The nucleoporin Nup60p functions as a Gsp1p-GTP-sensitive tether for Nup2pat the nuclear pore complex

The nucleoporins Nup60p, Nup2p, and Nup1p form part of the nuclear basket s tructure of the Saccharomyces cerevisiae nuclear pore complex (NPC). Here, we show that these necleoporins can be isolated from yeast extracts by affi nity chromatography on karyopherin Kap95p-coated beads. To characterize Nup 60p further, Nup60p-coated beads were used to capture its interacting prote ins from extracts. We find that Nup60p binds to Nup2p and serves as a clock ing site for Kap95p-Kap6Op heterodimers and Kap123p. Nup60p also binds Gsp1 p-GTP and its guanine nucleotide exchange factor Prp20p, and functions as a Gsp1p guanine nucleotide dissociation inhibitor by reducing the activity o f Prp20p. Yeast lacking Nup60p exhibit minor defects in nuclear export of K ap60p, nuclear import of Kap95 p-Kap6Op-dependent cargoes, and diffusion of small proteins across the NPC. Yeast lacking Nup60p also fail to anchor Nu p2p at the NPC, resulting in the mislocalization of Nup2p to the nucleoplas m and cytoplasm. Purified Nup60p and Nup2p bind each other directly, but th e stability of the complex is compromised when Kap60p binds Nup2p. Gsp1p-GT P enhances by 10fold the affinity between Nup60p and Nup2p, and restores bi nding of Nup2p-Kap6Op complexes to Nup60p. The results suggest a dynamic in teraction, controlled by the nucleoplasmic concentration of Gsp1p-GTP, betw een Nup60p and Nup2p at the NPC.