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

Abstract

The yeast karyopherin heterodimer Kap60p⅐Kap95p facilitates nuclear import of proteins bearing a classic nuclear localization signal (NLS). The ␣ 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 Cse1p⅐Gsp1p⅐GTP 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 Kap60p⅐NLS-cargo complexes and Kap60p⅐Nup2p complexes by increasing the affinity of Kap60p for its AIS motif. In contrast, Nup2p uses a conserved sequence motif (VMXXRKIA) coupled to an AISlike motif to accelerate dissociation of Kap60p⅐NLS complexes in a vectorial reaction mechanism. Mutation of either motif in Nup2p leads to a loss of KaRF activity and to the accumulation of Kap60p⅐NLS-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 NLScargos across the nuclear pore complex.

Single-molecule measurements of importin α/cargo complex dissociation at the nuclear pore

“…The FG-repeat domains can potentially serve as an assembly point for CAS/RanGTP complexes. A NUP50/ Imp ␣/cargo complex can be isolated (22,36), indicating that cargo release is not necessarily immediately induced by the binding of Imp ␣/cargo complexes to NUP50. Our data support the hypothesis that Imp ␣/cargo complexes do not dissociate upon binding to NUP50 in the absence of CAS and RanGTP.…”

“…Numerous groups have postulated that NUP50 (Nup2p) catalyzes Imp ␣1/cargo complex dissociation when it reaches the nuclear basket region of the NPC (16,(22)(23)(24)(25)(26)(27). In control experiments, Ϸ50% of EGFP2-mNup50 (fusion of 2ϫEGFP with mouse Nup50) was retained by the NPCs for at least 20 min after permeabilization (Fig.…”

“…This result was not predicted from biochemical studies. Although numerous investigators found that NUP50 destabilizes the Imp ␣/cargo interaction (16,22,23,25,26), this destabilization seems to be insufficient to cause Imp ␣/cargo complex dissociation in the context of a functioning NPC on the time scale of transport.…”

“…In principle, this mechanism could function within the NPC or within the nucleoplasm. According to a third mechanism, NUP50 (Nup2p in yeast), a nuclear pore protein, promotes dissociation of the Imp ␣/cargo complex (22)(23)(24)(25)(26)(27). Because NUP50 is located on the nuclear basket, this model predicts that the Imp ␣/cargo dissociation event occurs near the end of cargo translocation through the NPC.…”

“…In a bead binding assay, the mean dissociation time was much longer ( ϭ Ϸ9 min) (16), although this was probably due, at least in part, to a high avidity. According to a second model, binding of a CAS/RanGTP complex to Imp ␣ promotes dissociation of the Imp ␣/cargo complex (15,16,22). In principle, this mechanism could function within the NPC or within the nucleoplasm.…”