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

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 Ran-GAP 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.