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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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p10 mutants that cannot bind RanGDP do not support nuclear accumulation of FITC–RanGDP in digitonin-permeabilized cells. (A) Recombinant p10 proteins were dotted onto nitrocellulose, as described in the Materials and Methods, and the nitrocellulose was incubated with either Ran[α-32P]GDP or Ran[α-32P]GTP, washed, and exposed to film. (B) The ability of different p10 mutants to mediate the import of FITC–RanGDP (2 μM) in permeabilized BRL cells was assayed, as described in the legend to Fig. 2. Samples contained 0.5 mM GTP, 1 mM ATP plus a regenerating system, and 1 μM of the indicated p10 protein (dimer). Import was for 20 min at room temperature before washing and fixation.
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Figure 3: p10 mutants that cannot bind RanGDP do not support nuclear accumulation of FITC–RanGDP in digitonin-permeabilized cells. (A) Recombinant p10 proteins were dotted onto nitrocellulose, as described in the Materials and Methods, and the nitrocellulose was incubated with either Ran[α-32P]GDP or Ran[α-32P]GTP, washed, and exposed to film. (B) The ability of different p10 mutants to mediate the import of FITC–RanGDP (2 μM) in permeabilized BRL cells was assayed, as described in the legend to Fig. 2. Samples contained 0.5 mM GTP, 1 mM ATP plus a regenerating system, and 1 μM of the indicated p10 protein (dimer). Import was for 20 min at room temperature before washing and fixation.

Mentions: We had anticipated that a mutation in p10 would either have no effect on its biological activity or decrease it. To our surprise, we found that at low concentrations D23A p10 was markedly more efficient at supporting the nuclear accumulation of Ran than wt p10 (Fig. 2 C). In contrast, the mutant E42D p10 was unable to support Ran nuclear import. The reason for the observed inability of E42D p10 to support Ran import can be explained by the results shown in Fig. 3. The binding of wt and mutant p10s to RanGDP or RanGTP was analyzed by Ran overlay assays (Fig. 3 A). Of the p10 mutants tested, only D23A p10 retained its ability to bind RanGDP. Neither E42D nor Y19A p10 were observed to bind RanGDP in this assay. This inability to bind RanGDP explains why E42D and Y19A p10 are also unable to support Ran's nuclear accumulation (Fig. 2 C and 3 B). The crystal structure of p10 bound to RanGDP (Stewart et al. 1998) reveals that p10 amino acid E42 forms a salt bridge between Ran and p10 necessary for the stabilization of RanGDP binding. A different mutation in this amino acid, E42K, was shown previously to abolish Ran binding to p10, without altering the overall structure of p10 (Clarkson et al. 1997). In contrast, amino acids D23 and Y19 are not among the amino acids directly implicated in RanGDP binding by p10, although all of our data suggests that the Y19A mutation results in profound structural changes throughout the p10 molecule (see below).


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

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

p10 mutants that cannot bind RanGDP do not support nuclear accumulation of FITC–RanGDP in digitonin-permeabilized cells. (A) Recombinant p10 proteins were dotted onto nitrocellulose, as described in the Materials and Methods, and the nitrocellulose was incubated with either Ran[α-32P]GDP or Ran[α-32P]GTP, washed, and exposed to film. (B) The ability of different p10 mutants to mediate the import of FITC–RanGDP (2 μM) in permeabilized BRL cells was assayed, as described in the legend to Fig. 2. Samples contained 0.5 mM GTP, 1 mM ATP plus a regenerating system, and 1 μM of the indicated p10 protein (dimer). Import was for 20 min at room temperature before washing and fixation.
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Figure 3: p10 mutants that cannot bind RanGDP do not support nuclear accumulation of FITC–RanGDP in digitonin-permeabilized cells. (A) Recombinant p10 proteins were dotted onto nitrocellulose, as described in the Materials and Methods, and the nitrocellulose was incubated with either Ran[α-32P]GDP or Ran[α-32P]GTP, washed, and exposed to film. (B) The ability of different p10 mutants to mediate the import of FITC–RanGDP (2 μM) in permeabilized BRL cells was assayed, as described in the legend to Fig. 2. Samples contained 0.5 mM GTP, 1 mM ATP plus a regenerating system, and 1 μM of the indicated p10 protein (dimer). Import was for 20 min at room temperature before washing and fixation.
Mentions: We had anticipated that a mutation in p10 would either have no effect on its biological activity or decrease it. To our surprise, we found that at low concentrations D23A p10 was markedly more efficient at supporting the nuclear accumulation of Ran than wt p10 (Fig. 2 C). In contrast, the mutant E42D p10 was unable to support Ran nuclear import. The reason for the observed inability of E42D p10 to support Ran import can be explained by the results shown in Fig. 3. The binding of wt and mutant p10s to RanGDP or RanGTP was analyzed by Ran overlay assays (Fig. 3 A). Of the p10 mutants tested, only D23A p10 retained its ability to bind RanGDP. Neither E42D nor Y19A p10 were observed to bind RanGDP in this assay. This inability to bind RanGDP explains why E42D and Y19A p10 are also unable to support Ran's nuclear accumulation (Fig. 2 C and 3 B). The crystal structure of p10 bound to RanGDP (Stewart et al. 1998) reveals that p10 amino acid E42 forms a salt bridge between Ran and p10 necessary for the stabilization of RanGDP binding. A different mutation in this amino acid, E42K, was shown previously to abolish Ran binding to p10, without altering the overall structure of p10 (Clarkson et al. 1997). In contrast, amino acids D23 and Y19 are not among the amino acids directly implicated in RanGDP binding by p10, although all of our data suggests that the Y19A mutation results in profound structural changes throughout the p10 molecule (see below).

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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