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 prevents the formation of Ran/RanBP1– importin β complex. (A) ARAN1 was added to the immobilized  importin β–Ran-GTP complex and incubated for 30 min. RanBP1  was then added to the mixture. After 30 min of incubation, the  proteins bound to importin β were precipitated and analyzed  by SDS-PAGE followed by Coomassie brilliant blue staining. (B)  GST-tagged importin β was mixed with the Ran-GDP and  ARAN1 and then incubated with glutathione–Sepharose beads  for 1 h. After washing the beads, RanBP1 was added the mixture  and incubated for 1 h. The proteins bound to importin β were  precipitated and analyzed by SDS-PAGE followed by Coomassie  brilliant blue staining.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132938&req=5

Figure 5: ARAN1 prevents the formation of Ran/RanBP1– importin β complex. (A) ARAN1 was added to the immobilized importin β–Ran-GTP complex and incubated for 30 min. RanBP1 was then added to the mixture. After 30 min of incubation, the proteins bound to importin β were precipitated and analyzed by SDS-PAGE followed by Coomassie brilliant blue staining. (B) GST-tagged importin β was mixed with the Ran-GDP and ARAN1 and then incubated with glutathione–Sepharose beads for 1 h. After washing the beads, RanBP1 was added the mixture and incubated for 1 h. The proteins bound to importin β were precipitated and analyzed by SDS-PAGE followed by Coomassie brilliant blue staining.

Mentions: Both Ran-GTP and Ran-GDP form a ternary complex with RanBP1 and importin β in a solution binding assay. Although it has been assumed that RanBP1 plays a role in the dissociation of the Ran-GTP–importin β complex, the biological significance of the ternary complex has not yet been established. The RanBP1–Ran-GDP–importin β complex has been proposed to play a role in nuclear protein import as a ternary complex (Chi et al., 1997), although it has not yet demonstrated how the complex functions in the nuclear protein import. We therefore examined the effects of ARAN1 on the interaction of RanBP1 with both Ran-GTP– importin β and Ran-GDP–importin β complexes in the solution binding assay. In the absence of ARAN1, GST-tagged importin β efficiently precipitated both Ran-GTP and Ran-GDP together with RanBP1 in a molar ratio of 1:1 (Fig. 5, A and B, lane 1). In contrast, in the presence of ARAN1, the amount of RanBP1 precipitated with GST-tagged importin β apparently decreased, although the Ran was efficiently precipitated. These results indicate that RanBP1 is not able to interact with the Ran-GTP–importin β and Ran-GDP– importin β complexes, when bound to ARAN1. That is, the binding of ARAN1 to the Ran–importin β complex suppresses the ternary complex formation of both RanBP1– Ran-GTP–importin β and RanBP1–Ran-GDP–importin β in solution.


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 prevents the formation of Ran/RanBP1– importin β complex. (A) ARAN1 was added to the immobilized  importin β–Ran-GTP complex and incubated for 30 min. RanBP1  was then added to the mixture. After 30 min of incubation, the  proteins bound to importin β were precipitated and analyzed  by SDS-PAGE followed by Coomassie brilliant blue staining. (B)  GST-tagged importin β was mixed with the Ran-GDP and  ARAN1 and then incubated with glutathione–Sepharose beads  for 1 h. After washing the beads, RanBP1 was added the mixture  and incubated for 1 h. The proteins bound to importin β were  precipitated and analyzed by SDS-PAGE followed by Coomassie  brilliant blue staining.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: ARAN1 prevents the formation of Ran/RanBP1– importin β complex. (A) ARAN1 was added to the immobilized importin β–Ran-GTP complex and incubated for 30 min. RanBP1 was then added to the mixture. After 30 min of incubation, the proteins bound to importin β were precipitated and analyzed by SDS-PAGE followed by Coomassie brilliant blue staining. (B) GST-tagged importin β was mixed with the Ran-GDP and ARAN1 and then incubated with glutathione–Sepharose beads for 1 h. After washing the beads, RanBP1 was added the mixture and incubated for 1 h. The proteins bound to importin β were precipitated and analyzed by SDS-PAGE followed by Coomassie brilliant blue staining.
Mentions: Both Ran-GTP and Ran-GDP form a ternary complex with RanBP1 and importin β in a solution binding assay. Although it has been assumed that RanBP1 plays a role in the dissociation of the Ran-GTP–importin β complex, the biological significance of the ternary complex has not yet been established. The RanBP1–Ran-GDP–importin β complex has been proposed to play a role in nuclear protein import as a ternary complex (Chi et al., 1997), although it has not yet demonstrated how the complex functions in the nuclear protein import. We therefore examined the effects of ARAN1 on the interaction of RanBP1 with both Ran-GTP– importin β and Ran-GDP–importin β complexes in the solution binding assay. In the absence of ARAN1, GST-tagged importin β efficiently precipitated both Ran-GTP and Ran-GDP together with RanBP1 in a molar ratio of 1:1 (Fig. 5, A and B, lane 1). In contrast, in the presence of ARAN1, the amount of RanBP1 precipitated with GST-tagged importin β apparently decreased, although the Ran was efficiently precipitated. These results indicate that RanBP1 is not able to interact with the Ran-GTP–importin β and Ran-GDP– importin β complexes, when bound to ARAN1. That is, the binding of ARAN1 to the Ran–importin β complex suppresses the ternary complex formation of both RanBP1– Ran-GTP–importin β and RanBP1–Ran-GDP–importin β in solution.

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