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

在真核细胞中，分子的核质转运对于细胞内信号转导至关重要。然而，它仍然是未知的蛋白质如何穿越核孔定向。在这项研究中，为了阐明核孔复合物（NPC）的分子结构，我们将重点放在importin β上，它能够通过核孔在两个方向上自行移位。importin β的N-末端片段的未复合形式的结构已通过X射线晶体学解析。在此结构分析的基础上，我们构建了突变型importin β蛋白，其中从孔结合结构域的螺旋凸侧（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。当显微注射到培养细胞的细胞质或细胞核中时，与野生型相比，其中螺旋B4和B5突变的重组4 B5 B突变蛋白质在细胞核中显著积累。相反，其中螺旋A5和A6突变的重组5A 6A突变蛋白的核积累显著降低。通过使用透化细胞体外转运测定获得相同的结果。此外，我们发现5A 6A突变体将NLS-底物转运到细胞核的能力降低，这意味着importin β的转运能力与其自身的迁移活性平行。在未来，我们将能够通过分析突变的importin β蛋白与每个核孔蛋白的亲和力来识别NPC的关键组分（核孔蛋白），以使蛋白质通过核孔定向移动。

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.