Protein export from the nucleus requires the GTPase Ran and GTP hydrolysis.
Nuclei of digitonin-permeabilized cells that had been preloaded with a model transport substrate in a cytosol-dependent import reaction were subsequently incubated to investigate which conditions would result in export of transport substrate. We found that up to 80% of the imported substrate was exported when recombinant human Ran and GTP were present in the export reaction. Ran-mediated export was inhibited by nonhydrolyzable GTP analogs and also by wheat germ agglutinin but was unaffected by a nonhydrolyzable ATP analog. Moreover, a recombinant human Ran mutant that was deficient in its GTPase activity inhibited export. These data indicate that export of proteins from the nucleus requires Ran and GTP hydrolysis but not ATP hydrolysis. We also found that digitonin-permeabilized cells were depleted of their endogenous nuclear Ran, thus allowing detection of Ran as a limiting factor for export. In contrast, most endogenous karyopherin alpha was retained in nuclei of digitonin-permeabilized cells. Unexpectedly, exogenously added, fluorescently labeled Ran, although it accessed the nuclear interior, was found to dock at the nuclear rim in a punctate pattern, suggesting the existence of Ran-binding sites at the nuclear pore complex.
Moroianu
J
J
Laboratory of Cell Biology, Howard Hughes Medical Institute, Rockefeller University, New York, NY 10021, USA.
Blobel
G
G
D000255:Adenosine Triphosphate / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000737:chemistry; D002463:Cell Membrane Permeability; D002467:Cell Nucleus / Q000378:metabolism* / Q000648:ultrastructure; D004072:Digitonin; D016650:Fluorescein-5-isothiocyanate; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000187:drug effects / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D011915:Rats, Inbred BUF; D012709:Serum Albumin / Q000378:metabolism; D020931:ran GTP-Binding Protein
D000952:Antigens, Polyomavirus Transforming; D009687:Nuclear Proteins; D010446:Peptide Fragments; D012709:Serum Albumin; D000266:Adenylyl Imidodiphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020931:ran GTP-Binding Protein; D016650:Fluorescein-5-isothiocyanate; D004072:Digitonin