Molecular Basis for the Rapid Dissociation of Nuclear Localization Signals from Karyopherin α in the Nucleoplasm*

The yeast karyopherin heterodimer Kap60p·Kap95p facilitates nuclear import of proteins bearing a classic nuclear localization signal (NLS). The α subunit Kap60p binds to the NLS of cargo molecules in the cytoplasm, forming stable complexes that must ultimately dissociate in the nucleoplasm. Although Kap60p can release NLSs on its own using an autoinhibitory sequence (AIS) motif that can occupy the NLS binding site, that mechanism is too slow to support rapid nuclear import. We previously showed that the nuclear basket nucleoporin Nup2p and the exportin complex Cse1p·Gsp1p·GTP function as karyopherin release factors (KaRFs) because they can accelerate the rate of dissociation of NLSs from Kap60p. Here we dissect the molecular mechanics of their KaRF activity. We show that Cse1p accelerates dissociation of Kap60p·NLS-cargo complexes and Kap60p·Nup2p complexes by increasing the affinity of Kap60p for its AIS motif. In contrast, Nup2p uses a conserved sequence motif (VMXXRKIA) coupled to an AIS-like motif to accelerate dissociation of Kap60p·NLS complexes in a vectorial reaction mechanism. Mutation of either motif in Nup2p leads to a loss of KaRF activity and to the accumulation of Kap60p·NLS-cargo complexes in the nucleoplasm of yeast. We discuss a model whereby Nup2p, Cse1p, and Gsp1p cooperate to establish directionality in the movement of Kap60p and NLS-cargos across the nuclear pore complex.