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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 is a docking site for Kap95p–Kap60p heterodimers. (A) Nup60p binds Kap95p, but only in the absence of Gsp1p–GTP. GST–Nup60p (1 μg) was immobilized on beads and incubated with Kap95p (2 μg), Kap60p (3 μg), and/or Gsp1p–GTP Q71L (0.5 μg). After 1 h at 4°C, unbound and bound proteins were collected, resolved by SDS-PAGE, and stained with Coomassie blue. Note that Kap95p monomers bind to Nup60p, that Kap60p enhances binding of Kap95p to Nup60p, and that Gsp1p–GTP blocks binding of Kap95p and Kap95p–Kap60p heterodimers to Nup60p. (B) Nup60p plays a minor role in Kap95p–Kap60p-dependent import of cNLS-bearing cargo into the nucleus. Wild-type and nup60Δ yeast expressing the SV-40 T-antigen NLS fused to GFP were metabolically poisoned to deplete intracellular ATP and assayed for recovery of nuclear import upon removal of the poison (see Materials and methods). Values plotted at the indicated time points represent the mean fraction of yeast with predominantly nucleoplasmic NLS-GFP from six separate experiments (error bars represent SEM). The asterisks (***) indicate P < 0.01 for comparison of the two values at the indicated time points (unpaired, two-tailed t test). Note that yeast lacking Nup60p exhibit a slower initial rate of cNLS–GFP nuclear import.
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fig5: Nup60p is a docking site for Kap95p–Kap60p heterodimers. (A) Nup60p binds Kap95p, but only in the absence of Gsp1p–GTP. GST–Nup60p (1 μg) was immobilized on beads and incubated with Kap95p (2 μg), Kap60p (3 μg), and/or Gsp1p–GTP Q71L (0.5 μg). After 1 h at 4°C, unbound and bound proteins were collected, resolved by SDS-PAGE, and stained with Coomassie blue. Note that Kap95p monomers bind to Nup60p, that Kap60p enhances binding of Kap95p to Nup60p, and that Gsp1p–GTP blocks binding of Kap95p and Kap95p–Kap60p heterodimers to Nup60p. (B) Nup60p plays a minor role in Kap95p–Kap60p-dependent import of cNLS-bearing cargo into the nucleus. Wild-type and nup60Δ yeast expressing the SV-40 T-antigen NLS fused to GFP were metabolically poisoned to deplete intracellular ATP and assayed for recovery of nuclear import upon removal of the poison (see Materials and methods). Values plotted at the indicated time points represent the mean fraction of yeast with predominantly nucleoplasmic NLS-GFP from six separate experiments (error bars represent SEM). The asterisks (***) indicate P < 0.01 for comparison of the two values at the indicated time points (unpaired, two-tailed t test). Note that yeast lacking Nup60p exhibit a slower initial rate of cNLS–GFP nuclear import.

Mentions: As part of our ongoing effort to characterize the mechanism of Kap95p–Kap60p-mediated transport across the NPC, we isolated and identified Kap95p (karyopherin/importin β) binding proteins from yeast extracts. Kap95p is the yeast homologue of vertebrate karyopherin β1/importin β (Enenkel et al., 1995). Glutathione S-transferase (GST)–Kap95p was immobilized on glutathione-coated Sepharose beads (beads) and added to yeast extracts to capture interacting proteins. After washing the beads, bound proteins were eluted with 250 mM MgCl2, resolved by SDS-PAGE, and identified using mass spectrometry (MALDI-TOF and LC-MS). The most abundant proteins bound to Kap95p were identified as Nup1p, Nup2p, and Kap60p (Fig. 1 A); this was not surprising as these proteins bind Kap95p directly (Rexach and Blobel, 1995). The less abundant proteins bound were Ssa1p, Cdc19p, and Tef2p. Ssa1p plays a stimulatory role in Kap60p-dependent import reactions (Shulga et al., 1996), so its association may be specific. Cdc19p (pyruvate kinase) and Tef2p (translation elongation factor) are possible contaminants due to their high abundance in the yeast cytoplasm. Notably, Nup60p is also among the less abundant proteins that bind Kap95p, but its association is specific as it binds Kap95p monomers directly (see Figs. 5 A and 6 B). All of the captured Nups (Nup60p, Nup1p, and Nup2p) are components of the nuclear basket structure of the yeast NPC (Hood et al., 2000; Rout et al., 2000). Although much is known about Nup1p and Nup2p and their functions (see Introduction), the recently identified Nup60p is less well characterized.


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 is a docking site for Kap95p–Kap60p heterodimers. (A) Nup60p binds Kap95p, but only in the absence of Gsp1p–GTP. GST–Nup60p (1 μg) was immobilized on beads and incubated with Kap95p (2 μg), Kap60p (3 μg), and/or Gsp1p–GTP Q71L (0.5 μg). After 1 h at 4°C, unbound and bound proteins were collected, resolved by SDS-PAGE, and stained with Coomassie blue. Note that Kap95p monomers bind to Nup60p, that Kap60p enhances binding of Kap95p to Nup60p, and that Gsp1p–GTP blocks binding of Kap95p and Kap95p–Kap60p heterodimers to Nup60p. (B) Nup60p plays a minor role in Kap95p–Kap60p-dependent import of cNLS-bearing cargo into the nucleus. Wild-type and nup60Δ yeast expressing the SV-40 T-antigen NLS fused to GFP were metabolically poisoned to deplete intracellular ATP and assayed for recovery of nuclear import upon removal of the poison (see Materials and methods). Values plotted at the indicated time points represent the mean fraction of yeast with predominantly nucleoplasmic NLS-GFP from six separate experiments (error bars represent SEM). The asterisks (***) indicate P < 0.01 for comparison of the two values at the indicated time points (unpaired, two-tailed t test). Note that yeast lacking Nup60p exhibit a slower initial rate of cNLS–GFP nuclear import.
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Related In: Results  -  Collection

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fig5: Nup60p is a docking site for Kap95p–Kap60p heterodimers. (A) Nup60p binds Kap95p, but only in the absence of Gsp1p–GTP. GST–Nup60p (1 μg) was immobilized on beads and incubated with Kap95p (2 μg), Kap60p (3 μg), and/or Gsp1p–GTP Q71L (0.5 μg). After 1 h at 4°C, unbound and bound proteins were collected, resolved by SDS-PAGE, and stained with Coomassie blue. Note that Kap95p monomers bind to Nup60p, that Kap60p enhances binding of Kap95p to Nup60p, and that Gsp1p–GTP blocks binding of Kap95p and Kap95p–Kap60p heterodimers to Nup60p. (B) Nup60p plays a minor role in Kap95p–Kap60p-dependent import of cNLS-bearing cargo into the nucleus. Wild-type and nup60Δ yeast expressing the SV-40 T-antigen NLS fused to GFP were metabolically poisoned to deplete intracellular ATP and assayed for recovery of nuclear import upon removal of the poison (see Materials and methods). Values plotted at the indicated time points represent the mean fraction of yeast with predominantly nucleoplasmic NLS-GFP from six separate experiments (error bars represent SEM). The asterisks (***) indicate P < 0.01 for comparison of the two values at the indicated time points (unpaired, two-tailed t test). Note that yeast lacking Nup60p exhibit a slower initial rate of cNLS–GFP nuclear import.
Mentions: As part of our ongoing effort to characterize the mechanism of Kap95p–Kap60p-mediated transport across the NPC, we isolated and identified Kap95p (karyopherin/importin β) binding proteins from yeast extracts. Kap95p is the yeast homologue of vertebrate karyopherin β1/importin β (Enenkel et al., 1995). Glutathione S-transferase (GST)–Kap95p was immobilized on glutathione-coated Sepharose beads (beads) and added to yeast extracts to capture interacting proteins. After washing the beads, bound proteins were eluted with 250 mM MgCl2, resolved by SDS-PAGE, and identified using mass spectrometry (MALDI-TOF and LC-MS). The most abundant proteins bound to Kap95p were identified as Nup1p, Nup2p, and Kap60p (Fig. 1 A); this was not surprising as these proteins bind Kap95p directly (Rexach and Blobel, 1995). The less abundant proteins bound were Ssa1p, Cdc19p, and Tef2p. Ssa1p plays a stimulatory role in Kap60p-dependent import reactions (Shulga et al., 1996), so its association may be specific. Cdc19p (pyruvate kinase) and Tef2p (translation elongation factor) are possible contaminants due to their high abundance in the yeast cytoplasm. Notably, Nup60p is also among the less abundant proteins that bind Kap95p, but its association is specific as it binds Kap95p monomers directly (see Figs. 5 A and 6 B). All of the captured Nups (Nup60p, Nup1p, and Nup2p) are components of the nuclear basket structure of the yeast NPC (Hood et al., 2000; Rout et al., 2000). Although much is known about Nup1p and Nup2p and their functions (see Introduction), the recently identified Nup60p is less well characterized.

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