Analysis of the molecular organization of nuclear pore complexes using nuclear transport factor, importin β

使用核转运因子 importin β 分析核孔复合物的分子组织

基本信息

批准号：
12480215
负责人：
YONEDA Yoshihiro
金额：
$ 10.18万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12480215/
关键词：
importin β nuclear protein transport nuclear pore complex X-ray crystallography nucleoporin importin β importin α Ran

项目摘要

In eukaryotic cells, nucleo-cytoplasmic transport of molecules is crucial for intracellular signal transduction. However, it remains unknown how proteins traverse nuclear pores directionally. In this study, in order to elucidate the molecular organization of nuclear pore complexes (NPCs), we focused on importin β, which is able to translocate through the nuclear pores in both directions by itself. The structure of the uncomplexed form of the N-terminal fragment of importin β has been solved by X-ray crystallography. Based on this structural analysis, we constructed mutant importin β proteins, in which amino acid residues protruding from molecular surface of the convex side of helices (helix A) or the concave side of helices (helix B) of the pore-binding domain are substituted with alanines. When microinjected into the cytoplasm or the nucleus of cultured cells, recombinant 4B5B mutant proteins, in which the helices B4 and B5 are mutated, significantly accumulated in the nucleus compared with the wild type. In contrast, the nuclear accumulation of recombinant 5A6A mutant proteins, in which the helices A5 and A6 are mutated, dramatically decreased. The same results were obtained by using a permeabilized cell in vitro transport assay. In addition, we found that the ability of 5A6A mutants to import the NLS-substrates into the nucleus decreased, which means that the transport ability of importin β is parallel to its own migration activity. In the future, we will be able to identify key NPC components (nucleoporins) for directional movement of proteins through nuclear pores by analyzing the affinity of mutant importin β proteins with each nucleoporin.

在真核细胞中，在这项研究中，在核心孔复合物（NPC）的组织中，对细胞内信号转导的分子的核整合转运。 X射线晶体学的结构。孔结合结构域的螺旋（Helix a）螺旋侧（螺旋B）用丙氨酸取代，或者是培养细胞的核。与野生类型相比，重组5A6A突变蛋白的核积累，其中螺旋A5和A6被静音，通过在体外运输测定中使用透化细胞获得相同的结果突变体将NLS囊核导入核降低的转运能力与其自身的迁移活性平行，或者通过蛋白质通过mutantinβ蛋白与每个核孔蛋白的亲和力在核孔中的方向运动。