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J. Biol. Chem., Vol. 278, Issue 43, 42699-42709, October 24, 2003
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A Gradient of Affinity for the Karyopherin Kap95p along the Yeast Nuclear Pore Complex*
From the Department of Biological Sciences, Stanford University, Stanford, California 94305-5020
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 Nup1p
36 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.
Received for publication, July 3, 2003 , and in revised form, August 7, 2003.
* This work was supported by a Searle Scholars award (to M. R.) and by National Institutes of Health Grant RO1 GM61900-1A2. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 650-725-4814; Fax: 650-723-0155; E-mail: rexach{at}stanford.edu.
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