Unit of Biochemistry, The Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel.
Proc Natl Acad Sci U S A. 2013 Jun 25;110(26):10568-73. doi: 10.1073/pnas.1308928110. Epub 2013 Jun 10.
The mitotic (or spindle assembly) checkpoint system prevents premature separation of sister chromatids in mitosis. When the checkpoint is turned on, the mitotic checkpoint complex (MCC) inhibits the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C). MCC is composed of the checkpoint proteins BubR1, Bub3, and Mad2 associated with the APC/C activator Cdc20. The mechanisms of the assembly of MCC when the checkpoint is turned on, and of its disassembly when the checkpoint is inactivated, are not sufficiently understood. Previous reports indicated that APC/C-mediated polyubiquitylation of Cdc20 in MCC is required for the dissociation of APC/C-associated MCC, but not of free MCC. The pool of free MCC is disassembled by an ATP-dependent process stimulated by the Mad2-binding protein p31(comet). It remained unknown whether free MCC is the precursor or the dissociation product of APC/C-bound MCC. By characterizing the mechanisms of the disassembly of APC/C-bound MCC in a purified system, we find that it cannot be the source of free MCC, because it is bound at high affinity and is released only in ubiquitylated or partially disassembled forms. By the use of a cell-free system from Xenopus eggs that reproduces the mitotic checkpoint, we show that MCC can be assembled in the absence of APC/C in a checkpoint-dependent manner. We propose that when the checkpoint is turned on, free MCC is the precursor of APC/C-bound MCC. When the mitotic checkpoint is extinguished, both APC/C-bound and free MCC pools have to be disassembled to release APC/C from inhibition.
有丝分裂（或纺锤体组装）检查点系统可防止姐妹染色单体在有丝分裂中过早分离。当检查点开启时，有丝分裂检查点复合物（MCC）会抑制泛素连接酶后期促进复合物/环体（APC/C）。MCC 由与 APC/C 激活子 Cdc20 相关的检查点蛋白 BubR1、Bub3 和 Mad2 组成。当检查点开启时 MCC 组装的机制，以及当检查点失活时 MCC 解体的机制，尚未得到充分理解。以前的报告表明，MCC 中 APC/C 介导的 Cdc20 多泛素化对于 APC/C 相关 MCC 的解离是必需的，但对于游离 MCC 则不是必需的。游离的 MCC 池通过 Mad2 结合蛋白 p31(comet) 刺激的 ATP 依赖性过程进行解体。尚不清楚游离的 MCC 是 APC/C 结合的 MCC 的前体还是解离产物。通过在纯化系统中表征 APC/C 结合的 MCC 解体的机制，我们发现它不可能是游离 MCC 的来源，因为它以高亲和力结合，并且仅以泛素化或部分解体的形式释放。通过使用复制有丝分裂检查点的非洲爪蟾卵的无细胞系统，我们表明 MCC 可以在没有 APC/C 的情况下以检查点依赖的方式组装。我们提出，当检查点开启时，游离的 MCC 是 APC/C 结合的 MCC 的前体。当有丝分裂检查点熄灭时，APC/C 结合和游离的 MCC 池都必须解体，以释放 APC/C 以解除抑制。
