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The nucleoporin Nup60p functions as a Gsp1p-GTP-sensitive tether for Nup2p at the nuclear pore complex.

Denning D, Mykytka B, Allen NP, Huang L - J. Cell Biol. (2001)

Bottom Line: Yeast lacking Nup60p also fail to anchor Nup2p at the NPC, resulting in the mislocalization of Nup2p to the nucleoplasm and cytoplasm.Gsp1p-GTP enhances by 10-fold the affinity between Nup60p and Nup2p, and restores binding of Nup2p-Kap60p complexes to Nup60p.The results suggest a dynamic interaction, controlled by the nucleoplasmic concentration of Gsp1p-GTP, between Nup60p and Nup2p at the NPC.

View Article: PubMed Central - PubMed

Affiliation: Cancer Biology Program, Stanford Medical School, Stanford University, CA 94305, USA.

ABSTRACT
The nucleoporins Nup60p, Nup2p, and Nup1p form part of the nuclear basket structure of the Saccharomyces cerevisiae nuclear pore complex (NPC). Here, we show that these necleoporins can be isolated from yeast extracts by affinity chromatography on karyopherin Kap95p-coated beads. To characterize Nup60p further, Nup60p-coated beads were used to capture its interacting proteins from extracts. We find that Nup60p binds to Nup2p and serves as a docking site for Kap95p-Kap60p heterodimers and Kap123p. Nup60p also binds Gsp1p-GTP and its guanine nucleotide exchange factor Prp20p, and functions as a Gsp1p guanine nucleotide dissociation inhibitor by reducing the activity of Prp20p. Yeast lacking Nup60p exhibit minor defects in nuclear export of Kap60p, nuclear import of Kap95p-Kap60p-dependent cargoes, and diffusion of small proteins across the NPC. Yeast lacking Nup60p also fail to anchor Nup2p at the NPC, resulting in the mislocalization of Nup2p to the nucleoplasm and cytoplasm. Purified Nup60p and Nup2p bind each other directly, but the stability of the complex is compromised when Kap60p binds Nup2p. Gsp1p-GTP enhances by 10-fold the affinity between Nup60p and Nup2p, and restores binding of Nup2p-Kap60p complexes to Nup60p. The results suggest a dynamic interaction, controlled by the nucleoplasmic concentration of Gsp1p-GTP, between Nup60p and Nup2p at the NPC.

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Gsp1p–GTP and Kap60p modulate the interaction between Nup60p and Nup2p. (A) The interaction between Nup2p and Nup60p, and the effect of Kap60p. GST–Nup60p (1 μg) was immobilized on beads and incubated with Nup2p (0.5 μg), Nup2pΔ (aa 1–50) (0.5 μg), or Kap60p (1 μg) as indicated. After 1 h at 4°C, unbound and bound proteins were collected, resolved by SDS-PAGE, and visualized with Coomassie blue. Note that Nup2p binds Nup60p, that Kap60p prevents the interaction, and that the NH2 terminus of Nup2p is not required for binding Nup60p. (B) Effect of Gsp1p–GTP on the interaction between Nup2p and Nup60p. GST–Nup60p (1 μg) was immobilized on beads and incubated with Nup2p (0.5 μg), Kap60p (1 μg), or Gsp1p–GTP (His-Gsp1p Q71L) (1 μg) as before. Note that Kap60p interferes with the interaction of Nup2p with Nup60p, but that the presence of Gsp1p–GTP restores binding and promotes formation of Nup60p–Gsp1p–Nup2p–Kap60p complexes. (C) Gsp1p–GTP enhances binding of Nup2p to Nup60p in yeast extracts. GST-Nup60p (1 μg) was immobilized on beads and was incubated with yeast extract (∼1 mg) supplemented with 1.25 μM recombinant Gsp1p–GTP (Q71L), 0.5 μM recombinant Kap60p, or no additional protein. The amount of Nup2p bound to Nup60p-coated beads was determined by quantitative Western blotting as described in Materials and methods. The amount of Nup2p was expressed as the ratio of Nup2p bound per unit of immobilized GST–Nup60p, using the incubation of extract without additions as baseline. Shown are the mean ratios for two samples with error bars representing the SEM; this experiment was performed three times with similar results. The asterisks (***) indicate a P < 0.05 for comparison of mean Nup2p captured from extracts supplemented or not with additional Gsp1p–GTP (unpaired, two-tailed t test). Note that addition of Gsp1p–GTP to yeast extract increases by ∼65% the amount of Nup2p bound to Nup60p-coated beads. (D) Gsp1p–GTP increases the affinity between Nup60p and Nup2p. Nup60p-coated beads were incubated with various concentrations of radiolabeled Nup2p for 2 h at 25°C in binding buffer with 10 mg/ml BSA and protease inhibitors. The concentration of GST–Nup60p within the beads was 25 nM and 150 nM for experiments with or without Gsp1p–GTP, respectively. The dissociation constant (KD) of the Nup60p–Nup2p complex in the presence and absence of 3 μM Gsp1p–GTP Q71L was calculated as described in Materials and methods. To facilitate comparison, the results were plotted as a fraction of maximal Nup2p bound versus Nup2p concentration. Each data point was performed in duplicate and error bars represent SEM. Note the 10-fold higher affinity between Nup60p and Nup2p in the presence of Gsp1p–GTP.
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fig3: Gsp1p–GTP and Kap60p modulate the interaction between Nup60p and Nup2p. (A) The interaction between Nup2p and Nup60p, and the effect of Kap60p. GST–Nup60p (1 μg) was immobilized on beads and incubated with Nup2p (0.5 μg), Nup2pΔ (aa 1–50) (0.5 μg), or Kap60p (1 μg) as indicated. After 1 h at 4°C, unbound and bound proteins were collected, resolved by SDS-PAGE, and visualized with Coomassie blue. Note that Nup2p binds Nup60p, that Kap60p prevents the interaction, and that the NH2 terminus of Nup2p is not required for binding Nup60p. (B) Effect of Gsp1p–GTP on the interaction between Nup2p and Nup60p. GST–Nup60p (1 μg) was immobilized on beads and incubated with Nup2p (0.5 μg), Kap60p (1 μg), or Gsp1p–GTP (His-Gsp1p Q71L) (1 μg) as before. Note that Kap60p interferes with the interaction of Nup2p with Nup60p, but that the presence of Gsp1p–GTP restores binding and promotes formation of Nup60p–Gsp1p–Nup2p–Kap60p complexes. (C) Gsp1p–GTP enhances binding of Nup2p to Nup60p in yeast extracts. GST-Nup60p (1 μg) was immobilized on beads and was incubated with yeast extract (∼1 mg) supplemented with 1.25 μM recombinant Gsp1p–GTP (Q71L), 0.5 μM recombinant Kap60p, or no additional protein. The amount of Nup2p bound to Nup60p-coated beads was determined by quantitative Western blotting as described in Materials and methods. The amount of Nup2p was expressed as the ratio of Nup2p bound per unit of immobilized GST–Nup60p, using the incubation of extract without additions as baseline. Shown are the mean ratios for two samples with error bars representing the SEM; this experiment was performed three times with similar results. The asterisks (***) indicate a P < 0.05 for comparison of mean Nup2p captured from extracts supplemented or not with additional Gsp1p–GTP (unpaired, two-tailed t test). Note that addition of Gsp1p–GTP to yeast extract increases by ∼65% the amount of Nup2p bound to Nup60p-coated beads. (D) Gsp1p–GTP increases the affinity between Nup60p and Nup2p. Nup60p-coated beads were incubated with various concentrations of radiolabeled Nup2p for 2 h at 25°C in binding buffer with 10 mg/ml BSA and protease inhibitors. The concentration of GST–Nup60p within the beads was 25 nM and 150 nM for experiments with or without Gsp1p–GTP, respectively. The dissociation constant (KD) of the Nup60p–Nup2p complex in the presence and absence of 3 μM Gsp1p–GTP Q71L was calculated as described in Materials and methods. To facilitate comparison, the results were plotted as a fraction of maximal Nup2p bound versus Nup2p concentration. Each data point was performed in duplicate and error bars represent SEM. Note the 10-fold higher affinity between Nup60p and Nup2p in the presence of Gsp1p–GTP.

Mentions: The cellular mislocalization of Nup2p in yeast lacking Nup60p (Fig. 2) and the fact that Nup2p remains bound to Nup60p even in 1 M NaCl (Fig. 1 B, bottom) imply a direct association of Nup2p and Nup60p at the NPC. To test for a direct interaction, immobilized GST–Nup60p was incubated with purified recombinant Nup2p in solution. Nup2p binds tightly to Nup60p (KD ∼396 nM) in the absence of other proteins (Fig. 3, A and D) . To map the region of Nup60p that binds Nup2p, several Nup60p fragments were expressed as GST fusions and were incubated with recombinant Nup2p. As illustrated in Fig. 6 , Nup2p binds weakly to a central region of Nup60p (amino acids [aa] 188–388) and did not bind the NH2 terminus or COOH terminus of Nup60p alone. However, Nup60p fragments containing the middle region and the NH2 or COOH terminus bound Nup2p to the same levels as full-length Nup60p (Fig. 6 B). The same Nup60p fragments also captured Nup2p from yeast extracts with similar results (data not shown). Other Nup60p-interacting proteins bind to different regions of Nup60p; for example, Kap123p in yeast extracts binds selectively to the NH2 terminus of Nup60p (aa 1–187) and all Nup60p fragments containing the NH2 terminus (Fig. 6).


The nucleoporin Nup60p functions as a Gsp1p-GTP-sensitive tether for Nup2p at the nuclear pore complex.

Denning D, Mykytka B, Allen NP, Huang L - J. Cell Biol. (2001)

Gsp1p–GTP and Kap60p modulate the interaction between Nup60p and Nup2p. (A) The interaction between Nup2p and Nup60p, and the effect of Kap60p. GST–Nup60p (1 μg) was immobilized on beads and incubated with Nup2p (0.5 μg), Nup2pΔ (aa 1–50) (0.5 μg), or Kap60p (1 μg) as indicated. After 1 h at 4°C, unbound and bound proteins were collected, resolved by SDS-PAGE, and visualized with Coomassie blue. Note that Nup2p binds Nup60p, that Kap60p prevents the interaction, and that the NH2 terminus of Nup2p is not required for binding Nup60p. (B) Effect of Gsp1p–GTP on the interaction between Nup2p and Nup60p. GST–Nup60p (1 μg) was immobilized on beads and incubated with Nup2p (0.5 μg), Kap60p (1 μg), or Gsp1p–GTP (His-Gsp1p Q71L) (1 μg) as before. Note that Kap60p interferes with the interaction of Nup2p with Nup60p, but that the presence of Gsp1p–GTP restores binding and promotes formation of Nup60p–Gsp1p–Nup2p–Kap60p complexes. (C) Gsp1p–GTP enhances binding of Nup2p to Nup60p in yeast extracts. GST-Nup60p (1 μg) was immobilized on beads and was incubated with yeast extract (∼1 mg) supplemented with 1.25 μM recombinant Gsp1p–GTP (Q71L), 0.5 μM recombinant Kap60p, or no additional protein. The amount of Nup2p bound to Nup60p-coated beads was determined by quantitative Western blotting as described in Materials and methods. The amount of Nup2p was expressed as the ratio of Nup2p bound per unit of immobilized GST–Nup60p, using the incubation of extract without additions as baseline. Shown are the mean ratios for two samples with error bars representing the SEM; this experiment was performed three times with similar results. The asterisks (***) indicate a P < 0.05 for comparison of mean Nup2p captured from extracts supplemented or not with additional Gsp1p–GTP (unpaired, two-tailed t test). Note that addition of Gsp1p–GTP to yeast extract increases by ∼65% the amount of Nup2p bound to Nup60p-coated beads. (D) Gsp1p–GTP increases the affinity between Nup60p and Nup2p. Nup60p-coated beads were incubated with various concentrations of radiolabeled Nup2p for 2 h at 25°C in binding buffer with 10 mg/ml BSA and protease inhibitors. The concentration of GST–Nup60p within the beads was 25 nM and 150 nM for experiments with or without Gsp1p–GTP, respectively. The dissociation constant (KD) of the Nup60p–Nup2p complex in the presence and absence of 3 μM Gsp1p–GTP Q71L was calculated as described in Materials and methods. To facilitate comparison, the results were plotted as a fraction of maximal Nup2p bound versus Nup2p concentration. Each data point was performed in duplicate and error bars represent SEM. Note the 10-fold higher affinity between Nup60p and Nup2p in the presence of Gsp1p–GTP.
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fig3: Gsp1p–GTP and Kap60p modulate the interaction between Nup60p and Nup2p. (A) The interaction between Nup2p and Nup60p, and the effect of Kap60p. GST–Nup60p (1 μg) was immobilized on beads and incubated with Nup2p (0.5 μg), Nup2pΔ (aa 1–50) (0.5 μg), or Kap60p (1 μg) as indicated. After 1 h at 4°C, unbound and bound proteins were collected, resolved by SDS-PAGE, and visualized with Coomassie blue. Note that Nup2p binds Nup60p, that Kap60p prevents the interaction, and that the NH2 terminus of Nup2p is not required for binding Nup60p. (B) Effect of Gsp1p–GTP on the interaction between Nup2p and Nup60p. GST–Nup60p (1 μg) was immobilized on beads and incubated with Nup2p (0.5 μg), Kap60p (1 μg), or Gsp1p–GTP (His-Gsp1p Q71L) (1 μg) as before. Note that Kap60p interferes with the interaction of Nup2p with Nup60p, but that the presence of Gsp1p–GTP restores binding and promotes formation of Nup60p–Gsp1p–Nup2p–Kap60p complexes. (C) Gsp1p–GTP enhances binding of Nup2p to Nup60p in yeast extracts. GST-Nup60p (1 μg) was immobilized on beads and was incubated with yeast extract (∼1 mg) supplemented with 1.25 μM recombinant Gsp1p–GTP (Q71L), 0.5 μM recombinant Kap60p, or no additional protein. The amount of Nup2p bound to Nup60p-coated beads was determined by quantitative Western blotting as described in Materials and methods. The amount of Nup2p was expressed as the ratio of Nup2p bound per unit of immobilized GST–Nup60p, using the incubation of extract without additions as baseline. Shown are the mean ratios for two samples with error bars representing the SEM; this experiment was performed three times with similar results. The asterisks (***) indicate a P < 0.05 for comparison of mean Nup2p captured from extracts supplemented or not with additional Gsp1p–GTP (unpaired, two-tailed t test). Note that addition of Gsp1p–GTP to yeast extract increases by ∼65% the amount of Nup2p bound to Nup60p-coated beads. (D) Gsp1p–GTP increases the affinity between Nup60p and Nup2p. Nup60p-coated beads were incubated with various concentrations of radiolabeled Nup2p for 2 h at 25°C in binding buffer with 10 mg/ml BSA and protease inhibitors. The concentration of GST–Nup60p within the beads was 25 nM and 150 nM for experiments with or without Gsp1p–GTP, respectively. The dissociation constant (KD) of the Nup60p–Nup2p complex in the presence and absence of 3 μM Gsp1p–GTP Q71L was calculated as described in Materials and methods. To facilitate comparison, the results were plotted as a fraction of maximal Nup2p bound versus Nup2p concentration. Each data point was performed in duplicate and error bars represent SEM. Note the 10-fold higher affinity between Nup60p and Nup2p in the presence of Gsp1p–GTP.
Mentions: The cellular mislocalization of Nup2p in yeast lacking Nup60p (Fig. 2) and the fact that Nup2p remains bound to Nup60p even in 1 M NaCl (Fig. 1 B, bottom) imply a direct association of Nup2p and Nup60p at the NPC. To test for a direct interaction, immobilized GST–Nup60p was incubated with purified recombinant Nup2p in solution. Nup2p binds tightly to Nup60p (KD ∼396 nM) in the absence of other proteins (Fig. 3, A and D) . To map the region of Nup60p that binds Nup2p, several Nup60p fragments were expressed as GST fusions and were incubated with recombinant Nup2p. As illustrated in Fig. 6 , Nup2p binds weakly to a central region of Nup60p (amino acids [aa] 188–388) and did not bind the NH2 terminus or COOH terminus of Nup60p alone. However, Nup60p fragments containing the middle region and the NH2 or COOH terminus bound Nup2p to the same levels as full-length Nup60p (Fig. 6 B). The same Nup60p fragments also captured Nup2p from yeast extracts with similar results (data not shown). Other Nup60p-interacting proteins bind to different regions of Nup60p; for example, Kap123p in yeast extracts binds selectively to the NH2 terminus of Nup60p (aa 1–187) and all Nup60p fragments containing the NH2 terminus (Fig. 6).

Bottom Line: Yeast lacking Nup60p also fail to anchor Nup2p at the NPC, resulting in the mislocalization of Nup2p to the nucleoplasm and cytoplasm.Gsp1p-GTP enhances by 10-fold the affinity between Nup60p and Nup2p, and restores binding of Nup2p-Kap60p complexes to Nup60p.The results suggest a dynamic interaction, controlled by the nucleoplasmic concentration of Gsp1p-GTP, between Nup60p and Nup2p at the NPC.

View Article: PubMed Central - PubMed

Affiliation: Cancer Biology Program, Stanford Medical School, Stanford University, CA 94305, USA.

ABSTRACT
The nucleoporins Nup60p, Nup2p, and Nup1p form part of the nuclear basket structure of the Saccharomyces cerevisiae nuclear pore complex (NPC). Here, we show that these necleoporins can be isolated from yeast extracts by affinity chromatography on karyopherin Kap95p-coated beads. To characterize Nup60p further, Nup60p-coated beads were used to capture its interacting proteins from extracts. We find that Nup60p binds to Nup2p and serves as a docking site for Kap95p-Kap60p heterodimers and Kap123p. Nup60p also binds Gsp1p-GTP and its guanine nucleotide exchange factor Prp20p, and functions as a Gsp1p guanine nucleotide dissociation inhibitor by reducing the activity of Prp20p. Yeast lacking Nup60p exhibit minor defects in nuclear export of Kap60p, nuclear import of Kap95p-Kap60p-dependent cargoes, and diffusion of small proteins across the NPC. Yeast lacking Nup60p also fail to anchor Nup2p at the NPC, resulting in the mislocalization of Nup2p to the nucleoplasm and cytoplasm. Purified Nup60p and Nup2p bind each other directly, but the stability of the complex is compromised when Kap60p binds Nup2p. Gsp1p-GTP enhances by 10-fold the affinity between Nup60p and Nup2p, and restores binding of Nup2p-Kap60p complexes to Nup60p. The results suggest a dynamic interaction, controlled by the nucleoplasmic concentration of Gsp1p-GTP, between Nup60p and Nup2p at the NPC.

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Related in: MedlinePlus