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.