CENP-O class proteins form a stable complex and are required for proper kinetochore function
CENP-O class proteins form a stable complex and are required for proper kinetochore function
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DOI：
10.1091/mbc.e07-06-0556
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发表时间：
2008-03-01
影响因子：
3.3
通讯作者：
Fukagawa, Tatsuo
中科院分区：
文献类型：
--
作者：
Hori, Tetsuya;Okada, Masahiro;Fukagawa, Tatsuo
我们以前确定了一个多亚基复合物（CENP-H/I复合物）在人和鸡细胞的动粒。我们发现，CENP-H/I复合物分为三个功能类。在本研究中，我们研究了CENP-O类蛋白，包括CENP-O，-P，-Q，-R和-50（U）。我们创建了这些蛋白质的鸡DT 40细胞敲除，我们发现所有敲除细胞系都是可行的，但它们表现出缓慢的增殖和有丝分裂缺陷。CENP-O、-P、-Q和-50的动粒定位是相互依赖的，但在CENP-R缺陷细胞中观察到这些蛋白质的动粒定位。在细菌中的共表达测定显示CENP-0、-P、-Q和-50蛋白形成可与CENP-R缔合的稳定复合物。敲除细胞的表型分析表明，除了CENP-R之外的所有蛋白质都是从纺锤体损伤中恢复所需的，并且CENP-50的磷酸化对于从纺锤体损伤中恢复是必需的。我们还发现，用蛋白酶体抑制剂MG 132处理部分挽救了在CENP-50缺陷细胞中响应于诺考达唑阻断释放而观察到的严重有丝分裂表型。这表明CENP-O类蛋白质参与防止纺锤体损伤恢复期间过早的姐妹染色单体分离。
We previously identified a multisubunit complex (CENP-H/I complex) in kinetochores from human and chicken cells. We showed that the CENP-H/I complex is divided into three functional classes. In the present study, we investigated CENP-O class proteins, which include CENP-O, -P, -Q, -R, and -50 (U). We created chicken DT40 cell knockouts of each of these proteins, and we found that all knockout lines were viable, but that they showed slow proliferation and mitotic defects. Kinetochore localization of CENP-O, -P, -Q, and -50 was interdependent, but kinetochore localization of these proteins was observed in CENP-R -deficient cells. A coexpression assay in bacteria showed that CENP-O, -P, -Q, and -50 proteins form a stable complex that can associate with CENP-R. Phenotype analysis of knockout cells showed that all proteins except for CENP-R were required for recovery from spindle damage, and phosphorylation of CENP-50 was essential for recovery from spindle damage. We also found that treatment with the proteasome inhibitor MG132 partially rescued the severe mitotic phenotype observed in response to release from nocodazole block in CENP-50-deficient cells. This suggests that CENP-O class proteins are involved in the prevention of premature sister chromatid separation during recovery from spindle damage.