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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: 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.

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 is a dominant-negative inhibitor of the nuclear import of TRITC–BSA–NLS in vitro. (A) wt p10 or D23A p10 (both at 3.0 μM dimer concentration) were added to an import solution consisting of 10 μg/ml TRITC–BSA–NLS, 10 mg/ml Xenopus ovarian cytosol, 0.5 mM GTP, and 1 mM ATP plus a regenerating system. These mixtures were incubated with permeabilized BRL cells for 20 min at room temperature before washing and fixation. (B) The import assay was done as described in A, except purified transport factors were used to support import, rather than the cytosol, and the reaction was for 15 min. The import reactions contained 10 μg/ml TRITC–BSA–NLS, 0.5 μM Kap-α2, 0.25 μM Kap-β1, 2 μM RanGDP, 0.5 mM GTP, and 2 mg/ml BSA in addition to the indicated concentration of wt or D23A p10 dimer.
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Figure 4: D23A p10 is a dominant-negative inhibitor of the nuclear import of TRITC–BSA–NLS in vitro. (A) wt p10 or D23A p10 (both at 3.0 μM dimer concentration) were added to an import solution consisting of 10 μg/ml TRITC–BSA–NLS, 10 mg/ml Xenopus ovarian cytosol, 0.5 mM GTP, and 1 mM ATP plus a regenerating system. These mixtures were incubated with permeabilized BRL cells for 20 min at room temperature before washing and fixation. (B) The import assay was done as described in A, except purified transport factors were used to support import, rather than the cytosol, and the reaction was for 15 min. The import reactions contained 10 μg/ml TRITC–BSA–NLS, 0.5 μM Kap-α2, 0.25 μM Kap-β1, 2 μM RanGDP, 0.5 mM GTP, and 2 mg/ml BSA in addition to the indicated concentration of wt or D23A p10 dimer.

Mentions: Surprisingly, we found that although D23A p10 could support the nuclear import of Ran, this mutant p10 had a marked inhibitory effect on the import of a cNLS cargo (Fig. 4). In Fig. 4 A, the import of BSA–NLS into the nuclei of digitonin-permeabilized BRL cells was supported by Xenopus cytosol as a source of transport factors plus energy. This import substrate contains peptides containing the cNLS of the SV40 T antigen coupled to rhodamine-labeled BSA (Moore and Blobel 1992). The Xenopus cytosol used is known to contain endogenous p10 that is functional in permeabilized mammalian cells (Moore and Blobel 1994). wt p10 had no effect on the nuclear import of BSA–NLS when added to the cytosol at 3 μM (Fig. 4 A). In marked contrast, the addition of D23A p10 at the same concentration severely inhibited the nuclear import of BSA–NLS.


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

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

D23A p10 is a dominant-negative inhibitor of the nuclear import of TRITC–BSA–NLS in vitro. (A) wt p10 or D23A p10 (both at 3.0 μM dimer concentration) were added to an import solution consisting of 10 μg/ml TRITC–BSA–NLS, 10 mg/ml Xenopus ovarian cytosol, 0.5 mM GTP, and 1 mM ATP plus a regenerating system. These mixtures were incubated with permeabilized BRL cells for 20 min at room temperature before washing and fixation. (B) The import assay was done as described in A, except purified transport factors were used to support import, rather than the cytosol, and the reaction was for 15 min. The import reactions contained 10 μg/ml TRITC–BSA–NLS, 0.5 μM Kap-α2, 0.25 μM Kap-β1, 2 μM RanGDP, 0.5 mM GTP, and 2 mg/ml BSA in addition to the indicated concentration of wt or D23A p10 dimer.
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Figure 4: D23A p10 is a dominant-negative inhibitor of the nuclear import of TRITC–BSA–NLS in vitro. (A) wt p10 or D23A p10 (both at 3.0 μM dimer concentration) were added to an import solution consisting of 10 μg/ml TRITC–BSA–NLS, 10 mg/ml Xenopus ovarian cytosol, 0.5 mM GTP, and 1 mM ATP plus a regenerating system. These mixtures were incubated with permeabilized BRL cells for 20 min at room temperature before washing and fixation. (B) The import assay was done as described in A, except purified transport factors were used to support import, rather than the cytosol, and the reaction was for 15 min. The import reactions contained 10 μg/ml TRITC–BSA–NLS, 0.5 μM Kap-α2, 0.25 μM Kap-β1, 2 μM RanGDP, 0.5 mM GTP, and 2 mg/ml BSA in addition to the indicated concentration of wt or D23A p10 dimer.
Mentions: Surprisingly, we found that although D23A p10 could support the nuclear import of Ran, this mutant p10 had a marked inhibitory effect on the import of a cNLS cargo (Fig. 4). In Fig. 4 A, the import of BSA–NLS into the nuclei of digitonin-permeabilized BRL cells was supported by Xenopus cytosol as a source of transport factors plus energy. This import substrate contains peptides containing the cNLS of the SV40 T antigen coupled to rhodamine-labeled BSA (Moore and Blobel 1992). The Xenopus cytosol used is known to contain endogenous p10 that is functional in permeabilized mammalian cells (Moore and Blobel 1994). wt p10 had no effect on the nuclear import of BSA–NLS when added to the cytosol at 3 μM (Fig. 4 A). In marked contrast, the addition of D23A p10 at the same concentration severely inhibited the nuclear import of BSA–NLS.

Bottom Line: 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.

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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