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A monoclonal antibody to the COOH-terminal acidic portion of Ran inhibits both the recycling of Ran and nuclear protein import in living cells.

Hieda M, Tachibana T, Yokoya F, Kose S, Imamoto N, Yoneda Y - J. Cell Biol. (1999)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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

ABSTRACT
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.

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ARAN1 is exported from the nucleus to the cytoplasm.  ARAN1 (5 mg/ml) was injected into the cytoplasm (top) or nucleus (bottom) of BHK21 cells with FITC-BSA. After incubation  at 37°C for 30 min, cells were fixed and stained with Cy3-conjugated goat anti–mouse IgG antibodies (right). The localization of  FITC-BSA shows the injected site in the cells. (B) ARAN1 recognizes the Ran–importin β complex in Ehrlich ascites tumor cell  cytosolic extract. ARAN1 was added (lanes 1 and 3) or not (lanes  2 and 4) into the cytosolic extract and incubated for 1 h in the  presence of Q69L Ran-GTP. The proteins which were bound to  ARAN1 were precipitated with protein A–agarose beads and analyzed by immunoblotting with rabbit anti-Ran polyclonal antibodies (lanes 1 and 2), rabbit anti–importin β polyclonal antibodies (lanes 3 and 4), mouse anti-transportin monoclonal antibody,  and mouse anti-CAS monoclonal antibody (lanes 5 and 6, respectively).
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Figure 6: ARAN1 is exported from the nucleus to the cytoplasm. ARAN1 (5 mg/ml) was injected into the cytoplasm (top) or nucleus (bottom) of BHK21 cells with FITC-BSA. After incubation at 37°C for 30 min, cells were fixed and stained with Cy3-conjugated goat anti–mouse IgG antibodies (right). The localization of FITC-BSA shows the injected site in the cells. (B) ARAN1 recognizes the Ran–importin β complex in Ehrlich ascites tumor cell cytosolic extract. ARAN1 was added (lanes 1 and 3) or not (lanes 2 and 4) into the cytosolic extract and incubated for 1 h in the presence of Q69L Ran-GTP. The proteins which were bound to ARAN1 were precipitated with protein A–agarose beads and analyzed by immunoblotting with rabbit anti-Ran polyclonal antibodies (lanes 1 and 2), rabbit anti–importin β polyclonal antibodies (lanes 3 and 4), mouse anti-transportin monoclonal antibody, and mouse anti-CAS monoclonal antibody (lanes 5 and 6, respectively).

Mentions: As described above, it has been proposed that the Ran-GTP– importin β complex and Ran-GTP–importin β–related transport factor (such as transportin and CAS) complex are formed in the nucleus and exported to the cytoplasm. To determine whether these complexes are actually formed in the nucleus and migrate to the cytoplasm as a single entity in living cells, ARAN1, which binds to only the Ran complexed with importin β and importin β–related transport factors in solution, was injected into the cytoplasm or nucleus of cultured BHK cells. It was found that nuclear-injected ARAN1 was efficiently exported to the cytoplasm within 30 min of incubation (Fig. 6 A), whereas control mouse IgG was retained in the injected nucleus (data not shown). Since due to its size (Mr = 160,000), mouse IgG cannot passively traverse the nuclear envelope (Wen et al., 1995), these results indicate that ARAN1 migrates to the cytoplasm in a piggyback fashion. These findings suggest that it is most likely that ARAN1 binds to the Ran-GTP complexed with importin β–related transport factors in the nucleus, in which Ran would exist as the GTP-bound form, and is transported to the cytoplasm as a complex with them. That is, these results suggest that the Ran-GTP–importin β–related transport factor complex moves to the cytoplasm as a single entity without dissociation. On the other hand, cytoplasmically injected ARAN1 remained in the cytoplasm even after 3 h of incubation (data not shown).


A monoclonal antibody to the COOH-terminal acidic portion of Ran inhibits both the recycling of Ran and nuclear protein import in living cells.

Hieda M, Tachibana T, Yokoya F, Kose S, Imamoto N, Yoneda Y - J. Cell Biol. (1999)

ARAN1 is exported from the nucleus to the cytoplasm.  ARAN1 (5 mg/ml) was injected into the cytoplasm (top) or nucleus (bottom) of BHK21 cells with FITC-BSA. After incubation  at 37°C for 30 min, cells were fixed and stained with Cy3-conjugated goat anti–mouse IgG antibodies (right). The localization of  FITC-BSA shows the injected site in the cells. (B) ARAN1 recognizes the Ran–importin β complex in Ehrlich ascites tumor cell  cytosolic extract. ARAN1 was added (lanes 1 and 3) or not (lanes  2 and 4) into the cytosolic extract and incubated for 1 h in the  presence of Q69L Ran-GTP. The proteins which were bound to  ARAN1 were precipitated with protein A–agarose beads and analyzed by immunoblotting with rabbit anti-Ran polyclonal antibodies (lanes 1 and 2), rabbit anti–importin β polyclonal antibodies (lanes 3 and 4), mouse anti-transportin monoclonal antibody,  and mouse anti-CAS monoclonal antibody (lanes 5 and 6, respectively).
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Figure 6: ARAN1 is exported from the nucleus to the cytoplasm. ARAN1 (5 mg/ml) was injected into the cytoplasm (top) or nucleus (bottom) of BHK21 cells with FITC-BSA. After incubation at 37°C for 30 min, cells were fixed and stained with Cy3-conjugated goat anti–mouse IgG antibodies (right). The localization of FITC-BSA shows the injected site in the cells. (B) ARAN1 recognizes the Ran–importin β complex in Ehrlich ascites tumor cell cytosolic extract. ARAN1 was added (lanes 1 and 3) or not (lanes 2 and 4) into the cytosolic extract and incubated for 1 h in the presence of Q69L Ran-GTP. The proteins which were bound to ARAN1 were precipitated with protein A–agarose beads and analyzed by immunoblotting with rabbit anti-Ran polyclonal antibodies (lanes 1 and 2), rabbit anti–importin β polyclonal antibodies (lanes 3 and 4), mouse anti-transportin monoclonal antibody, and mouse anti-CAS monoclonal antibody (lanes 5 and 6, respectively).
Mentions: As described above, it has been proposed that the Ran-GTP– importin β complex and Ran-GTP–importin β–related transport factor (such as transportin and CAS) complex are formed in the nucleus and exported to the cytoplasm. To determine whether these complexes are actually formed in the nucleus and migrate to the cytoplasm as a single entity in living cells, ARAN1, which binds to only the Ran complexed with importin β and importin β–related transport factors in solution, was injected into the cytoplasm or nucleus of cultured BHK cells. It was found that nuclear-injected ARAN1 was efficiently exported to the cytoplasm within 30 min of incubation (Fig. 6 A), whereas control mouse IgG was retained in the injected nucleus (data not shown). Since due to its size (Mr = 160,000), mouse IgG cannot passively traverse the nuclear envelope (Wen et al., 1995), these results indicate that ARAN1 migrates to the cytoplasm in a piggyback fashion. These findings suggest that it is most likely that ARAN1 binds to the Ran-GTP complexed with importin β–related transport factors in the nucleus, in which Ran would exist as the GTP-bound form, and is transported to the cytoplasm as a complex with them. That is, these results suggest that the Ran-GTP–importin β–related transport factor complex moves to the cytoplasm as a single entity without dissociation. On the other hand, cytoplasmically injected ARAN1 remained in the cytoplasm even after 3 h of incubation (data not shown).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus