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

被引:55
作者
Otsuka, Shotaro [1 ]
Iwasaka, Shizuka [1 ]
Yoneda, Yoshihiro [2 ]
Takeyasu, Kunio [1 ]
Yoshimura, Shige H. [1 ,3 ]
机构
[1] Kyoto Univ, Lab Plasma Membrane & Nucl Signaling, Grad Sch Biostudies, Sakyo Ku, Kyoto 6268501, Japan
[2] Osaka Univ, Grad Sch Frontier Biosci, Biomol Dynam Grp, Suita, Osaka 5650871, Japan
[3] Japan Sci & Technol Agcy, Core Res Evolut Sci & Technol, Kawaguchi, Saitama 3320012, Japan
基金
日本科学技术振兴机构; 日本学术振兴会;
关键词
nuclear import; nuclear pore complex; nuclear transport; scanning probe microscopy; single-molecule force measurement;
D O I
10.1073/pnas.0802647105
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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 RanGTIP 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-p 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.
引用
收藏
页码:16101 / 16106
页数:6
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