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Proc Natl Acad Sci U S A
2008 Oct 21;10542:16101-6. doi: 10.1073/pnas.0802647105.
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Individual binding pockets of importin-beta for FG-nucleoporins have different binding properties and different sensitivities to RanGTP.
Otsuka S
,
Iwasaka S
,
Yoneda Y
,
Takeyasu K
,
Yoshimura SH
.
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Importin-beta mediates protein transport across the nuclear envelope through the nuclear pore complex (NPC) by interacting with components of the NPC, called nucleoporins, and a small G protein, Ran. Although there is accumulated knowledge on the specific interaction between importin-beta and the Phe-Gly (FG) motif in the nucleoporins as well as the effect of RanGTP on this interaction, the molecular mechanism by which importin-beta shuttles across the nuclear envelope through the NPC is unknown. In this study, we focused on four binding pockets of importin-beta for the FG motifs and characterized the interaction using a single-molecule force-measurement technique with atomic-force microscopy. The results from a series of importin-beta mutants containing amino acid substitutions within the FG-binding pockets demonstrate that the individual FG-binding pockets have different affinities to FG-Nups (Nup62 and Nup153) and different sensitivities to RanGTP; the binding of RanGTP to the amino-terminal domain of importin-beta induces the conformational change of the entire molecule and reduces the affinity of some of the pockets but not others. These heterogeneous characteristics of the multiple FG-binding pockets may play an important role in the behavior of importin-beta within the NPC. Single-molecule force measurement using the entire molecule of an NPC from a Xenopus oocyte also implies that the reduction of the affinity by RanGTP really occurs at the nucleoplasmic side of the entire NPC.
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