The nucleoporin Nup60p functions as a Gsp1p-GTP-sensitive tether for Nup2p at the nuclear pore complex.
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.
Denning
D
D
Cancer Biology Program, Stanford Medical School, Stanford University, CA 94305, USA.
Mykytka
B
B
Allen
N P
NP
Huang
L
L
Al Burlingame
Rexach
M
M
D021581:Active Transport, Cell Nucleus / Q000502:physiology; D017930:Genes, Reporter; D006160:Guanosine Triphosphate / Q000378:metabolism; D015151:Immunoblotting; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism*; D022022:Nuclear Pore / Q000378:metabolism*; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins / Q000378:metabolism*; D018272:Porins / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins; D028961:beta Karyopherins
C080324:GSP1 protein, S cerevisiae; C400753:NUP2 protein, S cerevisiae; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D018272:Porins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D028961:beta Karyopherins; D006160:Guanosine Triphosphate; D020559:Monomeric GTP-Binding Proteins