Interactions between Mad1p and the nuclear transport machinery in the yeast Saccharomyces cerevisiae
Interactions between Mad1p and the nuclear transport machinery in the yeast Saccharomyces cerevisiae
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DOI：
10.1091/mbc.e05-01-0011
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发表时间：
2005-09-01
影响因子：
3.3
通讯作者：
Wozniak, RW
中科院分区：
文献类型：
--
作者：
Scott, RJ;Lusk, CP;Wozniak, RW
除了在核质运输中的作用外，核孔复合体 (NPC) 还充当其表面主要细胞功能与核运输无关的蛋白质的对接位点，包括 Mad1p 和 Mad2p，这是纺锤体组装检查点 (SAC) 机制的两种蛋白质。为了理解这种关系，我们绘制了酿酒酵母 Mad1p 与核运输机制相互作用的结构域，包括进一步定义其与 NPC 的相互作用。我们表明，位于 Mad1p C 末端三分之一的 Kap95p/Kap6Op 依赖性核定位信号是其有效靶向 NPC 所必需的。在 NPC 中，Mad1p 通过其 N 末端内的两个卷曲线圈区域与 Nup53p 和假定的 Nup60p/Mlp1p/Mlp2p 复合物相互作用。当 SAC 被激活时，Mad1p 的一部分通过由 Mad1p C 末端区域介导的相互作用被招募到着丝粒，并且需要能量。我们使用光漂白分析表明，在诺考达唑抑制的细胞中，Mad1p 在 M1p 蛋白和着丝粒之间快速循环。我们的进一步分析还表明，SAC 功能仅需要 Mad1p 的 C 末端，并且 NPC 通过 Nup53p 可能起到调节 SAC 反应持续时间的作用。
In addition to its role in nucleocytoplasmic transport, the nuclear pore complex (NPC) acts as a docking site for proteins whose apparent primary cellular functions are unrelated to nuclear transport, including Mad1p and Mad2p, two proteins of the spindle assembly checkpoint (SAC) machinery. To understand this relationship, we have mapped domains of yeast Saccharomyces cerevisiae Mad1p that interact with the nuclear transport machinery, including further defining its interactions with the NPC. We showed that a Kap95p/Kap6Op-dependent nuclear localization signal, positioned in the C-terminal third of Mad1p, is required for its efficient targeting to the NPC. At the NPC, Mad1p interacts with Nup53p and a presumed Nup60p/Mlp1p/Mlp2p complex through two coiled coil regions within its N terminus. When the SAC is activated, a portion of Mad1p is recruited to kinetochores through an interaction that is mediated by the C-terminal region of Mad1p and requires energy. We showed using photobleaching analysis that in nocodazole-arrested cells Mad1p rapidly cycles between the M1p proteins and kinetochores. Our further analysis also showed that only the C terminus of Mad1p is required for SAC function and that the NPC, through Nup53p, may act to regulate the duration of the SAC response.