Selective disruption of nuclear import by a functional mutant nuclear transport carrier - PubMed (original) (raw)

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

C M Lane et al. J Cell Biol. 2000.

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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Figures

Figure 1

Purified recombinant p10 proteins. Purified recombinant p10 proteins were run on a 15% SDS-PAGE gel and stained with Coomassie blue. wt p10 has an apparent molecular mass of 10 kD, but a predicted molecular mass of 14 kD. Tagged p10s have an M r closer to their true predicted mass of 21 kD. Lanes: 1, untagged wt p10; 2, wt p10; 3, E42D p10; 4, Y19A p10; and 5, D23A p10.

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.

Figure 2

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.

Figure 3

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.

Figure 4

Figure 6

Interaction of p10 mutants with the NPC. (A) Digitonin-permeabilized HeLa cells were incubated with 0.25 μM (dimer) wt or mutant p10, as described in the Materials and Methods. After washing and fixation, the bound p10 was detected by indirect immunofluorescence microscopy with an anti-FLAG antibody followed by a TRITC-labeled anti–mouse second antibody. (B) Diagram indicates the fragments of Nup98 that were used in the overlay assay. Cell lysates from E. coli expressing different portions of the nucleoporin Nup98 (Nup98-1, Nup98-2, and Nup98-3) were separated on a 10% SDS-PAGE gel. Arrows indicate the migration position of the expressed pieces of Nup98 after staining of a gel with Coomassie blue. Other samples were transferred to nitrocellulose and overlaid with 0.5 μM wt or mutant p10, as described in the Materials and Methods. Bound p10 was detected by immunoblotting with an anti-Express antibody.

Figure 6

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.

Figure 7

wt p10 and D23A p10 binding to the nuclear envelopes of permeabilized cells in the presence of other nuclear transport factors. Binding of wt and mutant p10 to the nuclear envelope of digitonin-permeabilized HeLa cells was performed as described in the legend to Fig. 6. 0.25 μM (dimer) wt p10 or D23A p10 were added either alone (top), or with 20 μM RanGDP, 20 μM Kap-β1, or 20 μM untagged wt p10.

Figure 8

Inhibition of RanGDP import by high concentrations of Kap-β1. p10-mediated nuclear import of FITC–RanGDP was measured in the presence of increasing concentrations of Kap-β1. All samples contained 1.5 μM FITC–RanGDP, 0.5 mM GTP, 2 mg/ml BSA, and Kap-β1 at the indicated concentrations. In addition, samples contained 1.25 μM (dimer) WT p10 (○) or D23A p10 (•). Import was for 15 min before washing and fixation.

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Selective disruption of nuclear import by a functional mutant nuclear transport carrier - PubMed (original) (raw)