| Research Abstract |
Nuclear import of proteins arc initiated by nuclear localization signal (NLS)-dependent complex formation, termed the nuclear pore-targeting complex (PTAC), in the cytoplasm. PTAC is a stable protein complex composed of a karyophilic protein and two cytosolic components termed PTAC58/importin α and PTAC97/importin β. Importin α directly binds to NLS of karyophile and importin β provides nuclear pore complex (NPC) binding site for PTAC. We analyzed the properties of importin α and importin β, and obtained the following results : 1. We have identified three additional mouse genes homologous to the known importin α, and showed that the mouse importin α can be classified into three subfamilies (Rch1, NPI1, and Qip1), showing approximately 50% amino acid identity to one another. 2. We showed that importin α family proteins show some specificities in the recognition of substrates. For example, Qip1 recognizes a very restricted NLS, requiring both the upstream sequence and basic core sequence
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of Q1 ATPase for its substrate recognition. The other evidence is that tyrosine phosphorylated Stat his recognized by NPI1, but not Rch1. Stat 1 apparently does not possess typical basic-type NLS, and it binds to different portion of NPI1 from those of SV40 T-antigen basic NLS binding site. 3. We examined the behavior and functional domain of importin β Importin β rapidly shuttles between the cytoplasm and the nucleus through NPCs. The use of deletion mutants reveals that NPC-translocation of importin β requires neither Ran- nor importin α-binding but only the NPC-binding domain of this molecule. NPC translocation of importin β was found to require neither Ran nor its GTP hydrolysis. Furthermore, importin β possesses ability to translocate NPC on its own. This could be a common feature of transport factors that carries certain cargos through NPCs into and out of the nucleus. 4. We examined the nuclear import of β-catenin and found that b-catenin can translocate NPCs on it own without requiring soluble factors including Ran. Β-catenin is likely to share the same property with importin β for NPC translocation. Less
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