A monoclonal antibody to the COOH-terminal acidic portion of Ran inhibits both the recycling of Ran and nuclear protein import in living cells.
A small GTPase Ran is a key regulator for active nuclear transport. In immunoblotting analysis, a monoclonal antibody against recombinant human Ran, designated ARAN1, was found to recognize an epitope in the COOH-terminal domain of Ran. In a solution binding assay, ARAN1 recognized Ran when complexed with importin beta, transportin, and CAS, but not the Ran-GTP or the Ran-GDP alone, indicating that the COOH-terminal domain of Ran is exposed via its interaction with importin beta-related proteins. In addition, ARAN1 suppressed the binding of RanBP1 to the Ran-importin beta complex. When injected into the nucleus of BHK cells, ARAN1 was rapidly exported to the cytoplasm, indicating that the Ran-importin beta-related protein complex is exported as a complex from the nucleus to the cytoplasm in living cells. Moreover, ARAN1, when injected into the cultured cells induces the accumulation of endogenous Ran in the cytoplasm and prevents the nuclear import of SV-40 T-antigen nuclear localization signal substrates. From these findings, we propose that the binding of RanBP1 to the Ran-importin beta complex is required for the dissociation of the complex in the cytoplasm and that the released Ran is recycled to the nucleus, which is essential for the nuclear protein transport.
Hieda
M
M
Department of Anatomy and Cell Biology, Osaka University Medical School, Suita, Osaka 565-0871, Japan.
Tachibana
T
T
Yokoya
F
F
Kose
S
S
Imamoto
N
N
Yoneda
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D001693:Biological Transport, Active; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D000939:Epitopes / Q000737:chemistry / Q000235:genetics; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D028884:Karyopherins; D046911:Macromolecular Substances; D051379:Mice; D009687:Nuclear Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D020931:ran GTP-Binding Protein
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D000939:Epitopes; D028884:Karyopherins; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D011994:Recombinant Proteins; C091775:ran-binding protein 1; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020931:ran GTP-Binding Protein