RanGTP-mediated nuclear export of karyopherin alpha involves its interaction with the nucleoporin Nup153.
Using binding assays, we discovered an interaction between karyopherin alpha2 and the nucleoporin Nup153 and mapped their interacting domains. We also isolated a 15-kDa tryptic fragment of karyopherin beta1, termed beta1*, that contains a determinant for binding to the peptide repeat containing nucleoporin Nup98. In an in vitro assay in which export of endogenous nuclear karyopherin alpha from nuclei of digitonin-permeabilized cells was quantitatively monitored by indirect immunofluorescence with anti-karyopherin alpha antibodies, we found that karyopherin alpha export was stimulated by added GTPase Ran, required GTP hydrolysis, and was inhibited by wheat germ agglutinin. RanGTP-mediated export of karyopherin alpha was inhibited by peptides representing the interacting domains of Nup153 and karyopherin alpha2, indicating that the binding reactions detected in vitro are physiologically relevant and verifying our mapping data. Moreover, beta1*, although it inhibited import, did not inhibit export of karyopherin alpha. Hence, karyopherin alpha import into and export from nuclei are asymmetric processes.
Moroianu
J
J
Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10021, USA.
Blobel
G
G
Radu
A
A
D000595:Amino Acid Sequence; D001665:Binding Sites; D001692:Biological Transport; D002467:Cell Nucleus / Q000378:metabolism*; D019204:GTP-Binding Proteins; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins / Q000378:metabolism*; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000378:metabolism; D028901:alpha Karyopherins; D020931:ran GTP-Binding Protein
C079377:NUP153 protein, human; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D028901:alpha Karyopherins; D019204:GTP-Binding Proteins; D020931:ran GTP-Binding Protein