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Analysis of nuclear pore translocation mechanism : single molecule analysis and biochemical approach

核孔转位机制分析：单分子分析和生化方法

基本信息

批准号：
15370090
负责人：
IMAMOTO Naoko
金额：
$ 9.86万
依托单位：
RIKEN
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15370090/
关键词：
nuclear transport single molecule imaging nuclear pore comlex importin β semi-intact cells 70kDa heat-shock cognate protein Importinβ 低分子量GTPase Ran β-カテニン核局在化シグナル核膜孔複合体構成因子 importin β 輸送担体

项目摘要

Using novel microscopy based on total internal reflection fluorescent microscopy (TIRF), we were able to clearly visualize single fluorescent molecules of green fluorescent protein (GFP)-tagged importin β, a carrier protein, and GFP-tagged cargo protein during transport on the nuclear envelope in both permeabilized cells and in living cells. Image analysis of single nuclear pores showed that two point resolution of 70 nm was achieved. Kinetic parameters of the interactions between translocating molecules and NPCs were obtained through quantitative analysis. Two types of binding were found ; weaker binding sites, which gathers up to 〜100 molecules/NPC, concentrating substrates locally ; and stronger binding sites, where up to 〜8 molecules/NPC are bound. Retention times by single molecule analysis and translocation rates by single nuclear analysis showed a significant correlation with a coefficient of 〜8 molecules/NPC, which exhibits the stoichiometry of transport. We termed weaker binding sites as a "multiplex sites", which is likely to consist from FG-repeat containing nucleoporins. Based on correlation coefficient, we proposed presence of 8 "transit site" in nuclear pore complex. Whether this transit site is equivalent to strong binding sites observed remains to be elucidated in the further study. Whether the findings account for different pathways, which show a wide range of transport rates, also remains to be elucidated. In the parallel experiments, we have purified, and identified 70kDa heat-shock cognate protein (hsc70) as a molecule that facilitates recycling of importin β. The effect of hsc70 was observed, not only in the case of the nuclear export of importin β but also for that of another import receptors, transportin and importin α. These results suggest that hsc70 broadly modulates nucleocytoplasmic transport systems by regulating nuclear export of receptor proteins (Kose et al., J Cell Biol. 2005).

使用基于全内反射荧光显微镜 (TIRF) 的新型显微镜，我们能够清楚地观察透化细胞和活细胞中在核膜上运输过程中绿色荧光蛋白 (GFP) 标记的导入蛋白 β（一种载体蛋白）和 GFP 标记的货物蛋白的单个荧光分子。单核孔图像分析表明，两点分辨率达到了70 nm。通过定量分析获得易位分子与NPCs相互作用的动力学参数。发现了两种类型的结合；较弱的结合位点，可聚集约 100 个分子/NPC，局部集中底物；和更强的结合位点，其中最多可结合〜8 个分子/NPC。单分子分析的保留时间和单核分析的易位率显示与〜8分子/NPC的系数显着相关，这表现出运输的化学计量。我们将较弱的结合位点称为“多重位点”，它可能由含有核孔蛋白的 FG 重复序列组成。基于相关系数，我们提出核孔复合体中存在8个“转运位点”。该转运位点是否相当于观察到的强结合位点仍有待进一步研究阐明。这些发现是否解释了不同的途径（显示出广泛的运输速率）也有待阐明。在平行实验中，我们纯化并鉴定了 70kDa 热休克同源蛋白 (hsc70) 作为促进输入蛋白 β 循环的分子。观察到 hsc70 的影响不仅在输入蛋白 β 的核输出中，而且在另一种输入受体转运蛋白和输入蛋白 α 的核输出中也有影响。这些结果表明，hsc70 通过调节受体蛋白的核输出来广泛调节核细胞质转运系统（Kose 等人，J Cell Biol. 2005）。