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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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Mapping of Nup, karyopherin, Prp20p, and Gsp1p–GTP binding sites on Nup60p. (A) The cartoon depicts the Nup60p fragments used in this study provides a summary of the binding interactions observed in B. The + and − designations provide a qualitative assessment of the binding avidity. The absence of detectable binding is denoted by −, whereas +, ++, and +++ represent relative degrees of binding. (B) Binding of karyopherins, Nup2p, Prp20p, and Gsp1p–GTP to various Nup60p fragments. Each GST–Nup60p fragment (1 μg each) was immobilized on beads and incubated with purified Nup2p, Kap95p, Kap95p–Kap60p heterodimers, Prp20p, or Gsp1p–GTP (1 μg each), as indicated. After 1 h at 4°C, the beads were washed and bound proteins were collected, resolved by SDS-PAGE, and visualized with Coomassie blue stain. Alternatively, each GST–Nup60p fragment (5 μg each) was immobilized on beads and incubated with 10 mg of yeast extract for 2 h at 4°C. After washing the beads, bound proteins were eluted with 1 M NaCl, collected by precipitation with trichloroacetic acid and deoxycholate, resolved by SDS-PAGE, and stained with Coomassie blue. Asterisks designate GST–Nup60p fragments used as bait. The superscripts “a” and “b” denote the source of proteins used in the experiments: “a” marks cases where purified recombinant proteins where used, and “b” marks cases where yeast extracts were used. Note the shift in binding site selection of Kap95p in the presence and absence of Kap60p.
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fig6: Mapping of Nup, karyopherin, Prp20p, and Gsp1p–GTP binding sites on Nup60p. (A) The cartoon depicts the Nup60p fragments used in this study provides a summary of the binding interactions observed in B. The + and − designations provide a qualitative assessment of the binding avidity. The absence of detectable binding is denoted by −, whereas +, ++, and +++ represent relative degrees of binding. (B) Binding of karyopherins, Nup2p, Prp20p, and Gsp1p–GTP to various Nup60p fragments. Each GST–Nup60p fragment (1 μg each) was immobilized on beads and incubated with purified Nup2p, Kap95p, Kap95p–Kap60p heterodimers, Prp20p, or Gsp1p–GTP (1 μg each), as indicated. After 1 h at 4°C, the beads were washed and bound proteins were collected, resolved by SDS-PAGE, and visualized with Coomassie blue stain. Alternatively, each GST–Nup60p fragment (5 μg each) was immobilized on beads and incubated with 10 mg of yeast extract for 2 h at 4°C. After washing the beads, bound proteins were eluted with 1 M NaCl, collected by precipitation with trichloroacetic acid and deoxycholate, resolved by SDS-PAGE, and stained with Coomassie blue. Asterisks designate GST–Nup60p fragments used as bait. The superscripts “a” and “b” denote the source of proteins used in the experiments: “a” marks cases where purified recombinant proteins where used, and “b” marks cases where yeast extracts were used. Note the shift in binding site selection of Kap95p in the presence and absence of Kap60p.

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)

Mapping of Nup, karyopherin, Prp20p, and Gsp1p–GTP binding sites on Nup60p. (A) The cartoon depicts the Nup60p fragments used in this study provides a summary of the binding interactions observed in B. The + and − designations provide a qualitative assessment of the binding avidity. The absence of detectable binding is denoted by −, whereas +, ++, and +++ represent relative degrees of binding. (B) Binding of karyopherins, Nup2p, Prp20p, and Gsp1p–GTP to various Nup60p fragments. Each GST–Nup60p fragment (1 μg each) was immobilized on beads and incubated with purified Nup2p, Kap95p, Kap95p–Kap60p heterodimers, Prp20p, or Gsp1p–GTP (1 μg each), as indicated. After 1 h at 4°C, the beads were washed and bound proteins were collected, resolved by SDS-PAGE, and visualized with Coomassie blue stain. Alternatively, each GST–Nup60p fragment (5 μg each) was immobilized on beads and incubated with 10 mg of yeast extract for 2 h at 4°C. After washing the beads, bound proteins were eluted with 1 M NaCl, collected by precipitation with trichloroacetic acid and deoxycholate, resolved by SDS-PAGE, and stained with Coomassie blue. Asterisks designate GST–Nup60p fragments used as bait. The superscripts “a” and “b” denote the source of proteins used in the experiments: “a” marks cases where purified recombinant proteins where used, and “b” marks cases where yeast extracts were used. Note the shift in binding site selection of Kap95p in the presence and absence of Kap60p.
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fig6: Mapping of Nup, karyopherin, Prp20p, and Gsp1p–GTP binding sites on Nup60p. (A) The cartoon depicts the Nup60p fragments used in this study provides a summary of the binding interactions observed in B. The + and − designations provide a qualitative assessment of the binding avidity. The absence of detectable binding is denoted by −, whereas +, ++, and +++ represent relative degrees of binding. (B) Binding of karyopherins, Nup2p, Prp20p, and Gsp1p–GTP to various Nup60p fragments. Each GST–Nup60p fragment (1 μg each) was immobilized on beads and incubated with purified Nup2p, Kap95p, Kap95p–Kap60p heterodimers, Prp20p, or Gsp1p–GTP (1 μg each), as indicated. After 1 h at 4°C, the beads were washed and bound proteins were collected, resolved by SDS-PAGE, and visualized with Coomassie blue stain. Alternatively, each GST–Nup60p fragment (5 μg each) was immobilized on beads and incubated with 10 mg of yeast extract for 2 h at 4°C. After washing the beads, bound proteins were eluted with 1 M NaCl, collected by precipitation with trichloroacetic acid and deoxycholate, resolved by SDS-PAGE, and stained with Coomassie blue. Asterisks designate GST–Nup60p fragments used as bait. The superscripts “a” and “b” denote the source of proteins used in the experiments: “a” marks cases where purified recombinant proteins where used, and “b” marks cases where yeast extracts were used. Note the shift in binding site selection of Kap95p in the presence and absence of Kap60p.
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.

Show MeSH
Related in: MedlinePlus