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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 induces the intracellular localization rearrangement of Ran and inhibits the basic type NLS-dependent nuclear  protein import. (A) ARAN1 (bottom) or control mouse IgG (top,  both 50 mg/ml) was injected into the cytoplasm of the cultured  BHK21 cells and incubated at 37°C for 30 min. The cells were fixed  and probed with rabbit anti-Ran antibodies at 4°C overnight, and  then stained with Cy3-conjugated goat anti–rabbit antibodies. Injected ARAN1 and control mouse IgG was detected with FITC-conjugated goat anti–mouse IgG antibodies. Phase–contrast microscopy is shown in the right panels. (B) ARAN1 or control  mouse IgG (50 mg/ml) was injected into the cytoplasm of cultured  BHK21. After a 30-min incubation at 37°C, FITC-labeled T-BSA  was injected into the cytoplasm of the same cells. After incubation  at 37°C for 20 min, the cells were fixed and observed by fluorescent  microscopy. Injected IgG was detected with Cy3-labeled anti-mouse IgG.
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Figure 7: ARAN1 induces the intracellular localization rearrangement of Ran and inhibits the basic type NLS-dependent nuclear protein import. (A) ARAN1 (bottom) or control mouse IgG (top, both 50 mg/ml) was injected into the cytoplasm of the cultured BHK21 cells and incubated at 37°C for 30 min. The cells were fixed and probed with rabbit anti-Ran antibodies at 4°C overnight, and then stained with Cy3-conjugated goat anti–rabbit antibodies. Injected ARAN1 and control mouse IgG was detected with FITC-conjugated goat anti–mouse IgG antibodies. Phase–contrast microscopy is shown in the right panels. (B) ARAN1 or control mouse IgG (50 mg/ml) was injected into the cytoplasm of cultured BHK21. After a 30-min incubation at 37°C, FITC-labeled T-BSA was injected into the cytoplasm of the same cells. After incubation at 37°C for 20 min, the cells were fixed and observed by fluorescent microscopy. Injected IgG was detected with Cy3-labeled anti-mouse IgG.

Mentions: Ran is localized predominantly in the nucleus. If Ran continuously shuttles between two compartments, the nucleus and cytoplasm, and the binding of RanBP1 to the Ran-GTP–importin β complex in the cytoplasm is required for the recycling of Ran to the nucleus, ARAN1 would be expected to affect the distribution of Ran. To examine this hypothesis, ARAN1 was introduced into the cell and the distribution of endogenous Ran was observed. As shown in Fig. 6, 30 min after injection into either the nucleus or the cytoplasm, injected ARAN1 localized in the cytoplasm. 30 min after injection of ARAN1 into either the nucleus or the cytoplasm, the subcellular localization of endogenous Ran became apparently dominant in the cytoplasm of the ARAN1-injected cells, whereas control mouse IgG had no effect on this distribution (Fig. 7 A for cytoplasmic injection and data not shown for nuclear injection). The relocalization of Ran was caused irrespective of the injection sites of ARAN1.


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 induces the intracellular localization rearrangement of Ran and inhibits the basic type NLS-dependent nuclear  protein import. (A) ARAN1 (bottom) or control mouse IgG (top,  both 50 mg/ml) was injected into the cytoplasm of the cultured  BHK21 cells and incubated at 37°C for 30 min. The cells were fixed  and probed with rabbit anti-Ran antibodies at 4°C overnight, and  then stained with Cy3-conjugated goat anti–rabbit antibodies. Injected ARAN1 and control mouse IgG was detected with FITC-conjugated goat anti–mouse IgG antibodies. Phase–contrast microscopy is shown in the right panels. (B) ARAN1 or control  mouse IgG (50 mg/ml) was injected into the cytoplasm of cultured  BHK21. After a 30-min incubation at 37°C, FITC-labeled T-BSA  was injected into the cytoplasm of the same cells. After incubation  at 37°C for 20 min, the cells were fixed and observed by fluorescent  microscopy. Injected IgG was detected with Cy3-labeled anti-mouse IgG.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132938&req=5

Figure 7: ARAN1 induces the intracellular localization rearrangement of Ran and inhibits the basic type NLS-dependent nuclear protein import. (A) ARAN1 (bottom) or control mouse IgG (top, both 50 mg/ml) was injected into the cytoplasm of the cultured BHK21 cells and incubated at 37°C for 30 min. The cells were fixed and probed with rabbit anti-Ran antibodies at 4°C overnight, and then stained with Cy3-conjugated goat anti–rabbit antibodies. Injected ARAN1 and control mouse IgG was detected with FITC-conjugated goat anti–mouse IgG antibodies. Phase–contrast microscopy is shown in the right panels. (B) ARAN1 or control mouse IgG (50 mg/ml) was injected into the cytoplasm of cultured BHK21. After a 30-min incubation at 37°C, FITC-labeled T-BSA was injected into the cytoplasm of the same cells. After incubation at 37°C for 20 min, the cells were fixed and observed by fluorescent microscopy. Injected IgG was detected with Cy3-labeled anti-mouse IgG.
Mentions: Ran is localized predominantly in the nucleus. If Ran continuously shuttles between two compartments, the nucleus and cytoplasm, and the binding of RanBP1 to the Ran-GTP–importin β complex in the cytoplasm is required for the recycling of Ran to the nucleus, ARAN1 would be expected to affect the distribution of Ran. To examine this hypothesis, ARAN1 was introduced into the cell and the distribution of endogenous Ran was observed. As shown in Fig. 6, 30 min after injection into either the nucleus or the cytoplasm, injected ARAN1 localized in the cytoplasm. 30 min after injection of ARAN1 into either the nucleus or the cytoplasm, the subcellular localization of endogenous Ran became apparently dominant in the cytoplasm of the ARAN1-injected cells, whereas control mouse IgG had no effect on this distribution (Fig. 7 A for cytoplasmic injection and data not shown for nuclear injection). The relocalization of Ran was caused irrespective of the injection sites of ARAN1.

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