A monoclonal antibody to the COOH-terminal acidic portion of Ran inhibits both the recycling of Ran and nuclear protein import in living cells - PubMed (original) (raw)

A monoclonal antibody to the COOH-terminal acidic portion of Ran inhibits both the recycling of Ran and nuclear protein import in living cells

M Hieda et al. J Cell Biol. 1999.

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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Figures

Figure 1

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.

Figure 1

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.

Figure 2

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.

Figure 2

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.

Figure 3

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.

Figure 4

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.

Figure 4

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.

Figure 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.

Figure 6

Figure 6

ARAN1 is exported from the nucleus to the cytoplasm. ARAN1 (5 mg/ml) was injected into the cytoplasm (top) or nucleus (bottom) of BHK21 cells with FITC-BSA. After incubation at 37°C for 30 min, cells were fixed and stained with Cy3-conjugated goat anti–mouse IgG antibodies (right). The localization of FITC-BSA shows the injected site in the cells. (B) ARAN1 recognizes the Ran–importin β complex in Ehrlich ascites tumor cell cytosolic extract. ARAN1 was added (lanes 1 and 3) or not (lanes 2 and 4) into the cytosolic extract and incubated for 1 h in the presence of Q69L Ran-GTP. The proteins which were bound to ARAN1 were precipitated with protein A–agarose beads and analyzed by immunoblotting with rabbit anti-Ran polyclonal antibodies (lanes 1 and 2), rabbit anti–importin β polyclonal antibodies (lanes 3 and 4), mouse anti-transportin monoclonal antibody, and mouse anti-CAS monoclonal antibody (lanes 5 and 6, respectively).

Figure 6

Figure 6

ARAN1 is exported from the nucleus to the cytoplasm. ARAN1 (5 mg/ml) was injected into the cytoplasm (top) or nucleus (bottom) of BHK21 cells with FITC-BSA. After incubation at 37°C for 30 min, cells were fixed and stained with Cy3-conjugated goat anti–mouse IgG antibodies (right). The localization of FITC-BSA shows the injected site in the cells. (B) ARAN1 recognizes the Ran–importin β complex in Ehrlich ascites tumor cell cytosolic extract. ARAN1 was added (lanes 1 and 3) or not (lanes 2 and 4) into the cytosolic extract and incubated for 1 h in the presence of Q69L Ran-GTP. The proteins which were bound to ARAN1 were precipitated with protein A–agarose beads and analyzed by immunoblotting with rabbit anti-Ran polyclonal antibodies (lanes 1 and 2), rabbit anti–importin β polyclonal antibodies (lanes 3 and 4), mouse anti-transportin monoclonal antibody, and mouse anti-CAS monoclonal antibody (lanes 5 and 6, respectively).

Figure 7

Figure 7

ARAN1 induces the intracellular localization rearrangement of Ran and inhibits the basic type NLS-dependent nuclear protein import. (A) ARAN1 (bottom) or control mouse IgG (top, both 50 mg/ml) was injected into the cytoplasm of the cultured BHK21 cells and incubated at 37°C for 30 min. The cells were fixed and probed with rabbit anti-Ran antibodies at 4°C overnight, and then stained with Cy3-conjugated goat anti–rabbit antibodies. Injected ARAN1 and control mouse IgG was detected with FITC-conjugated goat anti–mouse IgG antibodies. Phase–contrast microscopy is shown in the right panels. (B) ARAN1 or control mouse IgG (50 mg/ml) was injected into the cytoplasm of cultured BHK21. After a 30-min incubation at 37°C, FITC-labeled T-BSA was injected into the cytoplasm of the same cells. After incubation at 37°C for 20 min, the cells were fixed and observed by fluorescent microscopy. Injected IgG was detected with Cy3-labeled anti-mouse IgG.

Figure 7

Figure 7

ARAN1 induces the intracellular localization rearrangement of Ran and inhibits the basic type NLS-dependent nuclear protein import. (A) ARAN1 (bottom) or control mouse IgG (top, both 50 mg/ml) was injected into the cytoplasm of the cultured BHK21 cells and incubated at 37°C for 30 min. The cells were fixed and probed with rabbit anti-Ran antibodies at 4°C overnight, and then stained with Cy3-conjugated goat anti–rabbit antibodies. Injected ARAN1 and control mouse IgG was detected with FITC-conjugated goat anti–mouse IgG antibodies. Phase–contrast microscopy is shown in the right panels. (B) ARAN1 or control mouse IgG (50 mg/ml) was injected into the cytoplasm of cultured BHK21. After a 30-min incubation at 37°C, FITC-labeled T-BSA was injected into the cytoplasm of the same cells. After incubation at 37°C for 20 min, the cells were fixed and observed by fluorescent microscopy. Injected IgG was detected with Cy3-labeled anti-mouse IgG.

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References

    1. Avis JM, Clarke PR. Ran, a GTPase involved in nuclear processes: its regulators and effectors. J Cell Sci. 1996;109:2423–2427. - PubMed
    1. Bischoff FR, Ponstingl H. Mitotic regulator protein RCC1 is complexed with a nuclear ras-related polypeptide. Proc Natl Acad Sci USA. 1991a;88:10830–10834. - PMC - PubMed
    1. Bischoff FR, Ponstingl H. Catalysis of guanine nucleotide exchange on Ran by the mitotic regulator RCC1. Nature. 1991b;354:80–82. - PubMed
    1. Bischoff FR, Klebe C, Kretschmer J, Wittinghofer A, Ponstingl H. RanGAP1 induces GTPase activity of nuclear ras-related Ran. Proc Natl Acad Sci USA. 1994;91:2587–2591. - PMC - PubMed
    1. Bischoff FR, Krebber H, Smirnova E, Dong W, Ponstingl H. Co-activation of Ran GTPase and inhibition of GTP dissociation by Ran-GTP binding protein RanBP1. EMBO (Eur Mol Biol Organ) J. 1995;14:705–715. - PMC - PubMed

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