期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 105, 期 42, 页码 16101-16106出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0802647105
关键词
nuclear import; nuclear pore complex; nuclear transport; scanning probe microscopy; single-molecule force measurement
资金
- Japanese Ministry of Education, Culture, Sports, Science and Technology
- Japan Society for the Promotion of Science (JSPS)
- Japan Science and Technology Agency
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.
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