Walczak Claire E, Gan Eugene C, Desai Arshad, Mitchison Timothy J, Kline-Smith Susan L
Medical Sciences, Indiana University, Bloomington, IN 47405, USA.
Curr Biol. 2002 Oct 29;12(21):1885-9. doi: 10.1016/s0960-9822(02)01227-7.
Xenopus kinesin catastrophe modulator-1 (XKCM1) is a Kin I kinesin family member that uses the energy of ATP hydrolysis to depolymerize microtubules. We demonstrated previously that XKCM1 is essential for mitotic-spindle assembly in vitro and acts by regulating microtubule dynamics as a pure protein, in extracts and in cells. A portion of the XKCM1 pool is specifically localized to centromeres during mitosis and may be important in chromosome movement. To selectively analyze the function of centromere-bound XKCM1, we generated glutathione-S-transferase (GST) fusion proteins containing the N-terminal globular domain (GST-NT), the centrally located catalytic domain (GST-CD), and the C-terminal alpha-helical tail (GST-CT) of XKCM1. The GST-NT protein targeted to centromeres during spindle assembly, suggesting that the N-terminal domain of XKCM1 is sufficient for centromere localization. Addition of GST-NT prior to or after spindle assembly replaced endogenous XKCM1, indicating that centromere targeting is a dynamic process. Loss of endogenous XKCM1 from centromeres caused a misalignment of chromosomes on the metaphase plate without affecting global spindle structure. These results suggest that centromere bound XKCM1 has an important role in chromosome positioning on the spindle.
非洲爪蟾驱动蛋白灾难调节因子1（XKCM1）是一种Kin I驱动蛋白家族成员，它利用ATP水解产生的能量使微管解聚。我们之前证明，XKCM1在体外对有丝分裂纺锤体组装至关重要，并且在提取物和细胞中作为一种纯蛋白通过调节微管动力学发挥作用。一部分XKCM1在有丝分裂期间特异性定位于着丝粒，可能在染色体移动中起重要作用。为了选择性分析着丝粒结合的XKCM1的功能，我们生成了包含XKCM1的N端球状结构域（GST-NT）、位于中央的催化结构域（GST-CD）和C端α螺旋尾（GST-CT）的谷胱甘肽-S-转移酶（GST）融合蛋白。GST-NT蛋白在纺锤体组装期间定位于着丝粒，这表明XKCM1的N端结构域足以实现着丝粒定位。在纺锤体组装之前或之后添加GST-NT替代了内源性XKCM1，这表明着丝粒靶向是一个动态过程。着丝粒上内源性XKCM1的缺失导致染色体在中期板上排列不齐，而不影响整体纺锤体结构。这些结果表明，着丝粒结合的XKCM1在染色体在纺锤体上的定位中起重要作用。
