Accelerating the rate of disassembly of karyopherin.cargo complexes.
Transport of macromolecules across the nuclear pore complex (NPC) occurs in seconds and involves assembly of a karyopherin.cargo complex and docking to the NPC, translocation of the complex across the NPC via interaction with nucleoporins (Nups), and dissociation of the complex in the nucleoplasm. To identify rate-limiting steps in the Kap95p.Kap60p-mediated nuclear import pathway of Saccharomyces cerevisiae, we reconstituted key intermediate complexes and measured their rates of dissociation and affinities of interaction. We found that a nuclear localization signal-containing protein (NLS-cargo) dissociates slowly from Kap60p monomers and Kap60p.Kap95p heterodimers with half-lives (t(12)) of 7 and 73 min, respectively; that Kap60p and Kap60p.NLS-cargo complexes dissociate slowly from Kap95p (t(12) = 36 and 73 min, respectively); and that Kap95p.Kap60p.NLS-cargo complexes and Kap95p.Kap60p heterodimers dissociate rapidly from the nucleoporin Nup1p (t(12) < or = 21 s) and other Nups. A search for factors that accelerate disassembly of the long-lived intermediates revealed that Nup1p and Nup2p accelerate 16- and 19-fold the rate of dissociation of NLS-cargo from Kap60p.Kap95p heterodimers; that Gsp1p-GTP accelerates > or = 447-fold the rate of dissociation of Kap60p.NLS-cargo from Kap95p; and that Nup2p and the Cse1p.Gsp1p-GTP complex independently accelerate > or = 22- and > or = 39-fold the rate of dissociation of NLS-cargo from Kap60p. We suggest that Nup1p, Nup2p, Cse1p, and Gsp1p accelerate disassembly of Kap95p.Kap60p.NLS-cargo complexes by triggering allosteric mechanisms within Kaps that cause rapid release of binding partners. In that way, Nup1p, Nup2p, Cse1p, and Gsp1p may function as karyopherin release factors (or KaRFs) in the nuclear basket structure of the S. cerevisiae NPC.
Gilchrist
Daniel
D
Department of Biological Sciences, Stanford University, Stanford, California 94305-5020, USA.
Mykytka
Brook
B
Rexach
Michael
M
D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D000818:Animals; D005656:Fungal Proteins / Q000378:metabolism; D028884:Karyopherins / Q000648:ultrastructure*; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism; D028861:Nuclear Pore Complex Proteins / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D029543:Nucleocytoplasmic Transport Proteins; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D028961:beta Karyopherins / Q000378:metabolism
C082274:CSE1 protein, S cerevisiae; D005656:Fungal Proteins; C080324:GSP1 protein, S cerevisiae; D028884:Karyopherins; C064550:NUP1 protein, S cerevisiae; C400753:NUP2 protein, S cerevisiae; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D029543:Nucleocytoplasmic Transport Proteins; D029701:Saccharomyces cerevisiae Proteins; D028961:beta Karyopherins; D020559:Monomeric GTP-Binding Proteins