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 the Ran–importin β–related transport factors complex but not the Ran molecule alone in solution.  (A) Recombinant Ran-GTP, Ran-GDP, and/or importin β were  incubated with ARAN1 for 1 h at 4°C and protein A–agarose was  then added. After a 1-h incubation, the bound proteins were analyzed by SDS-PAGE followed by Coomassie blue staining for  importin β and by immunoblotting for Ran using rabbit anti-Ran  polyclonal antibodies. Top and bottom panels show the bound  and unbound fractions, respectively. ARAN1 failed to precipitate Ran-GTP, Ran-GDP, and importin β alone, and precipitated  only the Ran-GTP–importin β complex and Ran-GDP–importin  β complex. (B) ARAN1 also recognizes the Ran/transportin  complex and Ran/CAS–importin α complex. Recombinant transportin or CAS and importin β were incubated with Ran-GTP and  ARAN1, then precipitated by protein A–agarose and analyzed  by SDS-PAGE.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132938&req=5

Figure 4: ARAN1 recognizes the Ran–importin β–related transport factors complex but not the Ran molecule alone in solution. (A) Recombinant Ran-GTP, Ran-GDP, and/or importin β were incubated with ARAN1 for 1 h at 4°C and protein A–agarose was then added. After a 1-h incubation, the bound proteins were analyzed by SDS-PAGE followed by Coomassie blue staining for importin β and by immunoblotting for Ran using rabbit anti-Ran polyclonal antibodies. Top and bottom panels show the bound and unbound fractions, respectively. ARAN1 failed to precipitate Ran-GTP, Ran-GDP, and importin β alone, and precipitated only the Ran-GTP–importin β complex and Ran-GDP–importin β complex. (B) ARAN1 also recognizes the Ran/transportin complex and Ran/CAS–importin α complex. Recombinant transportin or CAS and importin β were incubated with Ran-GTP and ARAN1, then precipitated by protein A–agarose and analyzed by SDS-PAGE.

Mentions: It is known that the stable Ran-GTP–importin β complex causes the inhibition of GTP hydrolysis on Ran stimulated by RanGAP1. In contrast, Ran-GDP shows a much lower affinity to importin β than Ran-GTP. To determine which form of Ran is recognized by ARAN1 in solution, immunoprecipitation analysis of ARAN1 using recombinant proteins was performed. Purified recombinant human Ran-GTP or Ran-GDP was incubated with ARAN1 in the presence or absence of importin β followed by the precipitation with protein A–conjugated agarose beads. The results showed that, whereas ARAN1 precipitated neither Ran-GTP nor Ran-GDP alone, it reacted with both Ran-GTP and Ran-GDP only in the presence of importin β and precipitated both Ran and importin β. To clearly confirm that ARAN1 can't recognize Ran molecule alone, precipitated proteins were analyzed by immunoblotting with anti-Ran polyclonal antibodies (Fig. 4 A). The molar ratio of the Ran and importin β precipitated with ARAN1 was estimated to be ∼1:1 (data not shown). Since ARAN1 did not bind to importin β directly in this assay, these results indicate that ARAN1 reacts only with Ran when bound to importin β, and suggest that Ran changes conformation when bound to importin β which, in turn, exposes its COOH-terminal portion.


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 the Ran–importin β–related transport factors complex but not the Ran molecule alone in solution.  (A) Recombinant Ran-GTP, Ran-GDP, and/or importin β were  incubated with ARAN1 for 1 h at 4°C and protein A–agarose was  then added. After a 1-h incubation, the bound proteins were analyzed by SDS-PAGE followed by Coomassie blue staining for  importin β and by immunoblotting for Ran using rabbit anti-Ran  polyclonal antibodies. Top and bottom panels show the bound  and unbound fractions, respectively. ARAN1 failed to precipitate Ran-GTP, Ran-GDP, and importin β alone, and precipitated  only the Ran-GTP–importin β complex and Ran-GDP–importin  β complex. (B) ARAN1 also recognizes the Ran/transportin  complex and Ran/CAS–importin α complex. Recombinant transportin or CAS and importin β were incubated with Ran-GTP and  ARAN1, then precipitated by protein A–agarose and analyzed  by SDS-PAGE.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: ARAN1 recognizes the Ran–importin β–related transport factors complex but not the Ran molecule alone in solution. (A) Recombinant Ran-GTP, Ran-GDP, and/or importin β were incubated with ARAN1 for 1 h at 4°C and protein A–agarose was then added. After a 1-h incubation, the bound proteins were analyzed by SDS-PAGE followed by Coomassie blue staining for importin β and by immunoblotting for Ran using rabbit anti-Ran polyclonal antibodies. Top and bottom panels show the bound and unbound fractions, respectively. ARAN1 failed to precipitate Ran-GTP, Ran-GDP, and importin β alone, and precipitated only the Ran-GTP–importin β complex and Ran-GDP–importin β complex. (B) ARAN1 also recognizes the Ran/transportin complex and Ran/CAS–importin α complex. Recombinant transportin or CAS and importin β were incubated with Ran-GTP and ARAN1, then precipitated by protein A–agarose and analyzed by SDS-PAGE.
Mentions: It is known that the stable Ran-GTP–importin β complex causes the inhibition of GTP hydrolysis on Ran stimulated by RanGAP1. In contrast, Ran-GDP shows a much lower affinity to importin β than Ran-GTP. To determine which form of Ran is recognized by ARAN1 in solution, immunoprecipitation analysis of ARAN1 using recombinant proteins was performed. Purified recombinant human Ran-GTP or Ran-GDP was incubated with ARAN1 in the presence or absence of importin β followed by the precipitation with protein A–conjugated agarose beads. The results showed that, whereas ARAN1 precipitated neither Ran-GTP nor Ran-GDP alone, it reacted with both Ran-GTP and Ran-GDP only in the presence of importin β and precipitated both Ran and importin β. To clearly confirm that ARAN1 can't recognize Ran molecule alone, precipitated proteins were analyzed by immunoblotting with anti-Ran polyclonal antibodies (Fig. 4 A). The molar ratio of the Ran and importin β precipitated with ARAN1 was estimated to be ∼1:1 (data not shown). Since ARAN1 did not bind to importin β directly in this assay, these results indicate that ARAN1 reacts only with Ran when bound to importin β, and suggest that Ran changes conformation when bound to importin β which, in turn, exposes its COOH-terminal portion.

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