The nucleoporin Nup98 is a site for GDP/GTP exchange on ran and termination of karyopherin β2-mediated nuclear import

Karyopherin β2 (Kapβ2, transportin) hinds the M9 sequence of human ribonucleoprotein A1 and mediates its nuclear import. Here we show a role for the nucleoporin Nup98 in the disassembly of Kapβ2 import complexes at the nuclear side of the nuclear pore complex (NPC). Kapβ2 bound to a region at the N terminus of Nup98 that contains an M9-like sequence. The human ribonucleoprotein A1 M9 sequence competed with Nup98 for binding to Kapβ2, indicating that Nup98 can dissociate Kapβ2 from its substrate. Binding of Kapβ2 to Nup98 was inhibited by Ran loaded with guanylyl imidophosphate, suggesting that RanGTP dissociates Kapβ2 from Nup98. RanGTP is produced from RanGDP through nucleotide exchange mediated by RanGEF (RCC1). Immunoelectron microscopy and nucleotide exchange assays revealed functional RanGEF on both sides of the NPC. On the nuclear side, the localization of RanGEF coincided with that of Nup98. RanGEF hound to Nup98 at a region adjacent to the Kapβ2-binding site. These findings suggest a model where 1) import substrate is released from Kapβ2 at the nucleoplasmic side of the NPC by competition with the Nup98 M9-like site, 2) Nup98-bound RanGEF catalyzes the formation of RanGTP, and 3) RanGTP dissociates Kapβ2 from Nup98 allowing repeated cycles of import.