Selective disruption of nuclear import by a functional mutant nuclear transport carrier.
p10/NTF2 is a nuclear transport carrier that mediates the uptake of cytoplasmic RanGDP into the nucleus. We constructed a point mutant of p10, D23A, that exhibited unexpected behavior both in digitonin-permeabilized and microinjected mammalian cells. D23A p10 was markedly more efficient than wild-type (wt) p10 at supporting Ran import, but simultaneously acted as a dominant-negative inhibitor of classical nuclear localization sequence (cNLS)-mediated nuclear import supported by karyopherins (Kaps) alpha and beta1. Binding studies indicated that these two nuclear transport carriers of different classes, p10 and Kap-beta1, compete for identical and/or overlapping binding sites at the nuclear pore complex (NPC) and that D23A p10 has an increased affinity relative to wt p10 and Kap-beta1 for these shared binding sites. Because of this increased affinity, D23A p10 is able to import its own cargo (RanGDP) more efficiently than wt p10, but Kap-beta1 can no longer compete efficiently for shared NPC docking sites, thus the import of cNLS cargo is inhibited. The competition of different nuclear carriers for shared NPC docking sites observed here predicts a dynamic equilibrium between multiple nuclear transport pathways inside the cell that could be easily shifted by a transient modification of one of the carriers.
Lane
C M
CM
Baylor College of Medicine, Department of Molecular and Cellular Biology, Houston, Texas 77030, USA.
Cushman
I
I
Moore
M S
MS
D021581:Active Transport, Cell Nucleus; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D019913:Nuclear Localization Signals / Q000037:antagonists & inhibitors; D022022:Nuclear Pore / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D029543:Nucleocytoplasmic Transport Proteins; D010446:Peptide Fragments / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D029701:Saccharomyces cerevisiae Proteins; D028961:beta Karyopherins; D020931:ran GTP-Binding Protein / Q000378:metabolism*
D002352:Carrier Proteins; C499531:NTF2 protein, S cerevisiae; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D029543:Nucleocytoplasmic Transport Proteins; D010446:Peptide Fragments; D029701:Saccharomyces cerevisiae Proteins; D028961:beta Karyopherins; D006153:Guanosine Diphosphate; D020931:ran GTP-Binding Protein