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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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Nup60p binds Gsp1p–GTP and Prp20p, and functions as a Gsp1p GDI. (A) Nup60p binds Gsp1p–GTP. GST-Nup60p (1 μg) was immobilized on beads and incubated with His-Gsp1p (2 μg) preloaded with GTP or GDP. After 1 h at 4°C, bound and unbound proteins were resolved by SDS-PAGE and visualized with Coomassie blue. Note that Nup60p binds Gsp1p–GTP, but not Gsp1p–GDP. (B) Affinity of Gsp1p–GTP to Nup60p, Nup2p, and Nup60p–Nup2p complexes. GST–Nup-coated beads were incubated with various concentrations of His-Gsp1p–[γ-32P]GTP for 2 h at 4°C in binding buffer with 10 mg/ml BSA and protease inhibitors. The concentrations of GST–Nup60p and GST–Nup2p within beads were 800 nM and 1.5 μM, respectively. The dissociation constants (KD) of the Nup2p–Gsp1p–GTP complex and the Nup60p–Gsp1p–GTP complex in the presence and absence of 500 nM Nup2p were calculated as described in Materials and methods. Results were plotted as a fraction of maximal Gsp1p–GTP bound versus Gsp1p–GTP concentration. Each data point was performed in duplicate and the error bars represent SEM. Note that Nup60p and Nup2p cooperate to bind Gsp1p–GTP. (C) Nup60p inhibits the Prp20p-stimulated release of GTP from Gsp1p. His-Gsp1p–[γ-32P]GTP immobilized on nickel-coated agarose beads (15 nM Gsp1p–GTP within the beads) was incubated with 0.9 nM Prp20p and 1 mM GDP, plus 4 μM GST–Nup60p (aa 188–539), Yrb1p, Nup2p, Kap95p, or GST. GST–Nup60p (aa 188–539) (indicated by asterisk) was used instead of full-length Nup60p due to its superior solubility and protease resistance. After 10 min, Prp20p activity was stopped with ice-cold buffer, beads were washed, and the [γ-32P]GTP that remained bound to the beads was quantified by scintillation counting. Each data point was performed in duplicate and error bars represent SEM. Note that Nup60p reduces (but does not abolish) the activity of Prp20p. (D) Nup60p binds Prp20p. GST–Nup60p (1 μg) was immobilized on beads and incubated with purified Prp20p (1 μg) in the presence or absence of DNAse I and RNAse I (1 U and 1 μg, respectively). After 1 h at 4°C, unbound and bound proteins were resolved by SDS-PAGE and visualized with Coomassie blue staining. Note that purified Prp20p binds Nup60p.
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fig4: Nup60p binds Gsp1p–GTP and Prp20p, and functions as a Gsp1p GDI. (A) Nup60p binds Gsp1p–GTP. GST-Nup60p (1 μg) was immobilized on beads and incubated with His-Gsp1p (2 μg) preloaded with GTP or GDP. After 1 h at 4°C, bound and unbound proteins were resolved by SDS-PAGE and visualized with Coomassie blue. Note that Nup60p binds Gsp1p–GTP, but not Gsp1p–GDP. (B) Affinity of Gsp1p–GTP to Nup60p, Nup2p, and Nup60p–Nup2p complexes. GST–Nup-coated beads were incubated with various concentrations of His-Gsp1p–[γ-32P]GTP for 2 h at 4°C in binding buffer with 10 mg/ml BSA and protease inhibitors. The concentrations of GST–Nup60p and GST–Nup2p within beads were 800 nM and 1.5 μM, respectively. The dissociation constants (KD) of the Nup2p–Gsp1p–GTP complex and the Nup60p–Gsp1p–GTP complex in the presence and absence of 500 nM Nup2p were calculated as described in Materials and methods. Results were plotted as a fraction of maximal Gsp1p–GTP bound versus Gsp1p–GTP concentration. Each data point was performed in duplicate and the error bars represent SEM. Note that Nup60p and Nup2p cooperate to bind Gsp1p–GTP. (C) Nup60p inhibits the Prp20p-stimulated release of GTP from Gsp1p. His-Gsp1p–[γ-32P]GTP immobilized on nickel-coated agarose beads (15 nM Gsp1p–GTP within the beads) was incubated with 0.9 nM Prp20p and 1 mM GDP, plus 4 μM GST–Nup60p (aa 188–539), Yrb1p, Nup2p, Kap95p, or GST. GST–Nup60p (aa 188–539) (indicated by asterisk) was used instead of full-length Nup60p due to its superior solubility and protease resistance. After 10 min, Prp20p activity was stopped with ice-cold buffer, beads were washed, and the [γ-32P]GTP that remained bound to the beads was quantified by scintillation counting. Each data point was performed in duplicate and error bars represent SEM. Note that Nup60p reduces (but does not abolish) the activity of Prp20p. (D) Nup60p binds Prp20p. GST–Nup60p (1 μg) was immobilized on beads and incubated with purified Prp20p (1 μg) in the presence or absence of DNAse I and RNAse I (1 U and 1 μg, respectively). After 1 h at 4°C, unbound and bound proteins were resolved by SDS-PAGE and visualized with Coomassie blue staining. Note that purified Prp20p binds Nup60p.

Mentions: An unexpectedly large amount of Gsp1p–GTP bound to Nup60p-coated beads containing substoichiometric amounts of Nup2p (Fig. 3 B, lane 3), more than could be explained by Gsp1p–GTP binding to Nup2p alone. To test whether Nup60p binds Gsp1p directly, Nup60p-coated beads were incubated with purified Gsp1p–GTP or Gsp1p–GDP. Surprisingly, Gsp1p–GTP (but not Gsp1p–GDP) binds to Nup60p in the absence of additional proteins (Fig. 4 A). We calculated the affinity of Gsp1p–GTP towards Nup60p as KD ∼5 μM, a value that is similar to the calculated affinity of Gsp1p–GTP towards Nup2p (KD ∼3.6 μM) (Fig. 4 B). These low affinities are physiologically relevant, as we estimate the concentration of Gsp1p–GTP in yeast nuclei to be ∼1–10 μM based on the reported number of Gsp1p molecules per cell (26,300) (Gygi et al., 1999) and assuming that most Gsp1p is concentrated in the nucleoplasm in the GTP-bound form. Therefore, it is possible that Nup60p and Nup2p “sense” minor fluctuations in the concentration of Gsp1p–GTP at the nuclear basket structure of the NPC.


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)

Nup60p binds Gsp1p–GTP and Prp20p, and functions as a Gsp1p GDI. (A) Nup60p binds Gsp1p–GTP. GST-Nup60p (1 μg) was immobilized on beads and incubated with His-Gsp1p (2 μg) preloaded with GTP or GDP. After 1 h at 4°C, bound and unbound proteins were resolved by SDS-PAGE and visualized with Coomassie blue. Note that Nup60p binds Gsp1p–GTP, but not Gsp1p–GDP. (B) Affinity of Gsp1p–GTP to Nup60p, Nup2p, and Nup60p–Nup2p complexes. GST–Nup-coated beads were incubated with various concentrations of His-Gsp1p–[γ-32P]GTP for 2 h at 4°C in binding buffer with 10 mg/ml BSA and protease inhibitors. The concentrations of GST–Nup60p and GST–Nup2p within beads were 800 nM and 1.5 μM, respectively. The dissociation constants (KD) of the Nup2p–Gsp1p–GTP complex and the Nup60p–Gsp1p–GTP complex in the presence and absence of 500 nM Nup2p were calculated as described in Materials and methods. Results were plotted as a fraction of maximal Gsp1p–GTP bound versus Gsp1p–GTP concentration. Each data point was performed in duplicate and the error bars represent SEM. Note that Nup60p and Nup2p cooperate to bind Gsp1p–GTP. (C) Nup60p inhibits the Prp20p-stimulated release of GTP from Gsp1p. His-Gsp1p–[γ-32P]GTP immobilized on nickel-coated agarose beads (15 nM Gsp1p–GTP within the beads) was incubated with 0.9 nM Prp20p and 1 mM GDP, plus 4 μM GST–Nup60p (aa 188–539), Yrb1p, Nup2p, Kap95p, or GST. GST–Nup60p (aa 188–539) (indicated by asterisk) was used instead of full-length Nup60p due to its superior solubility and protease resistance. After 10 min, Prp20p activity was stopped with ice-cold buffer, beads were washed, and the [γ-32P]GTP that remained bound to the beads was quantified by scintillation counting. Each data point was performed in duplicate and error bars represent SEM. Note that Nup60p reduces (but does not abolish) the activity of Prp20p. (D) Nup60p binds Prp20p. GST–Nup60p (1 μg) was immobilized on beads and incubated with purified Prp20p (1 μg) in the presence or absence of DNAse I and RNAse I (1 U and 1 μg, respectively). After 1 h at 4°C, unbound and bound proteins were resolved by SDS-PAGE and visualized with Coomassie blue staining. Note that purified Prp20p binds Nup60p.
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fig4: Nup60p binds Gsp1p–GTP and Prp20p, and functions as a Gsp1p GDI. (A) Nup60p binds Gsp1p–GTP. GST-Nup60p (1 μg) was immobilized on beads and incubated with His-Gsp1p (2 μg) preloaded with GTP or GDP. After 1 h at 4°C, bound and unbound proteins were resolved by SDS-PAGE and visualized with Coomassie blue. Note that Nup60p binds Gsp1p–GTP, but not Gsp1p–GDP. (B) Affinity of Gsp1p–GTP to Nup60p, Nup2p, and Nup60p–Nup2p complexes. GST–Nup-coated beads were incubated with various concentrations of His-Gsp1p–[γ-32P]GTP for 2 h at 4°C in binding buffer with 10 mg/ml BSA and protease inhibitors. The concentrations of GST–Nup60p and GST–Nup2p within beads were 800 nM and 1.5 μM, respectively. The dissociation constants (KD) of the Nup2p–Gsp1p–GTP complex and the Nup60p–Gsp1p–GTP complex in the presence and absence of 500 nM Nup2p were calculated as described in Materials and methods. Results were plotted as a fraction of maximal Gsp1p–GTP bound versus Gsp1p–GTP concentration. Each data point was performed in duplicate and the error bars represent SEM. Note that Nup60p and Nup2p cooperate to bind Gsp1p–GTP. (C) Nup60p inhibits the Prp20p-stimulated release of GTP from Gsp1p. His-Gsp1p–[γ-32P]GTP immobilized on nickel-coated agarose beads (15 nM Gsp1p–GTP within the beads) was incubated with 0.9 nM Prp20p and 1 mM GDP, plus 4 μM GST–Nup60p (aa 188–539), Yrb1p, Nup2p, Kap95p, or GST. GST–Nup60p (aa 188–539) (indicated by asterisk) was used instead of full-length Nup60p due to its superior solubility and protease resistance. After 10 min, Prp20p activity was stopped with ice-cold buffer, beads were washed, and the [γ-32P]GTP that remained bound to the beads was quantified by scintillation counting. Each data point was performed in duplicate and error bars represent SEM. Note that Nup60p reduces (but does not abolish) the activity of Prp20p. (D) Nup60p binds Prp20p. GST–Nup60p (1 μg) was immobilized on beads and incubated with purified Prp20p (1 μg) in the presence or absence of DNAse I and RNAse I (1 U and 1 μg, respectively). After 1 h at 4°C, unbound and bound proteins were resolved by SDS-PAGE and visualized with Coomassie blue staining. Note that purified Prp20p binds Nup60p.
Mentions: An unexpectedly large amount of Gsp1p–GTP bound to Nup60p-coated beads containing substoichiometric amounts of Nup2p (Fig. 3 B, lane 3), more than could be explained by Gsp1p–GTP binding to Nup2p alone. To test whether Nup60p binds Gsp1p directly, Nup60p-coated beads were incubated with purified Gsp1p–GTP or Gsp1p–GDP. Surprisingly, Gsp1p–GTP (but not Gsp1p–GDP) binds to Nup60p in the absence of additional proteins (Fig. 4 A). We calculated the affinity of Gsp1p–GTP towards Nup60p as KD ∼5 μM, a value that is similar to the calculated affinity of Gsp1p–GTP towards Nup2p (KD ∼3.6 μM) (Fig. 4 B). These low affinities are physiologically relevant, as we estimate the concentration of Gsp1p–GTP in yeast nuclei to be ∼1–10 μM based on the reported number of Gsp1p molecules per cell (26,300) (Gygi et al., 1999) and assuming that most Gsp1p is concentrated in the nucleoplasm in the GTP-bound form. Therefore, it is possible that Nup60p and Nup2p “sense” minor fluctuations in the concentration of Gsp1p–GTP at the nuclear basket structure of the NPC.

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