Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester, M13 9PT, UK.
J Cell Sci. 2011 Nov 15;124(Pt 22):3905-16. doi: 10.1242/jcs.093286. Epub 2011 Nov 18.
Accurate chromosome segregation requires the spindle assembly checkpoint to be active at the onset of mitosis, before being silenced following chromosome alignment. p31(comet) is a checkpoint antagonist in that its inhibition delays mitotic exit, whereas its overexpression overrides the checkpoint. How exactly p31(comet) antagonises the checkpoint is unclear. A prevalent model is that p31(comet) acts as a 'cap' by inhibiting recruitment of the open conformation form of Mad2 (O-Mad2) to the kinetochore-bound complex of Mad1-C-Mad2 (closed conformation Mad2), an essential step that is required for checkpoint activation. Here, we show that although p31(comet) localises to kinetochores in mitosis, modulation of its activity has no effect on recruitment of O-Mad2 to kinetochores. Rather, our observations support a checkpoint-silencing role for p31(comet) downstream of kinetochores. We show that p31(comet) binds Mad2 when it is bound to the mitotic checkpoint complex (MCC) components BubR1 and Cdc20. Furthermore, RNAi-mediated inhibition of p31(comet) results in more Mad2 bound to BubR1-Cdc20, and conversely, overexpression of p31(comet) results in less Mad2 bound to BubR1-Cdc20. Addition of recombinant p31(comet) to checkpoint-arrested extracts removes Mad2 from the MCC, whereas a p31(comet) mutant that cannot bind Mad2 has no effect. Significantly, expression of a Mad2 mutant that cannot bind p31(comet) prolongs the metaphase to anaphase transition. Taken together, our data support the notion that p31(comet) negatively regulates the spindle assembly checkpoint by extracting Mad2 from the MCC.
准确的染色体分离需要纺锤体组装检查点在有丝分裂开始时活跃，然后在染色体对齐后沉默。p31（彗星）是检查点的拮抗剂，因为它的抑制作用会延迟有丝分裂的退出，而其过度表达则会忽略检查点。p31（彗星）究竟如何拮抗检查点尚不清楚。一种流行的模型是，p31（彗星）通过抑制 Mad2 的开放构象形式（O-Mad2）与 Mad1-C-Mad2（封闭构象 Mad2）结合复合物的募集来发挥“帽子”作用，这是检查点激活所必需的。在这里，我们表明，尽管 p31（彗星）在有丝分裂时定位于动粒，但调节其活性对 O-Mad2 向动粒的募集没有影响。相反，我们的观察结果支持 p31（彗星）在动粒下游发挥检查点沉默作用。我们表明，p31（彗星）与有丝分裂检查点复合物（MCC）成分 BubR1 和 Cdc20 结合时与 Mad2 结合。此外，RNAi 介导的 p31（彗星）抑制导致更多的 Mad2 与 BubR1-Cdc20 结合，反之亦然，p31（彗星）的过表达导致更少的 Mad2 与 BubR1-Cdc20 结合。将重组 p31（彗星）添加到检查点停滞的提取物中会将 Mad2 从 MCC 中去除，而不能与 Mad2 结合的 p31（彗星）突变体则没有影响。重要的是，表达不能与 p31（彗星）结合的 Mad2 突变体延长了从有丝分裂中期到后期的过渡。总之，我们的数据支持了 p31（彗星）通过从 MCC 中提取 Mad2 来负调控纺锤体组装检查点的观点。
