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

在真核细胞中，分子的核质转运是细胞内信号转导的关键。然而，蛋白质如何定向穿越核孔仍是未知的。在本研究中，为了阐明核孔复合物（NPCs）的分子组织，我们重点研究了能够通过核孔在两个方向上自行转运的输入蛋白β。用x射线晶体学分析了进口蛋白β n端片段的非络合形式的结构。基于这一结构分析，我们构建了突变型输入蛋白β，其中从孔结合域的螺旋凸侧（螺旋A）或螺旋凹侧（螺旋B）的分子表面突出的氨基酸残基被丙氨酸取代。将重组4B5B突变蛋白微注射到培养细胞的细胞质或细胞核中，其中螺旋B4和B5发生突变，与野生型相比，重组4B5B突变蛋白在细胞核中显著积累。相比之下，重组5A6A突变蛋白的核积累显著减少，其中螺旋A5和A6发生突变。通过通透化细胞体外转运试验也得到了相同的结果。此外，我们发现5A6A突变体将nls底物导入细胞核的能力下降，这意味着导入β蛋白的转运能力与其自身的迁移活性是平行的。在未来，我们将能够通过分析突变的输入蛋白β蛋白与每个核孔蛋白的亲和力来识别蛋白质通过核孔定向运动的关键NPC成分（核孔蛋白）。

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.