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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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Anti-Ran monoclonal antibody,  ARAN1, mono-specifically recognizes Ran  molecule. (A) Cytosolic extract from mouse  Ehrlich ascites tumor cells (lanes 1 and 2),  and total extracts of HEL cells (lane 3) and  BHK21 cells (lane 4) were electrophoresed  on 12.5% polyacrylamide gels, transferred to  nitrocellulose, and then probed with anti-Ran  polyclonal antibodies (lane 1) and ARAN1  (lanes 2, 3, and 4). (B) BHK21 cells were double stained with ARAN1 and rabbit anti-Ran  polyclonal antibodies. ARAN1 and rabbit  anti-Ran polyclonal antibodies were detected with goat Cy3-conjugated anti–mouse IgG and goat FITC-conjugated anti–rabbit IgG, respectively. Phase–contrast microscopy is also shown.
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Figure 1: Anti-Ran monoclonal antibody, ARAN1, mono-specifically recognizes Ran molecule. (A) Cytosolic extract from mouse Ehrlich ascites tumor cells (lanes 1 and 2), and total extracts of HEL cells (lane 3) and BHK21 cells (lane 4) were electrophoresed on 12.5% polyacrylamide gels, transferred to nitrocellulose, and then probed with anti-Ran polyclonal antibodies (lane 1) and ARAN1 (lanes 2, 3, and 4). (B) BHK21 cells were double stained with ARAN1 and rabbit anti-Ran polyclonal antibodies. ARAN1 and rabbit anti-Ran polyclonal antibodies were detected with goat Cy3-conjugated anti–mouse IgG and goat FITC-conjugated anti–rabbit IgG, respectively. Phase–contrast microscopy is also shown.

Mentions: To study the functional domain(s) of Ran, monoclonal antibodies were produced against recombinant human Ran. The screening of the monoclonal antibodies was based on two criteria: (a) that they react with only Ran protein in immunoblotting; and (b) that they show the same staining pattern with polyclonal anti-Ran antibodies in immunofluorescence microscopy. Through this screening procedure, we isolated only one clone, designated ARAN1, for further characterization. ARAN1 was typed using a mouse monoclonal antibody isotyping kit as an immunoglobulin kappa G2b. On an immunoblot, ARAN1 detected recombinant human Ran and specifically reacted with a single 25-kD band in cell extracts from mouse Ehrlich ascites tumor cells, BHK21 cells, human embryonic lung (HEL) cells (Fig. 1 A), Madin-Darby bovine kidney-derived epithelial cells (MDBK), and Xenopus laevis oocytes (data not shown), which was also detected by affinity-purified rabbit anti-Ran polyclonal antibodies. Furthermore, ARAN1 recognized a single spot by immunoblotting analysis of HeLa total cell extracts after two-dimensional electrophoresis, which was also detected with the anti-Ran polyclonal antibodies (data not shown). Immunofluorescent image with ARAN1 in BHK cells showed the same typical staining pattern as that with the anti-Ran polyclonal antibodies (Fig. 1 B). From these findings, it was concluded that the monoclonal antibody ARAN1 specifically binds to Ran.


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)

Anti-Ran monoclonal antibody,  ARAN1, mono-specifically recognizes Ran  molecule. (A) Cytosolic extract from mouse  Ehrlich ascites tumor cells (lanes 1 and 2),  and total extracts of HEL cells (lane 3) and  BHK21 cells (lane 4) were electrophoresed  on 12.5% polyacrylamide gels, transferred to  nitrocellulose, and then probed with anti-Ran  polyclonal antibodies (lane 1) and ARAN1  (lanes 2, 3, and 4). (B) BHK21 cells were double stained with ARAN1 and rabbit anti-Ran  polyclonal antibodies. ARAN1 and rabbit  anti-Ran polyclonal antibodies were detected with goat Cy3-conjugated anti–mouse IgG and goat FITC-conjugated anti–rabbit IgG, respectively. Phase–contrast microscopy is also shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132938&req=5

Figure 1: Anti-Ran monoclonal antibody, ARAN1, mono-specifically recognizes Ran molecule. (A) Cytosolic extract from mouse Ehrlich ascites tumor cells (lanes 1 and 2), and total extracts of HEL cells (lane 3) and BHK21 cells (lane 4) were electrophoresed on 12.5% polyacrylamide gels, transferred to nitrocellulose, and then probed with anti-Ran polyclonal antibodies (lane 1) and ARAN1 (lanes 2, 3, and 4). (B) BHK21 cells were double stained with ARAN1 and rabbit anti-Ran polyclonal antibodies. ARAN1 and rabbit anti-Ran polyclonal antibodies were detected with goat Cy3-conjugated anti–mouse IgG and goat FITC-conjugated anti–rabbit IgG, respectively. Phase–contrast microscopy is also shown.
Mentions: To study the functional domain(s) of Ran, monoclonal antibodies were produced against recombinant human Ran. The screening of the monoclonal antibodies was based on two criteria: (a) that they react with only Ran protein in immunoblotting; and (b) that they show the same staining pattern with polyclonal anti-Ran antibodies in immunofluorescence microscopy. Through this screening procedure, we isolated only one clone, designated ARAN1, for further characterization. ARAN1 was typed using a mouse monoclonal antibody isotyping kit as an immunoglobulin kappa G2b. On an immunoblot, ARAN1 detected recombinant human Ran and specifically reacted with a single 25-kD band in cell extracts from mouse Ehrlich ascites tumor cells, BHK21 cells, human embryonic lung (HEL) cells (Fig. 1 A), Madin-Darby bovine kidney-derived epithelial cells (MDBK), and Xenopus laevis oocytes (data not shown), which was also detected by affinity-purified rabbit anti-Ran polyclonal antibodies. Furthermore, ARAN1 recognized a single spot by immunoblotting analysis of HeLa total cell extracts after two-dimensional electrophoresis, which was also detected with the anti-Ran polyclonal antibodies (data not shown). Immunofluorescent image with ARAN1 in BHK cells showed the same typical staining pattern as that with the anti-Ran polyclonal antibodies (Fig. 1 B). From these findings, it was concluded that the monoclonal antibody ARAN1 specifically binds to Ran.

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