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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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Related in: MedlinePlus

A cartoon depicting the NPC and the proposed role of Nup60p in nuclear import and export reactions.
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fig8: A cartoon depicting the NPC and the proposed role of Nup60p in nuclear import and export reactions.

Mentions: The cartoon in Fig. 8 depicts the proposed role of Nup60p in nuclear import and export reactions. It highlights the Nup60p–Gsp1p–Nup2p complex as an arrival terminal for incoming Kap95p–Kap60p cargo complexes (bottom half), and highlights the Nup60p–Gsp1p–Nup2p–Kap60p complex as a departure terminal for Kap60p–Cse1p–Gsp1p–GTP complexes (top half). Bottom half: an incoming Kap95p–Kap60p cargo complex binds to an FG Nup facing the nucleoplasmic side of the NPC (step 1); it then hops to a Nup60p–Gsp1p–GTP–Nup2p complex in the nuclear basket structure (step 2), where docking can occur via Kap95p–Kap60p binding to Nup60p or Nup2p. Prp20p bound to Nup60p generates Gsp1p–GTP locally and Gsp1p–GTP binds to Kap95p (step 3) to induce the release of the Kap60p–cargo complex from Kap95p (step 4; Rexach and Blobel, 1995). Upon dissociation from Kap95p, Kap60p lowers its affinity for cargo and binds Nup2p (step 5). In the absence of Gsp1p–GTP, Kap60p binding to Nup2p causes its dissociation from Nup60p (step 4), and the resulting Kap60p–Nup2p complex is free to diffuse in the nucleoplasm. Top half: Prp20p bound to Nup60p generates Gsp1p–GTP locally to promote binding of the Nup2p–Kap60p complexes to Nup60p, forming a Nup60p–Gsp1p–Nup2p–Kap60p complex (step 6). This complex functions as a platform for the assembly and departure of Kap60p–Cse1p–Gsp1p–GTP complexes from the nucleus (step 7). After donating Kap60p and Gsp1p–GTP to Cse1p, the resulting Nup60p–Nup2p complex becomes available for another round of Kap95p–Kap60p import and Kap60p export.


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)

A cartoon depicting the NPC and the proposed role of Nup60p in nuclear import and export reactions.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196189&req=5

fig8: A cartoon depicting the NPC and the proposed role of Nup60p in nuclear import and export reactions.
Mentions: The cartoon in Fig. 8 depicts the proposed role of Nup60p in nuclear import and export reactions. It highlights the Nup60p–Gsp1p–Nup2p complex as an arrival terminal for incoming Kap95p–Kap60p cargo complexes (bottom half), and highlights the Nup60p–Gsp1p–Nup2p–Kap60p complex as a departure terminal for Kap60p–Cse1p–Gsp1p–GTP complexes (top half). Bottom half: an incoming Kap95p–Kap60p cargo complex binds to an FG Nup facing the nucleoplasmic side of the NPC (step 1); it then hops to a Nup60p–Gsp1p–GTP–Nup2p complex in the nuclear basket structure (step 2), where docking can occur via Kap95p–Kap60p binding to Nup60p or Nup2p. Prp20p bound to Nup60p generates Gsp1p–GTP locally and Gsp1p–GTP binds to Kap95p (step 3) to induce the release of the Kap60p–cargo complex from Kap95p (step 4; Rexach and Blobel, 1995). Upon dissociation from Kap95p, Kap60p lowers its affinity for cargo and binds Nup2p (step 5). In the absence of Gsp1p–GTP, Kap60p binding to Nup2p causes its dissociation from Nup60p (step 4), and the resulting Kap60p–Nup2p complex is free to diffuse in the nucleoplasm. Top half: Prp20p bound to Nup60p generates Gsp1p–GTP locally to promote binding of the Nup2p–Kap60p complexes to Nup60p, forming a Nup60p–Gsp1p–Nup2p–Kap60p complex (step 6). This complex functions as a platform for the assembly and departure of Kap60p–Cse1p–Gsp1p–GTP complexes from the nucleus (step 7). After donating Kap60p and Gsp1p–GTP to Cse1p, the resulting Nup60p–Nup2p complex becomes available for another round of Kap95p–Kap60p import and Kap60p export.

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