Nucleotide-specific interaction of Ran/TC4 with nuclear transport factors NTF2 and p97.
The use of permeabilized cell models to study nuclear protein import has led to the identification of cytosolic components of the import machinery, including the NLS receptor, p97, Ran/TC4, and nuclear transport factor 2 (NTF2). These proteins are required to reconstitute docking of transport ligand at the nuclear pore complex and subsequent translocation through the nuclear pore. However, a detailed molecular understanding of how these factors mediate protein import is lacking. Here we describe the results of solution and solid phase binding assays, which demonstrate that the small GTPase Ran/TC4 interacts directly with the cytosolic transport factors p97 and NTF2. By preloading recombinant Ran/TC4 with [gamma-32P]GTP or [3H]GDP, we show that the interactions with p97 and NTF2 are specific for the GTP- and GDP-bound forms, respectively. These data together with previous studies lead us to suggest that the interaction of the GTP-bound form of Ran/TC4 with p97 is linked to an early step in the nuclear protein import pathway and that the association of the GDP-bound form of Ran/TC4 with NTF2 helps define vectorial transport.
Paschal
B M
BM
Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Delphin
C
C
Gerace
L
L
D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002463:Cell Membrane Permeability; D002467:Cell Nucleus / Q000378:metabolism; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents; D003600:Cytosol / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D005982:Glutathione Transferase; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism*; D029543:Nucleocytoplasmic Transport Proteins; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D028961:beta Karyopherins; D020931:ran GTP-Binding Protein
D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D009687:Nuclear Proteins; D029543:Nucleocytoplasmic Transport Proteins; D011993:Recombinant Fusion Proteins; D028961:beta Karyopherins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D005982:Glutathione Transferase; D019204:GTP-Binding Proteins; D020931:ran GTP-Binding Protein