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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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Ran-GTP production induces local cargo release at chromosomes and supports the chromosomal accumulation of hKid. Digitonin-permeabilized mitotic HeLa cells were incubated with 0.4 μM FLAG-hKid or 0.4 μM Cy3-BSA-NLS in the presence of 0.4 μM CFP–importin-α and 0.4 μM YFP–importin-β, with or without 4 μM Ran-GDP, an energy source, and 1.7 μM CAS. After incubation, the cells were fixed and subjected to indirect immunofluorescence staining. The images were processed as described in Fig 3. Bar, 10 μm.
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fig6: Ran-GTP production induces local cargo release at chromosomes and supports the chromosomal accumulation of hKid. Digitonin-permeabilized mitotic HeLa cells were incubated with 0.4 μM FLAG-hKid or 0.4 μM Cy3-BSA-NLS in the presence of 0.4 μM CFP–importin-α and 0.4 μM YFP–importin-β, with or without 4 μM Ran-GDP, an energy source, and 1.7 μM CAS. After incubation, the cells were fixed and subjected to indirect immunofluorescence staining. The images were processed as described in Fig 3. Bar, 10 μm.

Mentions: To understand how local Ran-GTP production stimulates the importin-β–mediated chromosome loading of hKid, the behavior of importin-α and -β during this process was dissected using CFP-tagged importin-α and YFP-tagged importin-β, both of which are capable of mediating the nuclear import of NLS-containing substrates (unpublished data). As shown in Fig. 6, importin-α and -β were cotargeted to mitotic chromosomes with hKid. The reaction was specifically induced upon the formation of a complex with hKid because a general NLS-containing cargo, SV40 T-antigen NLS conjugate, did not induce the same targeting (Fig. 6, first column). In the presence of Ran-GDP and an energy source, importin-β was released from the mitotic chromosomes (Fig. 6, third column). The addition of CAS, an export factor for importin-α (Kutay et al., 1997), released a significant amount of importin-α from the mitotic chromosomes (Fig. 6, fourth column), whereas hKid remained bound. These results indicate that a transient association of hKid with importin-α and -β is necessary for the initial targeting of hKid to mitotic chromosomes and that local Ran-GTP–mediated cargo release promotes the efficient accumulation of hKid at mitotic chromosomes.


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)

Ran-GTP production induces local cargo release at chromosomes and supports the chromosomal accumulation of hKid. Digitonin-permeabilized mitotic HeLa cells were incubated with 0.4 μM FLAG-hKid or 0.4 μM Cy3-BSA-NLS in the presence of 0.4 μM CFP–importin-α and 0.4 μM YFP–importin-β, with or without 4 μM Ran-GDP, an energy source, and 1.7 μM CAS. After incubation, the cells were fixed and subjected to indirect immunofluorescence staining. The images were processed as described in Fig 3. Bar, 10 μm.
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Related In: Results  -  Collection

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fig6: Ran-GTP production induces local cargo release at chromosomes and supports the chromosomal accumulation of hKid. Digitonin-permeabilized mitotic HeLa cells were incubated with 0.4 μM FLAG-hKid or 0.4 μM Cy3-BSA-NLS in the presence of 0.4 μM CFP–importin-α and 0.4 μM YFP–importin-β, with or without 4 μM Ran-GDP, an energy source, and 1.7 μM CAS. After incubation, the cells were fixed and subjected to indirect immunofluorescence staining. The images were processed as described in Fig 3. Bar, 10 μm.
Mentions: To understand how local Ran-GTP production stimulates the importin-β–mediated chromosome loading of hKid, the behavior of importin-α and -β during this process was dissected using CFP-tagged importin-α and YFP-tagged importin-β, both of which are capable of mediating the nuclear import of NLS-containing substrates (unpublished data). As shown in Fig. 6, importin-α and -β were cotargeted to mitotic chromosomes with hKid. The reaction was specifically induced upon the formation of a complex with hKid because a general NLS-containing cargo, SV40 T-antigen NLS conjugate, did not induce the same targeting (Fig. 6, first column). In the presence of Ran-GDP and an energy source, importin-β was released from the mitotic chromosomes (Fig. 6, third column). The addition of CAS, an export factor for importin-α (Kutay et al., 1997), released a significant amount of importin-α from the mitotic chromosomes (Fig. 6, fourth column), whereas hKid remained bound. These results indicate that a transient association of hKid with importin-α and -β is necessary for the initial targeting of hKid to mitotic chromosomes and that local Ran-GTP–mediated cargo release promotes the efficient accumulation of hKid at mitotic chromosomes.

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