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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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Role of the Nup62 complex in nuclear import. Shown are the effects of anti-Nup62 Fab fragment on (A) NLS-mediated import in NRK cells; (B) the solubilization of Nup358 and Nup153 (left) and the association of importin β with these Nups (right); and (C) the binding of importin β to GST-Nup62.
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Figure 3: Role of the Nup62 complex in nuclear import. Shown are the effects of anti-Nup62 Fab fragment on (A) NLS-mediated import in NRK cells; (B) the solubilization of Nup358 and Nup153 (left) and the association of importin β with these Nups (right); and (C) the binding of importin β to GST-Nup62.

Mentions: A role for the Nup62 complex in nuclear import has been suggested by the observation that transport is inhibited in nuclei assembled from Xenopus egg extracts from which the Nup62 complex has been depleted (Finlay et al. 1991). To obtain further evidence for a role of the Nup62 complex in importin β–mediated nuclear import, and to examine whether this protein may be an intermediate in the (direct or indirect) transfer of the import complex from Nup358 to Nup153, we carried out antibody inhibition experiments. We pretreated permeabilized NRK cells with the Fab fragment derived from anti-Nup62 IgG and then analyzed both nuclear import and the levels of importin β associated with Nup358 and Nup153. The anti-Nup62 antibodies reacted only with an ∼62-kD band on an immunoblot of NRK cells and showed strong nuclear rim staining in immunofluorescence, as expected (data not shown). As shown in Fig. 3 A, the anti-Nup62 Fab fragment inhibited import on average by 84% compared with a control reaction lacking the antibody, when transport is corrected for the 0°C control. This suggests that an interaction of importin β with Nup62 is essential for NLS-mediated import. Strong inhibition of import was also obtained with intact anti-Nup62 IgG, as well as with anti-Nup58 and anti-Nup54 IgG (data not shown). By contrast anti-Nup62 IgG and Fab do not inhibit in vitro nuclear export (our unpublished observations).


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

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

Role of the Nup62 complex in nuclear import. Shown are the effects of anti-Nup62 Fab fragment on (A) NLS-mediated import in NRK cells; (B) the solubilization of Nup358 and Nup153 (left) and the association of importin β with these Nups (right); and (C) the binding of importin β to GST-Nup62.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
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Figure 3: Role of the Nup62 complex in nuclear import. Shown are the effects of anti-Nup62 Fab fragment on (A) NLS-mediated import in NRK cells; (B) the solubilization of Nup358 and Nup153 (left) and the association of importin β with these Nups (right); and (C) the binding of importin β to GST-Nup62.
Mentions: A role for the Nup62 complex in nuclear import has been suggested by the observation that transport is inhibited in nuclei assembled from Xenopus egg extracts from which the Nup62 complex has been depleted (Finlay et al. 1991). To obtain further evidence for a role of the Nup62 complex in importin β–mediated nuclear import, and to examine whether this protein may be an intermediate in the (direct or indirect) transfer of the import complex from Nup358 to Nup153, we carried out antibody inhibition experiments. We pretreated permeabilized NRK cells with the Fab fragment derived from anti-Nup62 IgG and then analyzed both nuclear import and the levels of importin β associated with Nup358 and Nup153. The anti-Nup62 antibodies reacted only with an ∼62-kD band on an immunoblot of NRK cells and showed strong nuclear rim staining in immunofluorescence, as expected (data not shown). As shown in Fig. 3 A, the anti-Nup62 Fab fragment inhibited import on average by 84% compared with a control reaction lacking the antibody, when transport is corrected for the 0°C control. This suggests that an interaction of importin β with Nup62 is essential for NLS-mediated import. Strong inhibition of import was also obtained with intact anti-Nup62 IgG, as well as with anti-Nup58 and anti-Nup54 IgG (data not shown). By contrast anti-Nup62 IgG and Fab do not inhibit in vitro nuclear export (our unpublished observations).

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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