Mad2 and Mad3 cooperate to arrest budding yeast in mitosis.
Mad2 and Mad3 cooperate to arrest budding yeast in mitosis.
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DOI：
10.1016/j.cub.2011.12.029
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发表时间：
2012-02-07
期刊：
影响因子：
--
通讯作者：
Murray AW
中科院分区：
文献类型：
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作者：
Lau DT;Murray AW
纺锤体检查点通过延迟染色体分离确保准确的染色体传递，直到所有染色体在有丝分裂纺锤体上正确排列。检查点由未附着于微管或附着但不受张力的着丝点激活，并通过抑制后期促进复合体（APC）及其共激活物Cdc20在中期阻止细胞。尽管进行了大量的研究，但我们仍然不清楚检查点蛋白如何相互协调以抑制APCCdc20的活性。为了了解检查点成分是如何诱导中期停滞的，我们构建了检查点蛋白的融合体，并在出芽酵母（Saccharomyces cerevisiae）中表达它们，以模拟检查点激活过程中可能的蛋白质相互作用。我们发现Mad2-Mad3蛋白融合或非共价连接的Mad2和Mad3的表达，而不是两个单独蛋白的过表达，诱导独立于功能着丝点或其他检查点蛋白的中期阻滞。我们进一步表明，人工地将Mad2与Cdc20捆绑在一起也可以独立于其他检查点成分在中期阻止细胞。我们的研究结果表明，Mad3是体内Mad2与Cdc20稳定结合所必需的，这足以抑制APC活性，并且是纺锤体检查点激活中最下游的事件。
The spindle checkpoint ensures accurate chromosome transmission by delaying chromosome segregation until all chromosomes are correctly aligned on the mitotic spindle. The checkpoint is activated by kinetochores that are not attached to microtubules or are attached but not under tension and arrests cells at metaphase by inhibiting the anaphase-promoting complex (APC) and its co-activator Cdc20. Despite numerous studies, we still do not understand how the checkpoint proteins coordinate with each other to inhibit APCCdc20 activity. To ask how the checkpoint components induce metaphase arrest, we constructed fusions of checkpoint proteins and expressed them in the budding yeast, Saccharomyces cerevisiae, to mimic possible protein interactions during checkpoint activation. We found that expression of a Mad2-Mad3 protein fusion or non-covalently linked Mad2 and Mad3, but not the overexpression of the two separate proteins, induces metaphase arrest that is independent of functional kinetochores or other checkpoint proteins. We further showed that artificially tethering Mad2 to Cdc20 also arrests cells in metaphase independently of other checkpoint components. Our results suggest that Mad3 is required for the stable binding of Mad2 to Cdc20 in vivo, which is sufficient to inhibit APC activity and is the most downstream event in spindle checkpoint activation.