Selective Disruption of Nuclear Import by a Functional Mutant Nuclear Transport Carrier

• Published in: The Journal of cell biology 2000-10, Vol.151 (2), p.321-331
• Main Authors: Lane, Cynthia M., Cushman, Ian, Moore, Mary Shannon
• Format: Article
• Language: English
• Subjects: Active Transport, Cell Nucleus; Amino acids; Anatomy & physiology; beta Karyopherins; Binding Sites; Bulk carriers; Carrier Proteins - genetics; Carrier Proteins - metabolism; Cell nucleus; Cells; Dimers; Freight; Guanosine Diphosphate - metabolism; HeLa cells; Imports; karyopherins; nuclear import; Nuclear Localization Signals - antagonists & inhibitors; Nuclear pore; Nuclear Pore - metabolism; Nuclear pore complex proteins; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; Nucleocytoplasmic Transport Proteins; Original; p10/NTF2 protein; Peptide Fragments - metabolism; Point Mutation; Protein Binding; Proteins; ran GTP-Binding Protein - metabolism; RanGDP protein; Saccharomyces cerevisiae Proteins
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.151.2.321

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 digitoninpermeabilized 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) α and β1. Binding studies indicated that these two nuclear transport carriers of different classes, p10 and Kap-β1, 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-β1 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-β1 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.