Accurate mitosis is coordinated by the spindle assembly checkpoint (SAC) through the mitotic checkpoint complex (MCC), which inhibits the anaphase-promoting complex or cyclosome (APC/C). As an essential regulator, Cdc20 promotes mitotic exit through activating APC/C and monitors kinetochore-microtubule attachment through activating SAC. Cdc20 requires multiple interactions with APC/C and MCC subunits to elicit these functions. Functionally assessing these interactions within cells requires efficient depletion of endogenous Cdc20, which is highly difficult to achieve by RNA interference (RNAi). Here we generated Cdc20 RNAi-sensitive cell lines which display a penetrant metaphase arrest by a single RNAi treatment. In this null background, we accurately measured the contribution of each known motif of Cdc20 on APC/C and SAC activation. The CRY box, a previously identified degron, was found critical for SAC by promoting MCC formation and its interaction with APC/C. These data reveal additional regulation within the SAC and establish a novel method to interrogate Cdc20.

准确的有丝分裂由纺锤体组装检查点 （SAC） 通过有丝分裂检查点复合物 （MCC） 协调，MCC 抑制后期促进复合物或周期体 （APC/C）。作为一种必需的调节因子，Cdc20 通过激活 APC/C 促进有丝分裂退出，并通过激活 SAC 监测着丝粒-微管附着。Cdc20 需要与 APC/C 和 MCC 亚基的多次相互作用才能引发这些功能。从功能上评估细胞内的这些相互作用需要有效消耗内源性 Cdc20，而 RNA 干扰 （RNAi） 很难实现这一点。在这里，我们生成了 Cdc20 RNAi 敏感细胞系，其通过单一 RNAi 处理显示出渗透性中期阻滞。在这个零背景下，我们准确测量了 Cdc20 的每个已知基序对 APC/C 和 SAC 激活的贡献。CRY 盒是先前确定的 degron，被发现通过促进 MCC 形成及其与 APC/C 的相互作用对 SAC 至关重要。这些数据揭示了 SAC 内的额外调节，并建立了一种询问 Cdc20 的新方法。