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 immu noblotting analysis, a monoclonal antibody against recombinant human Ran, d esignated ARAN1, was found to recognize an epitope in the COOH-terminal dom ain of Ran. In a solution binding assay, ARAN1 recognized Ran when complexe d with importin beta, transportin, and GAS, but not the Ran-GTP or the Ran- GDP alone, indicating that the COOH-terminal domain of Ran is exposed via i ts interaction with importin beta-related proteins. In addition, ARAN1 supp ressed the binding of RanBP1 to the Ran-importin beta complex. When injecte d into the nucleus of BHK cells, ARAN1 was rapidly exported to the cytoplas m, indicating that the Ran-importin beta-related protein complex is exporte d as a complex from the nucleus to the cytoplasm in living cells. Moreover, ARAN1, when injected into the cultured cells induces the accumulation of e ndogenous Ran in the cytoplasm and prevents the nuclear import of SV-40 T-a ntigen nuclear localization signal substrates. From these findings, we prop ose 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.