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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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Mapping of the ARAN1 binding domain in the Ran  molecule. (A) Schematic presentation of Ran and a series of  truncated forms of Ran, which were expressed in E. coli as recombinant GST fusion proteins. Numbers at left, amino acid positions of the Ran sequence. A full length of human Ran consists  of 216 amino acid residues. (B) Immunoblotting analysis of full  and mutant Ran proteins using ARAN1 and rabbit anti-GST  polyclonal antibodies. E. coli was harvested after 6 h of induction  with IPTG and lysed in SDS-PAGE sample buffer. Samples were  electrophoresed on 12.5% polyacrylamide gels, transferred to nitrocellulose, and then probed with the ARAN1 and anti-GST  polyclonal antibodies. Right, a loading control stained with Coomassie brilliant blue.
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Figure 2: Mapping of the ARAN1 binding domain in the Ran molecule. (A) Schematic presentation of Ran and a series of truncated forms of Ran, which were expressed in E. coli as recombinant GST fusion proteins. Numbers at left, amino acid positions of the Ran sequence. A full length of human Ran consists of 216 amino acid residues. (B) Immunoblotting analysis of full and mutant Ran proteins using ARAN1 and rabbit anti-GST polyclonal antibodies. E. coli was harvested after 6 h of induction with IPTG and lysed in SDS-PAGE sample buffer. Samples were electrophoresed on 12.5% polyacrylamide gels, transferred to nitrocellulose, and then probed with the ARAN1 and anti-GST polyclonal antibodies. Right, a loading control stained with Coomassie brilliant blue.

Mentions: To determine the specific epitope recognized by ARAN1, we prepared a series of truncated forms of recombinant Ran fused with GST (Fig. 2 A) and analyzed the interaction of these with ARAN1 by immunoblotting. Although anti-GST polyclonal antibodies showed that all of the Ran mutants were appropriately expressed and transferred to nitrocellulose (Fig. 2 B), ARAN1 recognized only the full-length of GST-Ran. Removal of the COOH-terminal seven amino acids (210–216) of Ran completely abolished the reactivity with ARAN1 in immunoblotting, suggesting that the COOH-terminal domain, which consists of seven amino acid residues in Ran, represents the epitope for ARAN1. To confirm this, we prepared the 10-mer peptide of the COOH-terminal domain, 207–216, of Ran fused to GST (Fig. 3 A). As shown in Fig. 3, B and C, ARAN1 clearly reacted with the residues between 207 and 216 of human Ran not only in immunoblotting but also in a solution binding assay. From these findings, it was concluded that the epitope of ARAN1 is located in the COOH-terminal portion of Ran, which is highly negatively charged (−DEDDDL) and conserved among species (Fig. 3 D).


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)

Mapping of the ARAN1 binding domain in the Ran  molecule. (A) Schematic presentation of Ran and a series of  truncated forms of Ran, which were expressed in E. coli as recombinant GST fusion proteins. Numbers at left, amino acid positions of the Ran sequence. A full length of human Ran consists  of 216 amino acid residues. (B) Immunoblotting analysis of full  and mutant Ran proteins using ARAN1 and rabbit anti-GST  polyclonal antibodies. E. coli was harvested after 6 h of induction  with IPTG and lysed in SDS-PAGE sample buffer. Samples were  electrophoresed on 12.5% polyacrylamide gels, transferred to nitrocellulose, and then probed with the ARAN1 and anti-GST  polyclonal antibodies. Right, a loading control stained with Coomassie brilliant blue.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132938&req=5

Figure 2: Mapping of the ARAN1 binding domain in the Ran molecule. (A) Schematic presentation of Ran and a series of truncated forms of Ran, which were expressed in E. coli as recombinant GST fusion proteins. Numbers at left, amino acid positions of the Ran sequence. A full length of human Ran consists of 216 amino acid residues. (B) Immunoblotting analysis of full and mutant Ran proteins using ARAN1 and rabbit anti-GST polyclonal antibodies. E. coli was harvested after 6 h of induction with IPTG and lysed in SDS-PAGE sample buffer. Samples were electrophoresed on 12.5% polyacrylamide gels, transferred to nitrocellulose, and then probed with the ARAN1 and anti-GST polyclonal antibodies. Right, a loading control stained with Coomassie brilliant blue.
Mentions: To determine the specific epitope recognized by ARAN1, we prepared a series of truncated forms of recombinant Ran fused with GST (Fig. 2 A) and analyzed the interaction of these with ARAN1 by immunoblotting. Although anti-GST polyclonal antibodies showed that all of the Ran mutants were appropriately expressed and transferred to nitrocellulose (Fig. 2 B), ARAN1 recognized only the full-length of GST-Ran. Removal of the COOH-terminal seven amino acids (210–216) of Ran completely abolished the reactivity with ARAN1 in immunoblotting, suggesting that the COOH-terminal domain, which consists of seven amino acid residues in Ran, represents the epitope for ARAN1. To confirm this, we prepared the 10-mer peptide of the COOH-terminal domain, 207–216, of Ran fused to GST (Fig. 3 A). As shown in Fig. 3, B and C, ARAN1 clearly reacted with the residues between 207 and 216 of human Ran not only in immunoblotting but also in a solution binding assay. From these findings, it was concluded that the epitope of ARAN1 is located in the COOH-terminal portion of Ran, which is highly negatively charged (−DEDDDL) and conserved among species (Fig. 3 D).

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