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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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Nuclear accumulation of FITC–RanGDP. (A) Digitonin-permeabilized BRL cells were incubated with 2 μM FITC–RanGDP in TB containing 2 mg/ml BSA for 20 min at room temperature, washed, and fixed. All samples, except those labeled No Energy, also contained 0.5 mM GTP, 1 mM ATP, 5 mM phosphocreatine, and 20 U/ml creatine phosphokinase. Where indicated, the following proteins were also added: 1.0 μM wt p10 (dimer), and 0.25 μM Kap-β1 or Kap-β1(45–462). (B) Quantitation of the nuclear import of FITC–RanGDP in digitonin-permeabilized BRL cells was performed as described in the Materials and Methods. All samples contained 1.5 μM FITC–RanGDP and 2 mg/ml BSA in TB, and the import reaction was incubated for 15 min at room temperature before washing and fixation. Individual samples also contained the indicated concentration of wt p10 dimer and: ○, 0.5 mM GDP; •, 0.5 mM GTP; □, 0.5 mM GTP + 0.25 μM Kap-α2; and ▪, 0.5 mM GTP + 0.25 μM Kap-β1. (C) Quantitation of the nuclear import obtained of FITC–RanGDP in the presence of increasing concentrations of: ○, WT p10; •, D23A p10; and ▪, E42D p10. In addition to 1.5 μM FITC–RanGDP, all the samples contained 0.5 mM GTP, 0.25 μM Kap-β1, and 2 mg/ml BSA in TB. Import was for 7.5 min before washing and fixation.
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Figure 2: Nuclear accumulation of FITC–RanGDP. (A) Digitonin-permeabilized BRL cells were incubated with 2 μM FITC–RanGDP in TB containing 2 mg/ml BSA for 20 min at room temperature, washed, and fixed. All samples, except those labeled No Energy, also contained 0.5 mM GTP, 1 mM ATP, 5 mM phosphocreatine, and 20 U/ml creatine phosphokinase. Where indicated, the following proteins were also added: 1.0 μM wt p10 (dimer), and 0.25 μM Kap-β1 or Kap-β1(45–462). (B) Quantitation of the nuclear import of FITC–RanGDP in digitonin-permeabilized BRL cells was performed as described in the Materials and Methods. All samples contained 1.5 μM FITC–RanGDP and 2 mg/ml BSA in TB, and the import reaction was incubated for 15 min at room temperature before washing and fixation. Individual samples also contained the indicated concentration of wt p10 dimer and: ○, 0.5 mM GDP; •, 0.5 mM GTP; □, 0.5 mM GTP + 0.25 μM Kap-α2; and ▪, 0.5 mM GTP + 0.25 μM Kap-β1. (C) Quantitation of the nuclear import obtained of FITC–RanGDP in the presence of increasing concentrations of: ○, WT p10; •, D23A p10; and ▪, E42D p10. In addition to 1.5 μM FITC–RanGDP, all the samples contained 0.5 mM GTP, 0.25 μM Kap-β1, and 2 mg/ml BSA in TB. Import was for 7.5 min before washing and fixation.

Mentions: The ability of FLAG–p10 to support nuclear accumulation of Ran in digitonin-permeabilized cells is shown in Fig. 2. In these experiments, FITC–RanGDP was added to the permeabilized cells, either alone or with p10 and various additions. After washing and fixation, the nuclear accumulation of Ran was assessed and quantitated by fluorescence microscopy. In experiments not shown, the ability of FITC–RanGDP to support nuclear import of BSA–NLS in permeabilized cells was found to be unaffected by its labeling by fluorescein-maleimide, indicating that Ran was not damaged by the labeling procedure. Confirming the results of others (Ribbeck et al. 1998; Smith et al. 1998), we found that p10 would support the nuclear accumulation of Ran in permeabilized cells, but only under certain conditions. In the presence of p10, FITC–RanGDP, and an energy mix (consisting of GTP and ATP plus an ATP-regenerating system), nuclear accumulation of the added Ran was observed in ∼30% of the permeabilized cells (Fig. 2 A, top row). If the energy mix was omitted, this accumulation was not observed in any of the cells (Fig. 2 A, second row).


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

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

Nuclear accumulation of FITC–RanGDP. (A) Digitonin-permeabilized BRL cells were incubated with 2 μM FITC–RanGDP in TB containing 2 mg/ml BSA for 20 min at room temperature, washed, and fixed. All samples, except those labeled No Energy, also contained 0.5 mM GTP, 1 mM ATP, 5 mM phosphocreatine, and 20 U/ml creatine phosphokinase. Where indicated, the following proteins were also added: 1.0 μM wt p10 (dimer), and 0.25 μM Kap-β1 or Kap-β1(45–462). (B) Quantitation of the nuclear import of FITC–RanGDP in digitonin-permeabilized BRL cells was performed as described in the Materials and Methods. All samples contained 1.5 μM FITC–RanGDP and 2 mg/ml BSA in TB, and the import reaction was incubated for 15 min at room temperature before washing and fixation. Individual samples also contained the indicated concentration of wt p10 dimer and: ○, 0.5 mM GDP; •, 0.5 mM GTP; □, 0.5 mM GTP + 0.25 μM Kap-α2; and ▪, 0.5 mM GTP + 0.25 μM Kap-β1. (C) Quantitation of the nuclear import obtained of FITC–RanGDP in the presence of increasing concentrations of: ○, WT p10; •, D23A p10; and ▪, E42D p10. In addition to 1.5 μM FITC–RanGDP, all the samples contained 0.5 mM GTP, 0.25 μM Kap-β1, and 2 mg/ml BSA in TB. Import was for 7.5 min before washing and fixation.
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Figure 2: Nuclear accumulation of FITC–RanGDP. (A) Digitonin-permeabilized BRL cells were incubated with 2 μM FITC–RanGDP in TB containing 2 mg/ml BSA for 20 min at room temperature, washed, and fixed. All samples, except those labeled No Energy, also contained 0.5 mM GTP, 1 mM ATP, 5 mM phosphocreatine, and 20 U/ml creatine phosphokinase. Where indicated, the following proteins were also added: 1.0 μM wt p10 (dimer), and 0.25 μM Kap-β1 or Kap-β1(45–462). (B) Quantitation of the nuclear import of FITC–RanGDP in digitonin-permeabilized BRL cells was performed as described in the Materials and Methods. All samples contained 1.5 μM FITC–RanGDP and 2 mg/ml BSA in TB, and the import reaction was incubated for 15 min at room temperature before washing and fixation. Individual samples also contained the indicated concentration of wt p10 dimer and: ○, 0.5 mM GDP; •, 0.5 mM GTP; □, 0.5 mM GTP + 0.25 μM Kap-α2; and ▪, 0.5 mM GTP + 0.25 μM Kap-β1. (C) Quantitation of the nuclear import obtained of FITC–RanGDP in the presence of increasing concentrations of: ○, WT p10; •, D23A p10; and ▪, E42D p10. In addition to 1.5 μM FITC–RanGDP, all the samples contained 0.5 mM GTP, 0.25 μM Kap-β1, and 2 mg/ml BSA in TB. Import was for 7.5 min before washing and fixation.
Mentions: The ability of FLAG–p10 to support nuclear accumulation of Ran in digitonin-permeabilized cells is shown in Fig. 2. In these experiments, FITC–RanGDP was added to the permeabilized cells, either alone or with p10 and various additions. After washing and fixation, the nuclear accumulation of Ran was assessed and quantitated by fluorescence microscopy. In experiments not shown, the ability of FITC–RanGDP to support nuclear import of BSA–NLS in permeabilized cells was found to be unaffected by its labeling by fluorescein-maleimide, indicating that Ran was not damaged by the labeling procedure. Confirming the results of others (Ribbeck et al. 1998; Smith et al. 1998), we found that p10 would support the nuclear accumulation of Ran in permeabilized cells, but only under certain conditions. In the presence of p10, FITC–RanGDP, and an energy mix (consisting of GTP and ATP plus an ATP-regenerating system), nuclear accumulation of the added Ran was observed in ∼30% of the permeabilized cells (Fig. 2 A, top row). If the energy mix was omitted, this accumulation was not observed in any of the cells (Fig. 2 A, second row).

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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