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Gradient of increasing affinity of importin beta for nucleoporins along the pathway of nuclear import.

Ben-Efraim I, Gerace L - J. Cell Biol. (2001)

Bottom Line: Our results indicate that the importin beta 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 beta 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA.

ABSTRACT
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 beta 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 beta during import. Our results indicate that the importin beta 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 beta 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.

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The binding of importin β to nucleoporins in the solid phase assay is sensitive to RanGTP. The binding of 10 nM importin β to nucleoporins adsorbed to microtiter wells was analyzed in the absence of RanGTP, or in the presence of 20 or 100 nM RanGTP, as indicated. The bound importin β was quantified as described in the legend to Fig. 1.
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Figure 2: The binding of importin β to nucleoporins in the solid phase assay is sensitive to RanGTP. The binding of 10 nM importin β to nucleoporins adsorbed to microtiter wells was analyzed in the absence of RanGTP, or in the presence of 20 or 100 nM RanGTP, as indicated. The bound importin β was quantified as described in the legend to Fig. 1.

Mentions: Previous studies have shown that RanGTP strongly diminishes the binding of importin β to most nucleoporins as well as to intact NPCs, probably because importin β has an altered conformation when present in a complex with RanGTP (for reviews see Ohno et al. 1998; Gorlich and Kutay 1999). This suggests a role for RanGTP in regulating importin β–Nup interactions during import. In our assay, we found that the binding of importin β to the Nup62, Nup58, Nup54, and Nup153-C was strongly diminished by the addition of RanGTP at a 2:1 molar ratio to importin β (Fig. 2, A–D), conditions under which most of the importin β is expected to be complexed to Ran, based on its high binding affinity (for reviews see Ohno et al. 1998; Gorlich and Kutay 1999). The binding of importin β to these nucleoporins was further diminished with a 10:1 Ran/β ratio (Fig. 2, A–D). These data indicate that the importin β binding to this group of nucleoporins is inhibited by RanGTP, as is expected if the binding assays measure interactions relevant to nuclear import.


Gradient of increasing affinity of importin beta for nucleoporins along the pathway of nuclear import.

Ben-Efraim I, Gerace L - J. Cell Biol. (2001)

The binding of importin β to nucleoporins in the solid phase assay is sensitive to RanGTP. The binding of 10 nM importin β to nucleoporins adsorbed to microtiter wells was analyzed in the absence of RanGTP, or in the presence of 20 or 100 nM RanGTP, as indicated. The bound importin β was quantified as described in the legend to Fig. 1.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199621&req=5

Figure 2: The binding of importin β to nucleoporins in the solid phase assay is sensitive to RanGTP. The binding of 10 nM importin β to nucleoporins adsorbed to microtiter wells was analyzed in the absence of RanGTP, or in the presence of 20 or 100 nM RanGTP, as indicated. The bound importin β was quantified as described in the legend to Fig. 1.
Mentions: Previous studies have shown that RanGTP strongly diminishes the binding of importin β to most nucleoporins as well as to intact NPCs, probably because importin β has an altered conformation when present in a complex with RanGTP (for reviews see Ohno et al. 1998; Gorlich and Kutay 1999). This suggests a role for RanGTP in regulating importin β–Nup interactions during import. In our assay, we found that the binding of importin β to the Nup62, Nup58, Nup54, and Nup153-C was strongly diminished by the addition of RanGTP at a 2:1 molar ratio to importin β (Fig. 2, A–D), conditions under which most of the importin β is expected to be complexed to Ran, based on its high binding affinity (for reviews see Ohno et al. 1998; Gorlich and Kutay 1999). The binding of importin β to these nucleoporins was further diminished with a 10:1 Ran/β ratio (Fig. 2, A–D). These data indicate that the importin β binding to this group of nucleoporins is inhibited by RanGTP, as is expected if the binding assays measure interactions relevant to nuclear import.

Bottom Line: Our results indicate that the importin beta 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 beta 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA.

ABSTRACT
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 beta 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 beta during import. Our results indicate that the importin beta 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 beta 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.

Show MeSH
Related in: MedlinePlus