Friedrich Miescher Laboratory of the Max Planck Society, Tuebingen, Germany.
J Cell Sci. 2012 Oct 15;125(Pt 20):4720-7. doi: 10.1242/jcs.110387. Epub 2012 Jul 23.
The spindle assembly checkpoint (SAC) blocks entry into anaphase until all chromosomes have stably attached to the mitotic spindle through their kinetochores. The checkpoint signal originates from unattached kinetochores, where there is an enrichment of SAC proteins. Whether the enrichment of all SAC proteins is crucial for SAC signaling is unclear. Here, we provide evidence that, in fission yeast, recruitment of the kinase Mph1 is of vital importance for a stable SAC arrest. An Mph1 mutant that eliminates kinetochore enrichment abolishes SAC signaling, whereas forced recruitment of this mutant to kinetochores restores SAC signaling. In bub3Δ cells, the SAC is functional when only Mph1 and the Aurora kinase Ark1, but no other SAC proteins, are enriched at kinetochores. We analyzed the network of dependencies for SAC protein localization to kinetochores and identify a three-layered hierarchy with Ark1 and Mph1 on top, Bub1 and Bub3 in the middle, and Mad3 as well as the Mad1-Mad2 complex at the lower end of the hierarchy. If Mph1 is artificially recruited to kinetochores, Ark1 becomes dispensable for SAC activity. Our results highlight the crucial role of Mph1 at kinetochores and suggest that the Mad1-Mad2 complex does not necessarily need to be enriched at kinetochores for functional SAC signaling.
纺锤体组装检查点 (SAC) 阻止细胞进入后期，直到所有染色体通过其动粒稳定地附着到有丝分裂纺锤体上。检查点信号源自未连接的动粒，那里有 SAC 蛋白的富集。所有 SAC 蛋白的富集是否对 SAC 信号传导至关重要尚不清楚。在这里，我们提供的证据表明，在裂殖酵母中，激酶 Mph1 的募集对于稳定的 SAC 阻滞至关重要。消除动粒富集的 Mph1 突变体消除了 SAC 信号，而将这种突变体强制募集到动粒上则恢复了 SAC 信号。在 bub3Δ 细胞中，当仅在动粒上富集 Mph1 和 Aurora 激酶 Ark1，而没有其他 SAC 蛋白时，SAC 是有功能的。我们分析了 SAC 蛋白在动粒上定位的依赖网络，并确定了一个具有三层结构的层次结构，Ark1 和 Mph1 位于顶部，Bub1 和 Bub3 位于中间，Mad3 以及 Mad1-Mad2 复合物位于层次结构的低端。如果人为地将 Mph1 募集到动粒上，Ark1 对 SAC 活性就不再是必需的。我们的结果强调了 Mph1 在动粒上的关键作用，并表明 Mad1-Mad2 复合物不一定需要在动粒上富集才能进行功能性 SAC 信号传导。
