The Nuclear Transport Factor Karyopherin Binds Stoichiometrically to Ran-GTP and Inhibits the Ran GTPase Activating Protein

doi:10.1074/jbc.271.10.5313

The Nuclear Transport Factor Karyopherin Binds Stoichiometrically to Ran-GTP and Inhibits the Ran GTPase Activating Protein (*)

From the Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, New York 10021

^¶ To whom correspondence should be addressed. Tel.: 212-327-8096; Fax: 212-327-7880.

Abstract

The heterodimeric karyopherin functions in targeting a nuclear localization sequence (NLS)-containing protein to the nuclear pore complex followed by Ran-GTP and p10-mediated translocation of the NLS protein into the nucleoplasm. It was shown recently that Ran-GTP dissociated the karyopherin heterodimer and, in doing so, associated with karyopherin β (Rexach, M., and Blobel, G.(1995) Cell 83, 683-692). We show here, using all recombinant yeast proteins expressed in Escherichia coli, that karyopherin β binds to Ran-GTP and inhibits GTP hydrolysis stimulated by RanGAP (the Ran-specific GTPase activating protein). Inhibition of RanGAP-stimulated GTP hydrolysis by karyopherin β was dependent on karyopherin β concentration relative to Ran-GTP. Complete inhibition of RanGAP was observed at karyopherin β concentrations that were equimolar to Ran-GTP. In gel filtration experiments, we found Ran-GTP and karyopherin β to form a stoichiometric complex. Ran-GDP bound only weakly to karyopherin β. We propose that stoichiometric complex formation between karyopherin β and Ran-GTP renders Ran-GTP inaccessible to RanGAP.

Footnotes

↵^§ Supported by a Beckman Fellowship for predoctoral training.
↵* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

NLS

nuclear localization sequence

GAP

GTPase activating protein

PAGE

polyacrylamide gel electrophoresis

DTT

dithiothreitol

FPLC

fast protein liquid chromatography

GST

glutathione S-transferase

PBS

phosphate-buffered saline.
- Received October 16, 1995.
- Revision received January 16, 1996.