Molecular basis for the rapid disassembly of nuclear import complexes

To identify rate-limiting steps in the Kap95p·Kap60p-mediated nuclear import pathway of S. cerevisiae, key intermediate complexes were reconstituted and their rates of dissociation measured. The Kap95p-Kap60p-NLS cargo complex dissociates slowly; a nuclear localization signal-containing protein (NLS-cargo) dissociated from Kap60p and Kap60p·Kap95p heterodimers with half-lives (t_1/2) of 7 and 73 min, respectively, and Kap60p and Kap60p·NLS-cargo complexes dissociated from Kap95p with t_1/2 = 36 and 73 min, respectively. In contrast, Kap95p·Kap60p·NLS-cargo complexes and Kap95p·Kap60p heterodimers dissociated rapidly from the nucleoporin Nup1p (t_1/2 ≤ 21 sec) and other Nups. Proteins of the nucleoplasm and nuclear pore complex (NPC) nuclear basket accelerated disassembly of the Kap95p-Kap60p-NLS cargo complex. Nup1p and Nup2p accelerated the rate of dissociation of NLS-cargo from Kap60p·Kap95p heterodimers, Gsp1p-GTP accelerated the rate of dissociation of Kap60p·NLS-cargo from Kap95p, and Nup2p and the Cse1p·Gsp1p-GTP complex independently accelerated the rate of dissociation of NLS-cargo from Kap60p. These proteins thus serve as Karyopherin R&barbelow;elease F&barbelow;actors (KaRFs).; Kap60p can release NLSs slowly using an auto-inhibitory sequence (AIS) that occupies the NLS binding site; both Cse1p-Gsp1p and Nup2p employ the AIS to rapidly disassemble Kap60p-NLS cargo complexes. Cse1p-Gsp1p accelerates dissociation of Kap60p-NLS cargo complexes by increasing the affinity of Kap60p for its auto-inhibitory sequence. In contrast, Nup2p uses a conserved sequence, VMxxRKIA, together with an AIS-like motif in its N-terminus to accelerate dissociation of Kap60p-NLS complexes. Mutation of either motif in Nup2p abrogated KaRF activity and led to the accumulation of Kap60p-NLS complexes in the nucleoplasm of yeast. Following dissociation of Kap60p-NLS complexes by Nup2p, Kap60p's AIS sequence appeared to “cap” the NLS-binding site and make dissociation irreversible. Nup2p thus may assist Gsp1p in establishing the direction of cargo flux through the NPC.