The nuclear transport factor karyopherin beta binds stoichiometrically to Ran-GTP and inhibits the Ran GTPase activating protein.
The heterodimeric karyopherin functions in targeting a nuclear localization sequence (NLS)-containing protein to the nuclear pore complex followed by Ran-GTP and p10-mediated translocation of the NLS protein into the nucleoplasm. It was shown recently that Ran-GTP dissociated the karyopherin heterodimer and, in doing so, associated with karyopherin beta (Rexach, M., and Blobel, G. (1995) Cell 83, 683-692). We show here, using all recombinant yeast proteins expressed in Escherichia coli, that karyopherin beta binds to Ran-GTP and inhibits GTP hydrolysis stimulated by RanGAP (the Ran-specific GTPase activating protein). Inhibition of RanGAP-stimulated GTP hydrolysis by karyopherin beta was dependent on karyopherin beta concentration relative to Ran-GTP. Complete inhibition of RanGAP was observed at karyopherin beta concentrations that were equimolar to Ran-GTP. In gel filtration experiments, we found Ran-GTP and karyopherin beta to form a stoichiometric complex. Ran-GDP bound only weakly to karyopherin beta. We propose that stoichiometric complex formation between karyopherin beta and Ran-GTP renders Ran-GTP inaccessible to RanGAP.
Floer
M
M
Laboratory of Cell Biology, Howard Hughes Medical Institute, Rockefeller University, New York, New York 10021, USA.
Blobel
G
G
D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D017931:DNA Primers; D020690:GTPase-Activating Proteins; D005800:Genes, Fungal; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D014176:Protein Biosynthesis; D011506:Proteins / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D028961:beta Karyopherins; D020931:ran GTP-Binding Protein
D017931:DNA Primers; D020690:GTPase-Activating Proteins; D009687:Nuclear Proteins; D011506:Proteins; D011994:Recombinant Proteins; D028961:beta Karyopherins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D020931:ran GTP-Binding Protein