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© The Rockefeller University Press,
0021-9525/2001//411 $5.00
The Journal of Cell Biology, Volume 152, Number 2,
, 2001 411-418
Report |
Gradient of Increasing Affinity of Importin β for Nucleoporins along the Pathway of Nuclear Import
lgerace{at}scripps.edu
Nuclear import and export signals on macromolecules mediate directional, receptor-driven transport through the nuclear pore complex (NPC) by a process that is suggested to involve the sequential binding of transport complexes to different nucleoporins. The directionality of transport appears to be partly determined by the nucleocytoplasmic compartmentalization of components of the Ran GTPase system. We have analyzed whether the asymmetric localization of discrete nucleoporins can also contribute to transport directionality. To this end, we have used quantitative solid phase binding analysis to determine the affinity of an importin β cargo complex for Nup358, the Nup62 complex, and Nup153, which are in the cytoplasmic, central, and nucleoplasmic regions of the NPC, respectively. These nucleoporins are proposed to provide progressively more distal binding sites for importin β during import. Our results indicate that the importin β transport complex binds to nucleoporins with progressively increasing affinity as the complex moves from Nup358 to the Nup62 complex and to Nup153. Antibody inhibition studies support the possibility that importin β moves from Nup358 to Nup153 via the Nup62 complex during import. These results indicate that nucleoporins themselves, as well as the nucleocytoplasmic compartmentalization of the Ran system, are likely to play an important role in conferring directionality to nuclear protein import.
Key Words: nuclear import • nuclear pore complex • importin β • Nup62 complex • Nup153
© 2001 The Rockefeller University Press
Abbreviations used in this paper: FG, phenylalanine-glycine; GST, glutathione S-transferase; IBB, importin β binding; NLS, nuclear localization sequence; NPC, nuclear pore complex; RanBP, Ran binding protein; RBD, Ran binding domain.
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