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.
Laboratory of Plasma Membrane and Nuclear Signaling, Graduate School of Biostudies, Kyoto University, Yoshida Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan.
Shotaro
S
Otsuka
Shizuka
S
Iwasaka
Yoshihiro
Y
Yoneda
Kunio
K
Takeyasu
Shige H
SH
Yoshimura
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