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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 plays a role in the nuclear export of Kap60p. (A) The location of Kap60p in wild-type and nup60Δ yeast was detected by indirect immunofluorescence using affinity-purified anti-Kap60p antibodies. Yeast grown to early log phase at 30°C in rich media were fixed in 3.7% formaldehyde for 1 h and processed for immunofluorescence microscopy (left). Note the moderate accumulation of Kap60p in nuclei of nup60Δ yeast compared with wild-type. (B) Cse1p accepts Kap60p and Gsp1p–GTP from a donor Nup2p–Gsp1p–GTP–Nup2p–Kap60p complex in vitro. GST–Nup60p (1 μg) was immobilized on beads and incubated with Nup2p (2 μg), Gsp1p–GTP (Q71L) (2 μg), and Kap60p (2 μg) for 1 h at 4°C to form the Nup2p–Gsp1p–GTP–Nup2p–Kap60p complex. After washing the beads to remove unbound proteins, the quaternary complex was mixed with buffer of Cse1p (1 μg). After 1 h at 4°C, unbound and bound proteins were collected, resolved by SDS-PAGE, and stained with Coomassie blue. Note that when Cse1p is present, all of the Kap60p and some Gsp1p are lost from the immobilized Nup60p. Also note that Cse1p does not bind to Nup60p–Nup2p complexes.
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fig7: Nup60p plays a role in the nuclear export of Kap60p. (A) The location of Kap60p in wild-type and nup60Δ yeast was detected by indirect immunofluorescence using affinity-purified anti-Kap60p antibodies. Yeast grown to early log phase at 30°C in rich media were fixed in 3.7% formaldehyde for 1 h and processed for immunofluorescence microscopy (left). Note the moderate accumulation of Kap60p in nuclei of nup60Δ yeast compared with wild-type. (B) Cse1p accepts Kap60p and Gsp1p–GTP from a donor Nup2p–Gsp1p–GTP–Nup2p–Kap60p complex in vitro. GST–Nup60p (1 μg) was immobilized on beads and incubated with Nup2p (2 μg), Gsp1p–GTP (Q71L) (2 μg), and Kap60p (2 μg) for 1 h at 4°C to form the Nup2p–Gsp1p–GTP–Nup2p–Kap60p complex. After washing the beads to remove unbound proteins, the quaternary complex was mixed with buffer of Cse1p (1 μg). After 1 h at 4°C, unbound and bound proteins were collected, resolved by SDS-PAGE, and stained with Coomassie blue. Note that when Cse1p is present, all of the Kap60p and some Gsp1p are lost from the immobilized Nup60p. Also note that Cse1p does not bind to Nup60p–Nup2p complexes.

Mentions: Previous studies described a role for Nup2p in the Cse1p-dependent export of Kap60p from the nucleus (Booth et al., 1999; Hood et al., 2000; Solsbacher et al., 2000). As Nup60p tethers Nup2p to the NPC in vivo (Fig. 2 A), it may also influence the Cse1p-mediated export of Kap60p. The subcellular location of Kap60p in wild-type and nup60Δ yeast was examined by indirect immunofluorescence using specific anti-Kap60p antibodies. We find that Kap60p partially accumulates in the nucleoplasm of nup60Δ yeast, but not wild-type yeast (Fig. 7 A). Thus, yeast lacking Nup60p exhibit a mild defect in Kap60p export, likely caused by the loss of Nup2p from 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 plays a role in the nuclear export of Kap60p. (A) The location of Kap60p in wild-type and nup60Δ yeast was detected by indirect immunofluorescence using affinity-purified anti-Kap60p antibodies. Yeast grown to early log phase at 30°C in rich media were fixed in 3.7% formaldehyde for 1 h and processed for immunofluorescence microscopy (left). Note the moderate accumulation of Kap60p in nuclei of nup60Δ yeast compared with wild-type. (B) Cse1p accepts Kap60p and Gsp1p–GTP from a donor Nup2p–Gsp1p–GTP–Nup2p–Kap60p complex in vitro. GST–Nup60p (1 μg) was immobilized on beads and incubated with Nup2p (2 μg), Gsp1p–GTP (Q71L) (2 μg), and Kap60p (2 μg) for 1 h at 4°C to form the Nup2p–Gsp1p–GTP–Nup2p–Kap60p complex. After washing the beads to remove unbound proteins, the quaternary complex was mixed with buffer of Cse1p (1 μg). After 1 h at 4°C, unbound and bound proteins were collected, resolved by SDS-PAGE, and stained with Coomassie blue. Note that when Cse1p is present, all of the Kap60p and some Gsp1p are lost from the immobilized Nup60p. Also note that Cse1p does not bind to Nup60p–Nup2p complexes.
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fig7: Nup60p plays a role in the nuclear export of Kap60p. (A) The location of Kap60p in wild-type and nup60Δ yeast was detected by indirect immunofluorescence using affinity-purified anti-Kap60p antibodies. Yeast grown to early log phase at 30°C in rich media were fixed in 3.7% formaldehyde for 1 h and processed for immunofluorescence microscopy (left). Note the moderate accumulation of Kap60p in nuclei of nup60Δ yeast compared with wild-type. (B) Cse1p accepts Kap60p and Gsp1p–GTP from a donor Nup2p–Gsp1p–GTP–Nup2p–Kap60p complex in vitro. GST–Nup60p (1 μg) was immobilized on beads and incubated with Nup2p (2 μg), Gsp1p–GTP (Q71L) (2 μg), and Kap60p (2 μg) for 1 h at 4°C to form the Nup2p–Gsp1p–GTP–Nup2p–Kap60p complex. After washing the beads to remove unbound proteins, the quaternary complex was mixed with buffer of Cse1p (1 μg). After 1 h at 4°C, unbound and bound proteins were collected, resolved by SDS-PAGE, and stained with Coomassie blue. Note that when Cse1p is present, all of the Kap60p and some Gsp1p are lost from the immobilized Nup60p. Also note that Cse1p does not bind to Nup60p–Nup2p complexes.
Mentions: Previous studies described a role for Nup2p in the Cse1p-dependent export of Kap60p from the nucleus (Booth et al., 1999; Hood et al., 2000; Solsbacher et al., 2000). As Nup60p tethers Nup2p to the NPC in vivo (Fig. 2 A), it may also influence the Cse1p-mediated export of Kap60p. The subcellular location of Kap60p in wild-type and nup60Δ yeast was examined by indirect immunofluorescence using specific anti-Kap60p antibodies. We find that Kap60p partially accumulates in the nucleoplasm of nup60Δ yeast, but not wild-type yeast (Fig. 7 A). Thus, yeast lacking Nup60p exhibit a mild defect in Kap60p export, likely caused by the loss of Nup2p from 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.

Show MeSH
Related in: MedlinePlus