A gradient of affinity for the karyopherin Kap95p along the yeast nuclear pore complex.
Karyopherins (Kaps) transport cargo across the nuclear pore complex (NPC) by interacting with nucleoporins that contain phenylalanine-glycine (FG) peptide repeats (FG Nups). As a test of the "affinity gradient" model for Kap translocation, we measured the apparent affinity of Kap95p to FG Nups representing three distinct regions of the S. cerevisiae NPC. We find that the affinity of Kap95p-Kap60p-cargo complexes to Nup1p (a nuclear basket Nup) is 225-fold higher than to Nup100p (a central scaffold Nup) and 4000-fold higher than to Nup42p (a cytoplasmic filament Nup), revealing a steep gradient of affinity for Kap95p complexes along the yeast NPC. A high affinity binding site for a Kap95p import complex was mapped to the C terminus of Nup1p, and, surprisingly, deletion of all FG repeats in that region did not eliminate binding of the complex. Instead, a 36-amino acid truncation of the C terminus of Nup1p reduced its affinity for the Kap95p import complex by 450-fold. Mutant yeast that express Nup1pDelta36 instead of full-length Nup1p display specific defects in Kap95p localization and Kap95p-mediated nuclear import. We conclude that a high affinity binding site for Kap95p at the nuclear basket increases the translocation efficiency of Kap95p import complexes across the NPC.
Pyhtila
Brook
B
Department of Biological Sciences, Stanford University, Stanford, CA 94305-5020, USA.
Rexach
Michael
M
D021581:Active Transport, Cell Nucleus; D001665:Binding Sites; D020559:Monomeric GTP-Binding Proteins / Q000494:pharmacology; D022022:Nuclear Pore / Q000378:metabolism* / Q000502:physiology; D028861:Nuclear Pore Complex Proteins / Q000378:metabolism / Q000502:physiology; D009687:Nuclear Proteins / Q000494:pharmacology; D011485:Protein Binding; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D028961:beta Karyopherins / Q000378:metabolism*
C080324:GSP1 protein, S cerevisiae; C064550:NUP1 protein, S cerevisiae; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; D028961:beta Karyopherins; D020559:Monomeric GTP-Binding Proteins