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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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Importin-β–mediated chromosome loading of hKid is promoted by the production of Ran-GTP. (A) Digitonin-permeabilized mitotic HeLa cells were incubated with 0.4 μM FLAG-hKid in the presence or absence of 0.8 μM importin-α, 0.8 μM importin-β, and 4 μM Ran-GDP with or without an energy source. (B) Digitonin-permeabilized mitotic HeLa cells were incubated with 0.4 μM FLAG-hKid in the presence or absence of 0.8 μM importin-α, 0.8 μM importin-β, and 4 μM RanGDP or 4 μM T24N Ran with an energy source. After incubation, the cells were fixed and subjected to indirect immunofluorescence staining and the images were processed as described in Fig 3. Bars, 10 μm. Quantification of the fluorescent intensities of the images taken under the same conditions showed that the chromosomal accumulation of FLAG-hKid was reduced to 57% when Ran-GDP was replaced with T24N Ran.
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fig4: Importin-β–mediated chromosome loading of hKid is promoted by the production of Ran-GTP. (A) Digitonin-permeabilized mitotic HeLa cells were incubated with 0.4 μM FLAG-hKid in the presence or absence of 0.8 μM importin-α, 0.8 μM importin-β, and 4 μM Ran-GDP with or without an energy source. (B) Digitonin-permeabilized mitotic HeLa cells were incubated with 0.4 μM FLAG-hKid in the presence or absence of 0.8 μM importin-α, 0.8 μM importin-β, and 4 μM RanGDP or 4 μM T24N Ran with an energy source. After incubation, the cells were fixed and subjected to indirect immunofluorescence staining and the images were processed as described in Fig 3. Bars, 10 μm. Quantification of the fluorescent intensities of the images taken under the same conditions showed that the chromosomal accumulation of FLAG-hKid was reduced to 57% when Ran-GDP was replaced with T24N Ran.

Mentions: In mitosis, it is well accepted that chromosome-bound RCC1, a Ran guanine nucleotide exchange factor, constitutively generates Ran-GTP, producing a high Ran-GTP concentration around mitotic chromosomes (Kalab et al., 2002, 2006). Such a high local Ran-GTP concentration is considered to be critical for liberating various mitotic factors from the inhibitory effects of importins (Harel and Forbes, 2004). Along with this scenario, one may expect that importin-β does not bind hKid in the vicinity of the chromosomes; therefore, the physiological relevance of the importin-β–mediated chromosome loading of hKid is unknown. Q69L Ran-GTP reversed the effects of importin-β (Fig. 3 A, third column); however, under physiological conditions, Ran is continuously interconverted between its GTP and GDP forms at the mitotic chromosomes because of the actions of RCC1 and RanGAP (Melchior, 2001). We therefore partially reproduced these conditions in permeabilized cells by adding Ran-GDP together with an energy source and reexamined the behavior of hKid in that setting. Although the addition of Ran-GDP to the reaction did not overtly affect the behavior of hKid with regard to the chromosome and spindle (Figs. 3 A and 4 A, compare second columns), the addition of Ran-GDP together with an energy source enhanced the chromosome loading of hKid (Fig. 4 A, fourth column). The addition of Ran-GDP and an energy source in the absence of importin-α and -β also induced weak chromosome binding of hKid (Figs. 4 A and 5 A, third column). A chromosomal factor must have mediated this binding because mutant hKid that was defective in its NLSs was able to bind the chromosomes under these conditions (Fig. 5 B). The effect observed here was Ran independent because addition of the energy source alone also induced the same level of binding (unpublished data). When Ran-GDP was replaced with T24N Ran, a mutant Ran that inhibits the production of Ran-GTP by binding to RCC1 (Klebe et al., 1995), no increase in the chromosome loading of hKid was observed (Fig. 4 B). The addition of an energy source to permeabilized cells inevitably caused depolymerization of the spindle microtubules (Fig. 4 A, third row), raising the possibility that the increase in chromosome loading seen in the fourth column of Fig. 4 A was merely caused by a shift in the spindle population to the chromosomes. This possibility is unlikely, however, because the targeting of hKid was not enhanced in all settings involving disruption of the mitotic spindle in the presence of importin-α and β (Fig. 4 B, right; and Fig. 5). Collectively, hKid seems to be targeted as a complex, with importin-α and -β, to the vicinity of mitotic chromosomes where it is loaded in a Ran-GTP–dependent manner.


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)

Importin-β–mediated chromosome loading of hKid is promoted by the production of Ran-GTP. (A) Digitonin-permeabilized mitotic HeLa cells were incubated with 0.4 μM FLAG-hKid in the presence or absence of 0.8 μM importin-α, 0.8 μM importin-β, and 4 μM Ran-GDP with or without an energy source. (B) Digitonin-permeabilized mitotic HeLa cells were incubated with 0.4 μM FLAG-hKid in the presence or absence of 0.8 μM importin-α, 0.8 μM importin-β, and 4 μM RanGDP or 4 μM T24N Ran with an energy source. After incubation, the cells were fixed and subjected to indirect immunofluorescence staining and the images were processed as described in Fig 3. Bars, 10 μm. Quantification of the fluorescent intensities of the images taken under the same conditions showed that the chromosomal accumulation of FLAG-hKid was reduced to 57% when Ran-GDP was replaced with T24N Ran.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2234231&req=5

fig4: Importin-β–mediated chromosome loading of hKid is promoted by the production of Ran-GTP. (A) Digitonin-permeabilized mitotic HeLa cells were incubated with 0.4 μM FLAG-hKid in the presence or absence of 0.8 μM importin-α, 0.8 μM importin-β, and 4 μM Ran-GDP with or without an energy source. (B) Digitonin-permeabilized mitotic HeLa cells were incubated with 0.4 μM FLAG-hKid in the presence or absence of 0.8 μM importin-α, 0.8 μM importin-β, and 4 μM RanGDP or 4 μM T24N Ran with an energy source. After incubation, the cells were fixed and subjected to indirect immunofluorescence staining and the images were processed as described in Fig 3. Bars, 10 μm. Quantification of the fluorescent intensities of the images taken under the same conditions showed that the chromosomal accumulation of FLAG-hKid was reduced to 57% when Ran-GDP was replaced with T24N Ran.
Mentions: In mitosis, it is well accepted that chromosome-bound RCC1, a Ran guanine nucleotide exchange factor, constitutively generates Ran-GTP, producing a high Ran-GTP concentration around mitotic chromosomes (Kalab et al., 2002, 2006). Such a high local Ran-GTP concentration is considered to be critical for liberating various mitotic factors from the inhibitory effects of importins (Harel and Forbes, 2004). Along with this scenario, one may expect that importin-β does not bind hKid in the vicinity of the chromosomes; therefore, the physiological relevance of the importin-β–mediated chromosome loading of hKid is unknown. Q69L Ran-GTP reversed the effects of importin-β (Fig. 3 A, third column); however, under physiological conditions, Ran is continuously interconverted between its GTP and GDP forms at the mitotic chromosomes because of the actions of RCC1 and RanGAP (Melchior, 2001). We therefore partially reproduced these conditions in permeabilized cells by adding Ran-GDP together with an energy source and reexamined the behavior of hKid in that setting. Although the addition of Ran-GDP to the reaction did not overtly affect the behavior of hKid with regard to the chromosome and spindle (Figs. 3 A and 4 A, compare second columns), the addition of Ran-GDP together with an energy source enhanced the chromosome loading of hKid (Fig. 4 A, fourth column). The addition of Ran-GDP and an energy source in the absence of importin-α and -β also induced weak chromosome binding of hKid (Figs. 4 A and 5 A, third column). A chromosomal factor must have mediated this binding because mutant hKid that was defective in its NLSs was able to bind the chromosomes under these conditions (Fig. 5 B). The effect observed here was Ran independent because addition of the energy source alone also induced the same level of binding (unpublished data). When Ran-GDP was replaced with T24N Ran, a mutant Ran that inhibits the production of Ran-GTP by binding to RCC1 (Klebe et al., 1995), no increase in the chromosome loading of hKid was observed (Fig. 4 B). The addition of an energy source to permeabilized cells inevitably caused depolymerization of the spindle microtubules (Fig. 4 A, third row), raising the possibility that the increase in chromosome loading seen in the fourth column of Fig. 4 A was merely caused by a shift in the spindle population to the chromosomes. This possibility is unlikely, however, because the targeting of hKid was not enhanced in all settings involving disruption of the mitotic spindle in the presence of importin-α and β (Fig. 4 B, right; and Fig. 5). Collectively, hKid seems to be targeted as a complex, with importin-α and -β, to the vicinity of mitotic chromosomes where it is loaded in a Ran-GTP–dependent manner.

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
Related in: MedlinePlus