| Research Abstract |
In contrast to our growing knowledge of receptor molecules for nucleocytoplasmic transport, function of nuclear pore complex (NPC), as well as mechanism of translocation step of transport is still very poorly understood. In this study, we focused on the following study. (1) Structural study of importin p. We solved crystal structure of uncomplexed form of importin β. Precise structurall comparison of our structur and previously reported structure of complexed form of importin β has revealed the flexible nature of importin β. Since local conformation change and flexible motion of importin β resides within its NPC- binding domain in which we have determined by series of deletion mutants, this flexible nature could be important for its movement through nuclear pores. We are now making series of point mutants of importin p that behave differently at NPC. (2) In order to study mechanism of a directional movement of importin β at NPC, we examined a requirement ofATP in import and export of importin p, and found that only nuclear export of importin β involves energy-requiring step(s) in living cells. In vitro transport assay revealed that an energy-dependent nuclear export of importin β requires soluble factors. To address the issue of how the energy is utilized during the NPC-passage of importin β, we are now attempting to purify an energy-dependent nuclear export factor of importin β from mouse Ehrlich cytosolic extract.
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