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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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Model of the importin-β–mediated chromosome loading of hKid. The association of hKid with importin-β via importin-α triggers the initial targeting of hKid to mitotic chromosomes, whereas cargo release, mediated by the action of Ran-GTP, which is generated locally at the chromosomes, promotes the accumulation of hKid on the chromosomes. Cargo release is likely coupled with the deposition of hKid onto the chromosomes.
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fig8: Model of the importin-β–mediated chromosome loading of hKid. The association of hKid with importin-β via importin-α triggers the initial targeting of hKid to mitotic chromosomes, whereas cargo release, mediated by the action of Ran-GTP, which is generated locally at the chromosomes, promotes the accumulation of hKid on the chromosomes. Cargo release is likely coupled with the deposition of hKid onto the chromosomes.

Mentions: In this study, we found that importin-β and Ran actively mediate the chromosome loading of hKid during mitosis. We first identified the NLSs of hKid (Fig. 1) and established cell lines that stably expressed wt or NLS-deficient hKid fused to Venus (Fig. 2 A). We then carefully compared the behavior of the two proteins in live cells by FRAP and FLIP and found that hKid with functional NLSs had greater affinity for mitotic chromosomes but lower affinity for the mitotic spindle (Fig. 2). To dissect these phenomena more precisely, we examined how nucleocytoplasmic transport factors affect the mitotic behavior of hKid using digitonin-permeabilized mitotic cells (Fig. 3). In permeabilized cells, we found that chromosome loading of hKid could be reconstituted only in the presence of transport factors (Figs. 3–6), leading to the model depicted in Fig. 8. In this model, hKid is recruited to the vicinity of the chromosomes as a ternary complex with importin-α and -β and then deposited onto the chromosomes by the GTP-exchange reaction of Ran, which is mediated locally by chromosomal RCC1. Dissociation of importin-β occurs through the chromosomal generation of Ran-GTP, whereas CAS is required for the dissociation of importin-α. Based on these phenomena, we propose that importin-α and -β not only inhibit the activity of certain proteins, including SAFs, but also actively recruit proteins to chromosomes. With regard to the deposition of hKid on chromosomes, the Ran-GTP–induced release of importin-α and -β may be critical, as has been shown in the activation of other SAFs (Harel and Forbes, 2004).


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)

Model of the importin-β–mediated chromosome loading of hKid. The association of hKid with importin-β via importin-α triggers the initial targeting of hKid to mitotic chromosomes, whereas cargo release, mediated by the action of Ran-GTP, which is generated locally at the chromosomes, promotes the accumulation of hKid on the chromosomes. Cargo release is likely coupled with the deposition of hKid onto the chromosomes.
© Copyright Policy
Related In: Results  -  Collection

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fig8: Model of the importin-β–mediated chromosome loading of hKid. The association of hKid with importin-β via importin-α triggers the initial targeting of hKid to mitotic chromosomes, whereas cargo release, mediated by the action of Ran-GTP, which is generated locally at the chromosomes, promotes the accumulation of hKid on the chromosomes. Cargo release is likely coupled with the deposition of hKid onto the chromosomes.
Mentions: In this study, we found that importin-β and Ran actively mediate the chromosome loading of hKid during mitosis. We first identified the NLSs of hKid (Fig. 1) and established cell lines that stably expressed wt or NLS-deficient hKid fused to Venus (Fig. 2 A). We then carefully compared the behavior of the two proteins in live cells by FRAP and FLIP and found that hKid with functional NLSs had greater affinity for mitotic chromosomes but lower affinity for the mitotic spindle (Fig. 2). To dissect these phenomena more precisely, we examined how nucleocytoplasmic transport factors affect the mitotic behavior of hKid using digitonin-permeabilized mitotic cells (Fig. 3). In permeabilized cells, we found that chromosome loading of hKid could be reconstituted only in the presence of transport factors (Figs. 3–6), leading to the model depicted in Fig. 8. In this model, hKid is recruited to the vicinity of the chromosomes as a ternary complex with importin-α and -β and then deposited onto the chromosomes by the GTP-exchange reaction of Ran, which is mediated locally by chromosomal RCC1. Dissociation of importin-β occurs through the chromosomal generation of Ran-GTP, whereas CAS is required for the dissociation of importin-α. Based on these phenomena, we propose that importin-α and -β not only inhibit the activity of certain proteins, including SAFs, but also actively recruit proteins to chromosomes. With regard to the deposition of hKid on chromosomes, the Ran-GTP–induced release of importin-α and -β may be critical, as has been shown in the activation of other SAFs (Harel and Forbes, 2004).

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