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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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Nup2p is tethered to the NPC via Nup60p. (A) Direct visualization of Nup2p–GFP fusions in yeast. Various yeast strains that express NUP2-GFP from the NUP2 locus were grown in rich media at 30°C and were observed live under a fluorescence microscope. DAPI stain was used to visualize DNA in nuclei and pictures were taken using nuclei as the focal point. Note that in wild-type, nup170Δ, and nup100Δ yeast, Nup2p–GFP fusions accumulate in a punctate pattern at the nuclear periphery, but in nup60Δ yeast Nup2p–GFP is mislocalized to the nucleoplasm and cytoplasm. (B) Indirect immunofluorescence visualization of Nup2p, Nup1p, and Nup100p/Nup116p in nup60Δ yeast. nup60Δ yeast grown to early log phase in rich media at 30°C were fixed in 3.7% formaldehyde for 10 min and processed for immunofluorescence microscopy using affinity-purified anti-Nup antibodies and FITC-labeled secondary antibodies (left). DAPI was used to visualize nuclei (right). Note the mislocalization of Nup2p to the nucleoplasm in nup60Δ yeast in contrast to the normal punctate staining of Nup1p and Nup100p/Nup116p at the nuclear envelope.
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fig2: Nup2p is tethered to the NPC via Nup60p. (A) Direct visualization of Nup2p–GFP fusions in yeast. Various yeast strains that express NUP2-GFP from the NUP2 locus were grown in rich media at 30°C and were observed live under a fluorescence microscope. DAPI stain was used to visualize DNA in nuclei and pictures were taken using nuclei as the focal point. Note that in wild-type, nup170Δ, and nup100Δ yeast, Nup2p–GFP fusions accumulate in a punctate pattern at the nuclear periphery, but in nup60Δ yeast Nup2p–GFP is mislocalized to the nucleoplasm and cytoplasm. (B) Indirect immunofluorescence visualization of Nup2p, Nup1p, and Nup100p/Nup116p in nup60Δ yeast. nup60Δ yeast grown to early log phase in rich media at 30°C were fixed in 3.7% formaldehyde for 10 min and processed for immunofluorescence microscopy using affinity-purified anti-Nup antibodies and FITC-labeled secondary antibodies (left). DAPI was used to visualize nuclei (right). Note the mislocalization of Nup2p to the nucleoplasm in nup60Δ yeast in contrast to the normal punctate staining of Nup1p and Nup100p/Nup116p at the nuclear envelope.

Mentions: To test the proposed interaction between Nup60p and Nup2p in vivo, NUP2 was replaced with NUP2-GFP in wild-type yeast and yeast lacking Nup60p (nup60Δ). The Nup2p–GFP fusion was visualized by fluorescence microscopy. As expected for wild-type yeast, Nup2p–GFP localizes to the nuclear envelope in a punctate pattern typical of Nups (Fig. 2 A, top). In contrast, Nup2p–GFP clearly mislocalizes in nup60Δ yeast, and is observed primarily in the nucleoplasm and partially in the cytoplasm (Fig. 2 A, middle). A small fraction of Nup2p–GFP may have remained bound to the NPC in the nup60Δ yeast; however, as the majority of Nup2p–GFP fails to associate with the nuclear envelope in nup60Δ yeast, we conclude that Nup60p is the primary tethering site for Nup2p at the NPC (also see below).


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)

Nup2p is tethered to the NPC via Nup60p. (A) Direct visualization of Nup2p–GFP fusions in yeast. Various yeast strains that express NUP2-GFP from the NUP2 locus were grown in rich media at 30°C and were observed live under a fluorescence microscope. DAPI stain was used to visualize DNA in nuclei and pictures were taken using nuclei as the focal point. Note that in wild-type, nup170Δ, and nup100Δ yeast, Nup2p–GFP fusions accumulate in a punctate pattern at the nuclear periphery, but in nup60Δ yeast Nup2p–GFP is mislocalized to the nucleoplasm and cytoplasm. (B) Indirect immunofluorescence visualization of Nup2p, Nup1p, and Nup100p/Nup116p in nup60Δ yeast. nup60Δ yeast grown to early log phase in rich media at 30°C were fixed in 3.7% formaldehyde for 10 min and processed for immunofluorescence microscopy using affinity-purified anti-Nup antibodies and FITC-labeled secondary antibodies (left). DAPI was used to visualize nuclei (right). Note the mislocalization of Nup2p to the nucleoplasm in nup60Δ yeast in contrast to the normal punctate staining of Nup1p and Nup100p/Nup116p at the nuclear envelope.
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Related In: Results  -  Collection

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fig2: Nup2p is tethered to the NPC via Nup60p. (A) Direct visualization of Nup2p–GFP fusions in yeast. Various yeast strains that express NUP2-GFP from the NUP2 locus were grown in rich media at 30°C and were observed live under a fluorescence microscope. DAPI stain was used to visualize DNA in nuclei and pictures were taken using nuclei as the focal point. Note that in wild-type, nup170Δ, and nup100Δ yeast, Nup2p–GFP fusions accumulate in a punctate pattern at the nuclear periphery, but in nup60Δ yeast Nup2p–GFP is mislocalized to the nucleoplasm and cytoplasm. (B) Indirect immunofluorescence visualization of Nup2p, Nup1p, and Nup100p/Nup116p in nup60Δ yeast. nup60Δ yeast grown to early log phase in rich media at 30°C were fixed in 3.7% formaldehyde for 10 min and processed for immunofluorescence microscopy using affinity-purified anti-Nup antibodies and FITC-labeled secondary antibodies (left). DAPI was used to visualize nuclei (right). Note the mislocalization of Nup2p to the nucleoplasm in nup60Δ yeast in contrast to the normal punctate staining of Nup1p and Nup100p/Nup116p at the nuclear envelope.
Mentions: To test the proposed interaction between Nup60p and Nup2p in vivo, NUP2 was replaced with NUP2-GFP in wild-type yeast and yeast lacking Nup60p (nup60Δ). The Nup2p–GFP fusion was visualized by fluorescence microscopy. As expected for wild-type yeast, Nup2p–GFP localizes to the nuclear envelope in a punctate pattern typical of Nups (Fig. 2 A, top). In contrast, Nup2p–GFP clearly mislocalizes in nup60Δ yeast, and is observed primarily in the nucleoplasm and partially in the cytoplasm (Fig. 2 A, middle). A small fraction of Nup2p–GFP may have remained bound to the NPC in the nup60Δ yeast; however, as the majority of Nup2p–GFP fails to associate with the nuclear envelope in nup60Δ yeast, we conclude that Nup60p is the primary tethering site for Nup2p at the NPC (also see below).

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