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Selective disruption of nuclear import by a functional mutant nuclear transport carrier.

Lane CM, Cushman I, Moore MS - J. Cell Biol. (2000)

Bottom Line: We constructed a point mutant of p10, D23A, that exhibited unexpected behavior both in digitonin-permeabilized and microinjected mammalian cells.D23A p10 was markedly more efficient than wild-type (wt) p10 at supporting Ran import, but simultaneously acted as a dominant-negative inhibitor of classical nuclear localization sequence (cNLS)-mediated nuclear import supported by karyopherins (Kaps) alpha and beta1.Because of this increased affinity, D23A p10 is able to import its own cargo (RanGDP) more efficiently than wt p10, but Kap-beta1 can no longer compete efficiently for shared NPC docking sites, thus the import of cNLS cargo is inhibited.

View Article: PubMed Central - PubMed

Affiliation: Baylor College of Medicine, Department of Molecular and Cellular Biology, Houston, Texas 77030, USA.

ABSTRACT
p10/NTF2 is a nuclear transport carrier that mediates the uptake of cytoplasmic RanGDP into the nucleus. We constructed a point mutant of p10, D23A, that exhibited unexpected behavior both in digitonin-permeabilized and microinjected mammalian cells. D23A p10 was markedly more efficient than wild-type (wt) p10 at supporting Ran import, but simultaneously acted as a dominant-negative inhibitor of classical nuclear localization sequence (cNLS)-mediated nuclear import supported by karyopherins (Kaps) alpha and beta1. Binding studies indicated that these two nuclear transport carriers of different classes, p10 and Kap-beta1, compete for identical and/or overlapping binding sites at the nuclear pore complex (NPC) and that D23A p10 has an increased affinity relative to wt p10 and Kap-beta1 for these shared binding sites. Because of this increased affinity, D23A p10 is able to import its own cargo (RanGDP) more efficiently than wt p10, but Kap-beta1 can no longer compete efficiently for shared NPC docking sites, thus the import of cNLS cargo is inhibited. The competition of different nuclear carriers for shared NPC docking sites observed here predicts a dynamic equilibrium between multiple nuclear transport pathways inside the cell that could be easily shifted by a transient modification of one of the carriers.

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D23A p10 does not inhibit RanGDP nuclear accumulation, but does inhibit BSA–NLS nuclear accumulation in vivo. HeLa cells were coinjected in the cytoplasm with the following: FITC–RanGDP (1 mg/ml), TRITC-BSA–NLS (2 mg/ml), the injection marker Cascade blue–labeled BSA (Molecular Probes) (1 mg/ml), and unlabeled wt or mutant p10 (2.2 mg/ml). The cells were incubated 5 min at room temperature after microinjection, and then fixed for observation.
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Figure 5: D23A p10 does not inhibit RanGDP nuclear accumulation, but does inhibit BSA–NLS nuclear accumulation in vivo. HeLa cells were coinjected in the cytoplasm with the following: FITC–RanGDP (1 mg/ml), TRITC-BSA–NLS (2 mg/ml), the injection marker Cascade blue–labeled BSA (Molecular Probes) (1 mg/ml), and unlabeled wt or mutant p10 (2.2 mg/ml). The cells were incubated 5 min at room temperature after microinjection, and then fixed for observation.

Mentions: We found that D23A p10 has the same inhibitory effect on BSA–NLS nuclear import in vivo as in permeabilized cells (Fig. 5). We had determined previously that FITC–RanGDP injected into the cytoplasm of HeLa cells could be observed to rapidly (within 5 min) accumulate inside the nucleus (data not shown). To simultaneously examine the effects of the p10 mutants on Ran uptake and NLS-mediated import, the following were coinjected into the cytoplasm: FITC–RanGDP, TRITC–BSA–NLS, Cascade blue–labeled BSA (marker), and unlabeled p10 (wt or mutant). Neither the wt nor mutant p10s had any effect on the nuclear accumulation of Ran when coinjected with it into the cytoplasm (Fig. 5, left column); the injected cells would of course contain endogenous wt p10. However, as seen previously in the permeabilized cells, D23A p10 inhibited the nuclear import of coinjected BSA–NLS at the same time that the nuclear accumulation of coinjected Ran was unaffected. As these whole cells at the time of injection obviously contain enough nuclear Ran to support NLS-mediated nuclear import, and also because this mutant appears fully capable of supporting Ran nuclear import in vitro, we concluded that D23A p10 must be inhibiting BSA–NLS import by a mechanism unrelated to the nuclear uptake of Ran.


Selective disruption of nuclear import by a functional mutant nuclear transport carrier.

Lane CM, Cushman I, Moore MS - J. Cell Biol. (2000)

D23A p10 does not inhibit RanGDP nuclear accumulation, but does inhibit BSA–NLS nuclear accumulation in vivo. HeLa cells were coinjected in the cytoplasm with the following: FITC–RanGDP (1 mg/ml), TRITC-BSA–NLS (2 mg/ml), the injection marker Cascade blue–labeled BSA (Molecular Probes) (1 mg/ml), and unlabeled wt or mutant p10 (2.2 mg/ml). The cells were incubated 5 min at room temperature after microinjection, and then fixed for observation.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2192641&req=5

Figure 5: D23A p10 does not inhibit RanGDP nuclear accumulation, but does inhibit BSA–NLS nuclear accumulation in vivo. HeLa cells were coinjected in the cytoplasm with the following: FITC–RanGDP (1 mg/ml), TRITC-BSA–NLS (2 mg/ml), the injection marker Cascade blue–labeled BSA (Molecular Probes) (1 mg/ml), and unlabeled wt or mutant p10 (2.2 mg/ml). The cells were incubated 5 min at room temperature after microinjection, and then fixed for observation.
Mentions: We found that D23A p10 has the same inhibitory effect on BSA–NLS nuclear import in vivo as in permeabilized cells (Fig. 5). We had determined previously that FITC–RanGDP injected into the cytoplasm of HeLa cells could be observed to rapidly (within 5 min) accumulate inside the nucleus (data not shown). To simultaneously examine the effects of the p10 mutants on Ran uptake and NLS-mediated import, the following were coinjected into the cytoplasm: FITC–RanGDP, TRITC–BSA–NLS, Cascade blue–labeled BSA (marker), and unlabeled p10 (wt or mutant). Neither the wt nor mutant p10s had any effect on the nuclear accumulation of Ran when coinjected with it into the cytoplasm (Fig. 5, left column); the injected cells would of course contain endogenous wt p10. However, as seen previously in the permeabilized cells, D23A p10 inhibited the nuclear import of coinjected BSA–NLS at the same time that the nuclear accumulation of coinjected Ran was unaffected. As these whole cells at the time of injection obviously contain enough nuclear Ran to support NLS-mediated nuclear import, and also because this mutant appears fully capable of supporting Ran nuclear import in vitro, we concluded that D23A p10 must be inhibiting BSA–NLS import by a mechanism unrelated to the nuclear uptake of Ran.

Bottom Line: We constructed a point mutant of p10, D23A, that exhibited unexpected behavior both in digitonin-permeabilized and microinjected mammalian cells.D23A p10 was markedly more efficient than wild-type (wt) p10 at supporting Ran import, but simultaneously acted as a dominant-negative inhibitor of classical nuclear localization sequence (cNLS)-mediated nuclear import supported by karyopherins (Kaps) alpha and beta1.Because of this increased affinity, D23A p10 is able to import its own cargo (RanGDP) more efficiently than wt p10, but Kap-beta1 can no longer compete efficiently for shared NPC docking sites, thus the import of cNLS cargo is inhibited.

View Article: PubMed Central - PubMed

Affiliation: Baylor College of Medicine, Department of Molecular and Cellular Biology, Houston, Texas 77030, USA.

ABSTRACT
p10/NTF2 is a nuclear transport carrier that mediates the uptake of cytoplasmic RanGDP into the nucleus. We constructed a point mutant of p10, D23A, that exhibited unexpected behavior both in digitonin-permeabilized and microinjected mammalian cells. D23A p10 was markedly more efficient than wild-type (wt) p10 at supporting Ran import, but simultaneously acted as a dominant-negative inhibitor of classical nuclear localization sequence (cNLS)-mediated nuclear import supported by karyopherins (Kaps) alpha and beta1. Binding studies indicated that these two nuclear transport carriers of different classes, p10 and Kap-beta1, compete for identical and/or overlapping binding sites at the nuclear pore complex (NPC) and that D23A p10 has an increased affinity relative to wt p10 and Kap-beta1 for these shared binding sites. Because of this increased affinity, D23A p10 is able to import its own cargo (RanGDP) more efficiently than wt p10, but Kap-beta1 can no longer compete efficiently for shared NPC docking sites, thus the import of cNLS cargo is inhibited. The competition of different nuclear carriers for shared NPC docking sites observed here predicts a dynamic equilibrium between multiple nuclear transport pathways inside the cell that could be easily shifted by a transient modification of one of the carriers.

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