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Importin-beta and the small guanosine triphosphatase Ran mediate chromosome loading of the human chromokinesin Kid.

Tahara K, Takagi M, Ohsugi M, Sone T, Nishiumi F, Maeshima K, Horiuchi Y, Tokai-Nishizumi N, Imamoto F, Yamamoto T, Kose S, Imamoto N - J. Cell Biol. (2008)

Bottom Line: Upon the loss of its functional NLSs, hKid exhibited reduced interactions with the mitotic chromosomes of living cells.Our results indicate that the association of importin-beta and -alpha with hKid triggers the initial targeting of hKid to mitotic chromosomes and that local Ran-GTP-mediated cargo release promotes the accumulation of hKid on chromosomes.Thus, this study demonstrates a novel nucleocytoplasmic transport factor-mediated mechanism for targeting proteins to mitotic chromosomes.

View Article: PubMed Central - PubMed

Affiliation: Cellular Dynamics Laboratory, Discovery Research Institute, Institute of Physical and Chemical Research, Wako, Saitama, 351-0198, Japan.

ABSTRACT
Nucleocytoplasmic transport factors mediate various cellular processes, including nuclear transport, spindle assembly, and nuclear envelope/pore formation. In this paper, we identify the chromokinesin human kinesin-like DNA binding protein (hKid) as an import cargo of the importin-alpha/beta transport pathway and determine its nuclear localization signals (NLSs). Upon the loss of its functional NLSs, hKid exhibited reduced interactions with the mitotic chromosomes of living cells. In digitonin-permeabilized mitotic cells, hKid was bound only to the spindle and not to the chromosomes themselves. Surprisingly, hKid bound to importin-alpha/beta was efficiently targeted to mitotic chromosomes. The addition of Ran-guanosine diphosphate and an energy source, which generates Ran-guanosine triphosphate (GTP) locally at mitotic chromosomes, enhanced the importin-beta-mediated chromosome loading of hKid. Our results indicate that the association of importin-beta and -alpha with hKid triggers the initial targeting of hKid to mitotic chromosomes and that local Ran-GTP-mediated cargo release promotes the accumulation of hKid on chromosomes. Thus, this study demonstrates a novel nucleocytoplasmic transport factor-mediated mechanism for targeting proteins to mitotic chromosomes.

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hKid possesses two functional NLSs that bind importin-β via importin-α. (A) 30 pmol of recombinant GST–importin-β, 60 pmol FLAG-hKid, and 30 pmol importin-α were incubated in 50 μl of solution and then pulled down with glutathione-Sepharose beads. The bound proteins were analyzed by SDS-PAGE followed by Coomassie brilliant blue staining. hKid bound importin-β in the presence of importin-α. (B) 20 pmol of recombinant GST–importin-α or its ED mutant (defective in basic NLS binding) was incubated with 40 pmol FLAG-hKid, 40 pmol importin-β, or both in 50 μl of solution and then pulled down with glutathione-Sepharose beads. Coomassie brilliant blue staining of the bound proteins analyzed by SDS-PAGE is shown. Importin-α bound hKid directly, whereas the mutant version of importin-α did not bind hKid. Importin-β binding was unaffected by the mutations introduced in importin-α. (C, top) Nuclear migration of 0.4 μM of recombinant FLAG-hKid was examined in digitonin-permeabilized HeLa cells in the presence of 0.4 μM importin-α, 0.4 μM importin-β, 4 μM Ran-GDP, and an energy source. (C, bottom) Localization of EGFP-hKid in HeLa cells 3 h after nuclear injection of the expression plasmid. Mutations in both NLSs abolished the nuclear localization of hKid. Bars, 10 μm. (D) Schematic diagram of hKid. The positions of the two NLSs where mutations were introduced are indicated by arrows.
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fig1: hKid possesses two functional NLSs that bind importin-β via importin-α. (A) 30 pmol of recombinant GST–importin-β, 60 pmol FLAG-hKid, and 30 pmol importin-α were incubated in 50 μl of solution and then pulled down with glutathione-Sepharose beads. The bound proteins were analyzed by SDS-PAGE followed by Coomassie brilliant blue staining. hKid bound importin-β in the presence of importin-α. (B) 20 pmol of recombinant GST–importin-α or its ED mutant (defective in basic NLS binding) was incubated with 40 pmol FLAG-hKid, 40 pmol importin-β, or both in 50 μl of solution and then pulled down with glutathione-Sepharose beads. Coomassie brilliant blue staining of the bound proteins analyzed by SDS-PAGE is shown. Importin-α bound hKid directly, whereas the mutant version of importin-α did not bind hKid. Importin-β binding was unaffected by the mutations introduced in importin-α. (C, top) Nuclear migration of 0.4 μM of recombinant FLAG-hKid was examined in digitonin-permeabilized HeLa cells in the presence of 0.4 μM importin-α, 0.4 μM importin-β, 4 μM Ran-GDP, and an energy source. (C, bottom) Localization of EGFP-hKid in HeLa cells 3 h after nuclear injection of the expression plasmid. Mutations in both NLSs abolished the nuclear localization of hKid. Bars, 10 μm. (D) Schematic diagram of hKid. The positions of the two NLSs where mutations were introduced are indicated by arrows.

Mentions: hKid was previously shown to contain at least two NLSs that bind to importin-β either directly or indirectly in a Ran-GTP–sensitive manner (Trieselmann et al., 2003). We assessed the binding of hKid to various transport receptors using bacterially expressed full-length recombinant hKid. As shown in Fig. 1 A, purified recombinant hKid did not bind importin-β directly but associated with importin-β in the presence of importin-α. Using a digitonin-permeabilized cell-free transport assay (Adam et al., 1990), hKid was found to migrate into the nucleus only in the presence of importin-α, importin-β, and Ran, which is consistent with the behavior of proteins that migrate into the nucleus via the conventional importin-α/β pathway (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200708003/DC1).


Importin-beta and the small guanosine triphosphatase Ran mediate chromosome loading of the human chromokinesin Kid.

Tahara K, Takagi M, Ohsugi M, Sone T, Nishiumi F, Maeshima K, Horiuchi Y, Tokai-Nishizumi N, Imamoto F, Yamamoto T, Kose S, Imamoto N - J. Cell Biol. (2008)

hKid possesses two functional NLSs that bind importin-β via importin-α. (A) 30 pmol of recombinant GST–importin-β, 60 pmol FLAG-hKid, and 30 pmol importin-α were incubated in 50 μl of solution and then pulled down with glutathione-Sepharose beads. The bound proteins were analyzed by SDS-PAGE followed by Coomassie brilliant blue staining. hKid bound importin-β in the presence of importin-α. (B) 20 pmol of recombinant GST–importin-α or its ED mutant (defective in basic NLS binding) was incubated with 40 pmol FLAG-hKid, 40 pmol importin-β, or both in 50 μl of solution and then pulled down with glutathione-Sepharose beads. Coomassie brilliant blue staining of the bound proteins analyzed by SDS-PAGE is shown. Importin-α bound hKid directly, whereas the mutant version of importin-α did not bind hKid. Importin-β binding was unaffected by the mutations introduced in importin-α. (C, top) Nuclear migration of 0.4 μM of recombinant FLAG-hKid was examined in digitonin-permeabilized HeLa cells in the presence of 0.4 μM importin-α, 0.4 μM importin-β, 4 μM Ran-GDP, and an energy source. (C, bottom) Localization of EGFP-hKid in HeLa cells 3 h after nuclear injection of the expression plasmid. Mutations in both NLSs abolished the nuclear localization of hKid. Bars, 10 μm. (D) Schematic diagram of hKid. The positions of the two NLSs where mutations were introduced are indicated by arrows.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2234231&req=5

fig1: hKid possesses two functional NLSs that bind importin-β via importin-α. (A) 30 pmol of recombinant GST–importin-β, 60 pmol FLAG-hKid, and 30 pmol importin-α were incubated in 50 μl of solution and then pulled down with glutathione-Sepharose beads. The bound proteins were analyzed by SDS-PAGE followed by Coomassie brilliant blue staining. hKid bound importin-β in the presence of importin-α. (B) 20 pmol of recombinant GST–importin-α or its ED mutant (defective in basic NLS binding) was incubated with 40 pmol FLAG-hKid, 40 pmol importin-β, or both in 50 μl of solution and then pulled down with glutathione-Sepharose beads. Coomassie brilliant blue staining of the bound proteins analyzed by SDS-PAGE is shown. Importin-α bound hKid directly, whereas the mutant version of importin-α did not bind hKid. Importin-β binding was unaffected by the mutations introduced in importin-α. (C, top) Nuclear migration of 0.4 μM of recombinant FLAG-hKid was examined in digitonin-permeabilized HeLa cells in the presence of 0.4 μM importin-α, 0.4 μM importin-β, 4 μM Ran-GDP, and an energy source. (C, bottom) Localization of EGFP-hKid in HeLa cells 3 h after nuclear injection of the expression plasmid. Mutations in both NLSs abolished the nuclear localization of hKid. Bars, 10 μm. (D) Schematic diagram of hKid. The positions of the two NLSs where mutations were introduced are indicated by arrows.
Mentions: hKid was previously shown to contain at least two NLSs that bind to importin-β either directly or indirectly in a Ran-GTP–sensitive manner (Trieselmann et al., 2003). We assessed the binding of hKid to various transport receptors using bacterially expressed full-length recombinant hKid. As shown in Fig. 1 A, purified recombinant hKid did not bind importin-β directly but associated with importin-β in the presence of importin-α. Using a digitonin-permeabilized cell-free transport assay (Adam et al., 1990), hKid was found to migrate into the nucleus only in the presence of importin-α, importin-β, and Ran, which is consistent with the behavior of proteins that migrate into the nucleus via the conventional importin-α/β pathway (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200708003/DC1).

Bottom Line: Upon the loss of its functional NLSs, hKid exhibited reduced interactions with the mitotic chromosomes of living cells.Our results indicate that the association of importin-beta and -alpha with hKid triggers the initial targeting of hKid to mitotic chromosomes and that local Ran-GTP-mediated cargo release promotes the accumulation of hKid on chromosomes.Thus, this study demonstrates a novel nucleocytoplasmic transport factor-mediated mechanism for targeting proteins to mitotic chromosomes.

View Article: PubMed Central - PubMed

Affiliation: Cellular Dynamics Laboratory, Discovery Research Institute, Institute of Physical and Chemical Research, Wako, Saitama, 351-0198, Japan.

ABSTRACT
Nucleocytoplasmic transport factors mediate various cellular processes, including nuclear transport, spindle assembly, and nuclear envelope/pore formation. In this paper, we identify the chromokinesin human kinesin-like DNA binding protein (hKid) as an import cargo of the importin-alpha/beta transport pathway and determine its nuclear localization signals (NLSs). Upon the loss of its functional NLSs, hKid exhibited reduced interactions with the mitotic chromosomes of living cells. In digitonin-permeabilized mitotic cells, hKid was bound only to the spindle and not to the chromosomes themselves. Surprisingly, hKid bound to importin-alpha/beta was efficiently targeted to mitotic chromosomes. The addition of Ran-guanosine diphosphate and an energy source, which generates Ran-guanosine triphosphate (GTP) locally at mitotic chromosomes, enhanced the importin-beta-mediated chromosome loading of hKid. Our results indicate that the association of importin-beta and -alpha with hKid triggers the initial targeting of hKid to mitotic chromosomes and that local Ran-GTP-mediated cargo release promotes the accumulation of hKid on chromosomes. Thus, this study demonstrates a novel nucleocytoplasmic transport factor-mediated mechanism for targeting proteins to mitotic chromosomes.

Show MeSH