Individual binding pockets of importin-beta for FG-nucleoporins have different binding properties and different sensitivities to RanGTP.
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.
Otsuka
Shotaro
S
Laboratory of Plasma Membrane and Nuclear Signaling, Graduate School of Biostudies, Kyoto University, Yoshida Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan.
Iwasaka
Shizuka
S
Yoneda
Yoshihiro
Y
Takeyasu
Kunio
K
Yoshimura
Shige H
SH
D000818:Animals; D001665:Binding Sites; D005260:Female; D005998:Glycine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D028861:Nuclear Pore Complex Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D009865:Oocytes; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D014982:Xenopus laevis; D028961:beta Karyopherins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D020931:ran GTP-Binding Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D028861:Nuclear Pore Complex Proteins; D028961:beta Karyopherins; D010649:Phenylalanine; D020931:ran GTP-Binding Protein; D005998:Glycine