase in securin-knockout cells. Acute loss of securin and SGO2, but not of either protein individually, resulted in separase deregulation associated with premature cohesin cleavage and cytotoxicity. Similar to securin^8,9, SGO2 is a competitive inhibitor that uses a pseudo-substrate sequence to block the active site of separase. APC/C-dependent ubiquitylation and action of the AAA-ATPase TRIP13 in conjunction with the MAD2-specific adaptor p31^comet liberate separase from SGO2–MAD2 in vitro. The latter mechanism facilitates a considerable degree of sister chromatid separation in securin-knockout cells that lack APC/C activity. Thus, our results identify an unexpected function of SGO2 in mitotically dividing cells and a mechanism of separase regulation that is independent of securin but still supervised by the SAC.

细胞周期中真核姐妹染色单体的分离由纺锤体组装检查点 (SAC) 计时，并最终在分离酶裂解粘连介导的粘连蛋白^1,2,3时触发。中期 SAC 的沉默会激活泛素连接酶 APC/C（后期促进复合物，也称为环体），并导致分离酶抑制剂 securin ¹的蛋白酶体破坏。在没有 securin 的情况下，哺乳动物染色体仍然按计划分离，但尚不清楚在这些条件下如何调节分离酶^4,5 。在这里，我们表明，人 shugoshin 2 (SGO2) 是减数分裂粘连蛋白的重要保护剂，在体细胞中功能未知^6,7 ，在与 SAC 激活的 MAD2 结合后转变成分离酶抑制剂。 SGO2-MAD2 可以在功能上替代 securin，并隔离 securin 敲除细胞中的大多数分离酶。 securin 和 SGO2 的急性缺失（但不是其中任何一种蛋白质单独缺失）会导致与过早粘连蛋白裂解和细胞毒性相关的分离酶失调。与 securin ^8,9类似，SGO2 是一种竞争性抑制剂，它使用伪底物序列来阻断分离酶的活性位点。 APC/C 依赖性泛素化和 AAA-ATPase TRIP13 与 MAD2 特异性接头 p31^彗星结合的作用在体外从 SGO2-MAD2 中释放分离酶。后一种机制有利于缺乏 APC/C 活性的 securin 敲除细胞中相当程度的姐妹染色单体分离。因此，我们的结果确定了 SGO2 在有丝分裂细胞中的意外功能，以及独立于 securin 但仍受 SAC 监督的分离酶调节机制。