Limits...
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.

Show MeSH

Related in: MedlinePlus

ARAN1 recognizes an epitope located within 10 amino  acid residues of the COOH terminus of Ran. (A) Schematic  drawing of the recombinant fusion protein of the COOH-terminal tail fragment of Ran expressed in E. coli. (B) Immunoblotting  analysis of the E. coli extract which expresses the GST fused Ran  COOH-terminal tail, 207–216 with ARAN1 (lane 1). Lane 2  shows negative control without ARAN1. The right panel shows  an SDS-PAGE profile of E. coli extract expressing GST-fused  COOH-terminal tail fragment of Ran, applied in the same  amount as was used for immunoblotting. (C) Recombinant GST-fused COOH-terminal portion (207–216) of Ran (lane 1) or GST  (lane 2) is incubated with ARAN1 and protein A–bound agarose  beads for 1 h. The beads were then washed and the bound  proteins were analyzed by SDS-PAGE followed by Coomassie  brilliant blue staining. (D) Amino acid comparison of the COOH- terminal domain of Ran in human, mouse, C. elegans and S. cerevisiae. The COOH-terminal domain of human RhoA is also  shown. Negatively charged residues are shown in boldface.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132938&req=5

Figure 3: ARAN1 recognizes an epitope located within 10 amino acid residues of the COOH terminus of Ran. (A) Schematic drawing of the recombinant fusion protein of the COOH-terminal tail fragment of Ran expressed in E. coli. (B) Immunoblotting analysis of the E. coli extract which expresses the GST fused Ran COOH-terminal tail, 207–216 with ARAN1 (lane 1). Lane 2 shows negative control without ARAN1. The right panel shows an SDS-PAGE profile of E. coli extract expressing GST-fused COOH-terminal tail fragment of Ran, applied in the same amount as was used for immunoblotting. (C) Recombinant GST-fused COOH-terminal portion (207–216) of Ran (lane 1) or GST (lane 2) is incubated with ARAN1 and protein A–bound agarose beads for 1 h. The beads were then washed and the bound proteins were analyzed by SDS-PAGE followed by Coomassie brilliant blue staining. (D) Amino acid comparison of the COOH- terminal domain of Ran in human, mouse, C. elegans and S. cerevisiae. The COOH-terminal domain of human RhoA is also shown. Negatively charged residues are shown in boldface.

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)

ARAN1 recognizes an epitope located within 10 amino  acid residues of the COOH terminus of Ran. (A) Schematic  drawing of the recombinant fusion protein of the COOH-terminal tail fragment of Ran expressed in E. coli. (B) Immunoblotting  analysis of the E. coli extract which expresses the GST fused Ran  COOH-terminal tail, 207–216 with ARAN1 (lane 1). Lane 2  shows negative control without ARAN1. The right panel shows  an SDS-PAGE profile of E. coli extract expressing GST-fused  COOH-terminal tail fragment of Ran, applied in the same  amount as was used for immunoblotting. (C) Recombinant GST-fused COOH-terminal portion (207–216) of Ran (lane 1) or GST  (lane 2) is incubated with ARAN1 and protein A–bound agarose  beads for 1 h. The beads were then washed and the bound  proteins were analyzed by SDS-PAGE followed by Coomassie  brilliant blue staining. (D) Amino acid comparison of the COOH- terminal domain of Ran in human, mouse, C. elegans and S. cerevisiae. The COOH-terminal domain of human RhoA is also  shown. Negatively charged residues are shown in boldface.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: ARAN1 recognizes an epitope located within 10 amino acid residues of the COOH terminus of Ran. (A) Schematic drawing of the recombinant fusion protein of the COOH-terminal tail fragment of Ran expressed in E. coli. (B) Immunoblotting analysis of the E. coli extract which expresses the GST fused Ran COOH-terminal tail, 207–216 with ARAN1 (lane 1). Lane 2 shows negative control without ARAN1. The right panel shows an SDS-PAGE profile of E. coli extract expressing GST-fused COOH-terminal tail fragment of Ran, applied in the same amount as was used for immunoblotting. (C) Recombinant GST-fused COOH-terminal portion (207–216) of Ran (lane 1) or GST (lane 2) is incubated with ARAN1 and protein A–bound agarose beads for 1 h. The beads were then washed and the bound proteins were analyzed by SDS-PAGE followed by Coomassie brilliant blue staining. (D) Amino acid comparison of the COOH- terminal domain of Ran in human, mouse, C. elegans and S. cerevisiae. The COOH-terminal domain of human RhoA is also shown. Negatively charged residues are shown in boldface.
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