Ran-binding Protein 1 (RanBP1) Forms a Ternary Complex with Ran and Karyopherin β and Reduces Ran GTPase-activating Protein (RanGAP) Inhibition by Karyopherin β

doi:10.1074/jbc.272.1.551

Ran-binding Protein 1 (RanBP1) Forms a Ternary Complex with Ran and Karyopherin β and Reduces Ran GTPase-activating Protein (RanGAP) Inhibition by Karyopherin β*

From the Department of Pathology, University of Vermont, and the Vermont Cancer Center, Burlington, Vermont 05405

^§ To whom correspondence should be addressed. Current address:
Center for Cell Signaling, Health Sciences Center, University of Virginia, Charlottesville, VA 22908
. Tel: 804-982-0074; Fax: 804-924-1236.

Abstract

The nuclear accumulation of proteins containing nuclear localization signals requires the Ran GTPase and a complex of proteins assembled at the nuclear pore. RanBP1 is a cytosolic Ran-binding protein that inhibits RCC1-stimulated release of GTP from Ran. RanBP1 also promotes the binding of Ran to karyopherin β (also called importin β and p97) and is a co-stimulator of RanGAP activity. Yeast karyopherin β inhibits the GTP hydrolysis by Ran catalyzed by RanGAP. To further define the roles of RanBP1 and karyopherin β in Ran function, we explored the effects of RanBP1 and karyopherin β on mammalian proteins known to regulate Ran. Like RanBP1, karyopherin β prevented the release of GTP from Ran stimulated by RCC1 or EDTA. As with the yeast protein, mammalian karyopherin β completely blocked RanGAP activity. However, the addition of RanBP1 to this assay partially rescued the inhibited RanGAP activity. Kinetic analysis of the effects on RanGAP activity by karyopherin β and RanBP1 revealed a combination of competitive and noncompetitive interactions. Solution binding assays confirmed the ability of RanBP1 to associate with Ran and karyopherin β in a ternary complex, and RanBP1 binding was not competed out by the addition of karyopherin β. These results demonstrate that RanBP1 and karyopherin β interact with distinct sites of Ran and suggest that RanBP1 plays an essential role in nuclear transport by permitting RanGAP-mediated hydrolysis of GTP on Ran complexed to karyopherin β.

Footnotes

↵^‡ Supported by National Research Service Award F32CA63801 from the NCI, National Institutes of Health. Current address: UVM Medical Research Facility, 55A South Park Dr., Colchester, VT 05446.
↵* This work was supported in part by United States Public Service Award GM50526 from the National Institutes of Health, DHHS. 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.
↵¹ The abbreviations used are:

NLS

nuclear localization signal

RanBP

Ran-binding protein

RanBD

Ran-binding domain

GST

glutathione S-transferase

GAP

GTPase-activating protein

PAGE

polyacrylamide gel electrophoresis

MOPS

3-(N-morpholino)propanesulfonic acid.
- Received October 22, 1996.