Disassembly of RanGTP-Karyopherin β Complex, an Intermediate in Nuclear Protein Import*
- From the Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, New York 10021
Abstract
We previously showed that RanGTP forms a 1:1 complex with karyopherin β that renders RanGTP inaccessible to RanGAP (Floer, M., and Blobel, G. (1996) J. Biol. Chem. 271, 5313–5316) and karyopherin β functionally inactive (Rexach, M., and Blobel, G. (1995) Cell 83, 683–692). Recycling of both factors for another round of function requires dissociation of the RanGTP-karyopherin β complex. Here we show using BIAcore™, a solution binding assay, and GTP hydrolysis and exchange assays, with yeast proteins, that karyopherin β and RanGTP are recycled efficiently in a reaction that involves karyopherin α, RanBP1, RanGAP, and the C terminus of the nucleoporin Nup1. We find that karyopherin α first releases RanGTP from karyopherin β in a reaction that does not require GTP hydrolysis. The released RanGTP is then sequestered by RanBP1, and the newly formed karyopherin αβ binds to the C terminus of Nup1. Finally, RanGTP is converted to RanGDP via nucleotide hydrolysis when RanGAP is present. Conversion of RanGTP to RanGDP can also occur via nucleotide exchange in the presence of RanGEF, an excess of GDP, and if RanBP1 is absent. Additional nucleoporin domains that bind karyopherin αβ stimulate recycling of karyopherin β and Ran in a manner similar to the C terminus of Nup1.
Footnotes
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↵* This work was supported in part by a postdoctoral fellowship from the Jane Coffin Childs Memorial Fund for Medical Research (to M. R.) and a Beckman Fellowship for predoctoral training (to M. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom correspondence should be addressed. Tel.: 212-327-8096; Fax: 212-327-7880; E-mail: blobel{at}rockvax.rockefeller.edu.
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↵1 The abbreviations used are: NLS, nuclear localization sequence; GAP, GTPase-activating protein; GEF, guanine nucleotide exchange factor; C-Nup1, C terminus of Nup1 (amino acids 963–1076); AA, amino acids; GMP-PCP, guanylyl-(β,γ-methylene) diphosphonate; GST, glutathione S-transferase; RU, resonance units (44); NPC, nuclear pore complex; PAGE, polyacrylamide gel electrophoresis.
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↵2 M. Floer and G. Blobel, unpublished data.
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↵3 M. Rexach, and G. Blobel, submitted for publication.
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↵4 M. Floer, U. Nehrbass, and G. Blobel, unpublished data.
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↵5 U. Nehrbass and G. Blobel, unpublished data.
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- Received April 16, 1997.
- Revision received May 23, 1997.
