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© The Rockefeller University Press, 0021-9525/1999//645 $5.00
The Journal of Cell Biology, Volume 144, Number 4, , 1999 645-655

Regular Articles

A Monoclonal Antibody to the COOH-terminal Acidic Portion of Ran Inhibits Both the Recycling of Ran and Nuclear Protein Import in Living Cells



Miki Hieda, Taro Tachibana, Fumihiko Yokoya, Shingo Kose, Naoko Imamoto, and Yoshihiro Yoneda

Department of Anatomy and Cell Biology, Osaka University Medical School, Suita, Osaka 565-0871, Japan

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 β, 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 β–related proteins. In addition, ARAN1 suppressed the binding of RanBP1 to the Ran–importin β complex. When injected into the nucleus of BHK cells, ARAN1 was rapidly exported to the cytoplasm, indicating that the Ran–importin β–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 β 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.

Key Words: nuclear protein import • Ran • monoclonal antibody • importin • microinjection

Abbreviations used in this paper: BSA, bovine serum albumin; CAS, cellular apoptosis susceptibility gene; GFP, green fluorescent protein; GST, glutathione-S-transferase; NES, nuclear export signal; NLS, nuclear localization signal; NPC, nuclear pore complex; RanBP, Ran-binding protein; TB, transport buffer.

M. Hieda and T. Tachibana contributed equally to this work.

T. Tachibana's present address is Department of Neurochemistry and Neuropharmacology, Biomedical Research Center, Osaka University Medical School, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan.


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