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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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Model for the directional movement of an importin β cargo complex through the NPC. See text for details.
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Figure 4: Model for the directional movement of an importin β cargo complex through the NPC. See text for details.

Mentions: Several models for the movement of transport complexes through the NPC have been discussed (Rexach and Blobel 1995; Nachury and Weis 1999). We believe that the simplest model for importin β–mediated nuclear import that is consistent with the observations presented in this study is an “affinity gradient” mechanism (Fig. 4). Our antibody inhibition and biochemical analyses support the possibility that movement of importin β through the NPC involves its transfer from Nup358 to Nup153 via the Nup62 complex. Although other unidentified nucleoporin intermediates may also be involved, it is possible that Nup358 and Nup153, which are components of the flexible cyto/nucleoplasmic fibrils, might be able to directly interact with the Nup62 complex. Based on the progressive increase in the affinity of importin β for Nup358, Nup62 complex proteins, and Nup153, we suggest that the movement of the importin β cargo complex through the NPC has a strong cytoplasmic-to-nuclear directional bias due in part to increasing affinity of the transport complex for the nucleoporin binding sites that it sequentially encounters. In the simplest situation, transfer between nucleoporin pairs could occur in either a forward or backward direction at each step, but forward movement would be favored by an increase in the affinity of transport complexes for more distal nucleoporins. Release from the terminal nucleoporin binding site could be mediated by RanGTP (Gorlich et al. 1996). It is striking that two different FG repeat regions of Nup358 bound to importin β with nearly identical affinity, as did three different subunits of the Nup62 complex. This suggests that the affinity of import complexes for a specific region of the NPC may be an important parameter in specifying directionality (see below).


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

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

Model for the directional movement of an importin β cargo complex through the NPC. See text for details.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Model for the directional movement of an importin β cargo complex through the NPC. See text for details.
Mentions: Several models for the movement of transport complexes through the NPC have been discussed (Rexach and Blobel 1995; Nachury and Weis 1999). We believe that the simplest model for importin β–mediated nuclear import that is consistent with the observations presented in this study is an “affinity gradient” mechanism (Fig. 4). Our antibody inhibition and biochemical analyses support the possibility that movement of importin β through the NPC involves its transfer from Nup358 to Nup153 via the Nup62 complex. Although other unidentified nucleoporin intermediates may also be involved, it is possible that Nup358 and Nup153, which are components of the flexible cyto/nucleoplasmic fibrils, might be able to directly interact with the Nup62 complex. Based on the progressive increase in the affinity of importin β for Nup358, Nup62 complex proteins, and Nup153, we suggest that the movement of the importin β cargo complex through the NPC has a strong cytoplasmic-to-nuclear directional bias due in part to increasing affinity of the transport complex for the nucleoporin binding sites that it sequentially encounters. In the simplest situation, transfer between nucleoporin pairs could occur in either a forward or backward direction at each step, but forward movement would be favored by an increase in the affinity of transport complexes for more distal nucleoporins. Release from the terminal nucleoporin binding site could be mediated by RanGTP (Gorlich et al. 1996). It is striking that two different FG repeat regions of Nup358 bound to importin β with nearly identical affinity, as did three different subunits of the Nup62 complex. This suggests that the affinity of import complexes for a specific region of the NPC may be an important parameter in specifying directionality (see below).

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