| Research Abstract |
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.
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