| .用酵母双杂交系统筛选CENP-E相互作用蛋白 |
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| 引用本文： | 都 建）,陈立建）,沈继龙）,花沙沙）,姚雪彪）. .用酵母双杂交系统筛选CENP-E相互作用蛋白[J]. 中国生物化学与分子生物学报, 2009, 25(8): 719-726 |
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| 作者姓名： | 都 建） 陈立建） 沈继龙） 花沙沙） 姚雪彪） |
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| 作者单位： | 1）安徽医科大学基础医学院生化与分子生物学教研室,合肥230032;2）安徽医科大学第一附属医院麻醉科,合肥230032;3）安徽医科大学病原生物学教研室,合肥230032;4）中国科学技术大学细胞动力学实验室,合肥230027） |
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| 摘 要： | 纺锤体检验点(spindle checkpoint)是一个重要的细胞分裂生化调节通路, 可监督染色体正确分离和传代．着丝粒相关蛋白E (centromere-associated protein E, CENP-E)是一个分子量为312 kD的微管马达驱动蛋白，可以衔接纺锤体微管与动点并参与纺锤体检验点调控．为研究CENP-E的作用机理,以其动点结合区域为诱饵蛋白,用酵母双杂交技术从人HeLa细胞 cDNA 文库中筛选出了Nuf2蛋白．体外的pull-down实验和体内的免疫共沉淀实验表明, Nuf2蛋白通过其卷曲螺旋(coiled-coil) 功能域特异结合CENP-E的 C 末端区域，间接免疫荧光显示Nuf2与CENP-E共定位于细胞有丝分裂期染色体的动点．由此推论, CENP-E 通过Nuf2的直接作用参与构筑动点-微管界面，进而参与细胞有丝分裂纺锤体检验点信号转导通路，为染色体正确分离发挥调控作用．
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| 关 键 词： | 酵母双杂交 着丝粒相关蛋白E(CENP-E) 马达驱动蛋白 |
| 收稿时间： | 2009-02-24 |
Screening Proteins that Interact with Centromere-associated Protein E viaYeast Two-hybrid System |
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| DU Jian),CHEN Li-Jian),SHEN Ji-Long),HUA Sha-Sha),YAO Xue-Biao). Screening Proteins that Interact with Centromere-associated Protein E viaYeast Two-hybrid System[J]. Chinese Journal of Biochemistry and Molecular Biology, 2009, 25(8): 719-726 |
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| Authors: | DU Jian) CHEN Li-Jian) SHEN Ji-Long) HUA Sha-Sha) YAO Xue-Biao) |
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| Affiliation: | 1) Department of Biochemistry and Molecular Biology, Anhui Medical University, Hefei 230032, China; 2) Department of Anesthesiology, First Affiliated Hospital of Anhui Medical University, Hefei 230032, China; 3) Department of Microbiology and Parasitology , Anhui Medical University, Hefei 230032, China; 4) Laboratory of Cellular Dynamics, University of Science and Technology of China, National Laboratory for Physical Sciences, Hefei 230027, China) |
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| Abstract: | This study is to isolate and identify proteins that interact with centromere-associated protein E(CENP-E), providing new clues for exploring the function of CENP-E in cell cycle control and the pathogenesis of tumor. Yeast two hybrid screening (MATCH-MAKER GAL4 Two Hybrid System 3) and regular molecular biologic techniques were used to screen human HeLa cDNA library with the kinetochore binding domain of CENP-E. Thebait from the C-terminus of CENP-E was created by subcloned methods to find out optimal candidate proteins that interact with the kinetochore binding domain of CENP-E. Six novel CENP-E interacting proteins including Homo sapiensNuf2 were obtained. The interaction was confirmed by in vitro of glutathione S-transferase pull-down and in vivo co-immunoprecipitation. In yeast two-hybrid assay, the last coiled-coil domain of Nuf2 was found to be necessary and sufficient for the interaction with the C-terminus of CENP-E. In the kinetochore of human HeLa cell Nuf2 co-localized with the endogenous CENP-E. Our finding of the interaction of CENP-E with Nuf2 demonstrates that CENP-E and Nuf2 may play important roles in the functional regulation of microtubule-kinetochore attachment and spindle checkpoint pathway.Screening Proteins that Interact with Centromere-associated Protein E via Yeast Two-hybrid System |
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| Keywords: | yeast-two-hybrid centromere-associated protein E(CENP-E) kinesin-related motor protein mitosis |
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