The pseudo GTPase CENP-M drives human kinetochore assembly.
The pseudo GTPase CENP-M drives human kinetochore assembly.
复制标题
伪GTPASE CENP-M驱动人类动物组件。
DOI：
10.7554/elife.02978
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发表时间：
2014-07-08
期刊：
影响因子：
7.7
通讯作者：
Musacchio A
中科院分区：
文献类型：
--
作者：
Basilico F;Maffini S;Weir JR;Prumbaum D;Rojas AM;Zimniak T;De Antoni A;Jeganathan S;Voss B;van Gerwen S;Krenn V;Massimiliano L;Valencia A;Vetter IR;Herzog F;Raunser S;Pasqualato S;Musacchio A
着丝粒是一种多亚基复合体，在与着丝粒的界面处组装，结合纺锤体微管以确保在细胞分裂期间染色体的忠实递送。着丝粒-着丝粒界面的结构和功能知之甚少。我们报告说，在这个接口，CENP-M，蛋白质的结构和进化相关的小GTP酶，但不能GTP结合和构象转换。我们表明，CENP-M是至关重要的四聚体的组装和稳定性，也包括CENP-I，CENP-H，和CENP-K，HIKM复合物，我们通过结构，生物化学和细胞生物学方法的组合广泛表征。影响CENP-M/CENP-I相互作用的点突变体阻碍动粒组装和染色体对齐，并阻止CENP-T/W复合物的动粒募集，质疑CENP-T/W作为动粒组装独立轴的创始人的作用。我们的研究确定了一个单一的途径，CENP-C作为创始人，CENP-H/I/K/M和CENP-T/W作为CENP-C依赖的追随者。DOI：http://dx.doi.org/10.7554/eLife.02978.001当人类细胞分裂产生新细胞时，它的46条染色体必须复制，然后在两个子细胞之间均匀分离。分离的过程是由纺锤体完成的，纺锤体是在细胞内形成的纤维网络，附着在染色体上，将复制品拉到细胞的两端。纺锤体纤维附着在一个叫做动粒的结构上，动粒形成在染色体的一个叫做着丝粒的区域。着丝粒具有多层结构，含有许多蛋白质的多个拷贝，内层由至少16个着丝粒蛋白质组成。这些蛋白质直接与着丝粒相互作用，并影响其余动粒和纺锤体纤维的形成。虽然一些着丝粒蛋白之间的相互作用已经被发现，其中几个的作用，包括一个称为CENP-M仍然未知。现在，Basilico，Maffini，Weir等人揭示了CENP-M对于组装和稳定动粒内层至关重要。然而，虽然它在结构上和进化上与称为GTP酶的酶有关，但CENP-M不是酶。相反，CENP-M蛋白与其他三种着丝粒蛋白相互作用，形成复合物，成为动粒内层的一部分。Basilico，Maffini，Weir等人还发现另一种着丝粒蛋白CENP-C似乎启动了内层的组装。然后，这种蛋白质将由其他着丝粒蛋白质组成的两个复合物招募到动粒，包括含有CENP-M的复合物。下一个挑战将是重组包含更多来自动粒内层的蛋白质的更大的蛋白质复合物，以便可以更详细地研究这些组装。研究CENP-C如何作为骨架组织动粒和着丝粒之间的界面也将是重要的。DOI：http://dx.doi.org/10.7554/eLife.02978.002网站
Kinetochores, multi-subunit complexes that assemble at the interface with centromeres, bind spindle microtubules to ensure faithful delivery of chromosomes during cell division. The configuration and function of the kinetochore–centromere interface is poorly understood. We report that a protein at this interface, CENP-M, is structurally and evolutionarily related to small GTPases but is incapable of GTP-binding and conformational switching. We show that CENP-M is crucially required for the assembly and stability of a tetramer also comprising CENP-I, CENP-H, and CENP-K, the HIKM complex, which we extensively characterize through a combination of structural, biochemical, and cell biological approaches. A point mutant affecting the CENP-M/CENP-I interaction hampers kinetochore assembly and chromosome alignment and prevents kinetochore recruitment of the CENP-T/W complex, questioning a role of CENP-T/W as founder of an independent axis of kinetochore assembly. Our studies identify a single pathway having CENP-C as founder, and CENP-H/I/K/M and CENP-T/W as CENP-C-dependent followers. DOI: http://dx.doi.org/10.7554/eLife.02978.001 When a human cell divides to make new cells, its 46 chromosomes must be replicated and then separated evenly between the two daughter cells. The process of separation is performed by the spindle—a network of fibres that form inside the cell, attach to the chromosomes and pull the copies to the opposite ends of the cell. The spindle fibres attach to a structure called a kinetochore, which forms at a region of the chromosomes called the centromere. The kinetochore has a layered structure with multiple copies of many proteins, and the inner layer is composed of at least 16 centromeric proteins. These proteins interact directly with the centromere and influence the formation of the rest of the kinetochore and the spindle fibres. While some of the interactions between centromeric proteins have been uncovered, the roles of several of them—including one called CENP-M—remain unknown. Now, Basilico, Maffini, Weir et al. reveal that CENP-M is essential for assembling and stabilizing the inner layer of the kinetochore. However, while it is structurally and evolutionarily related to enzymes called GTPases, CENP-M is not an enzyme. Instead, the CENP-M protein interacts with three other centromeric proteins to form a complex that becomes part of the inner layer of the kinetochore. Basilico, Maffini, Weir et al. also find that another centromeric protein, CENP-C, appears to start the assembly of the inner layer. This protein then recruits two complexes made of other centromeric proteins to the kinetochore, including the complex that contains CENP-M. The next challenge will be to reconstitute larger protein complexes that contain more proteins from the inner layer of the kinetochore, so that these assemblies can be studied in greater detail. It will also be important to investigate how CENP-C acts as a scaffold to organize the interface between the kinetochore and the centromere. DOI: http://dx.doi.org/10.7554/eLife.02978.002