Individual binding pockets of importin-β for FG-nucleoporins have different binding properties and different sensitivities to RanGTP

Importin-β 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-β and the Phe-Gly...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2008-10, Vol.105 (42), p.16101-16106
Main Authors: Otsuka, Shotaro, Iwasaka, Shizuka, Yoneda, Yoshihiro, Takeyasu, Kunio, Yoshimura, Shige H
Format: Article
Language:English
Subjects:
Amino acids
Animals
beta Karyopherins - chemistry
beta Karyopherins - genetics
beta Karyopherins - metabolism
beta Karyopherins - ultrastructure
Binding Sites
Biological Sciences
Cantilevers
Crystal structure
Cytoplasm
Female
Glycine - genetics
Glycine - metabolism
Histograms
Humans
Imaging
Mice
Microscopy, Atomic Force
Models, Molecular
Molecules
Mutation - genetics
Nuclear pore
Nuclear pore complex proteins
Nuclear Pore Complex Proteins - chemistry
Nuclear Pore Complex Proteins - genetics
Nuclear Pore Complex Proteins - metabolism
Nuclear Pore Complex Proteins - ultrastructure
Oocytes
Phenylalanine - genetics
Phenylalanine - metabolism
Protein Binding
Protein Structure, Tertiary
ran GTP-Binding Protein - chemistry
ran GTP-Binding Protein - genetics
ran GTP-Binding Protein - metabolism
Substrate Specificity
Xenopus laevis
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Summary:Importin-β 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-β 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-β shuttles across the nuclear envelope through the NPC is unknown. In this study, we focused on four binding pockets of importin-β 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-β 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-β 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-β 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.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.0802647105