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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-α/β inhibits spindle binding but facilitates chromosome loading of hKid in digitonin-permeabilized cells. (A) Digitonin-permeabilized mitotic HeLa cells were incubated with 0.4 μM FLAG-hKid or FLAG–ΔNLSs hKid in the absence or presence of 0.8 μM each importin-α and -β, with or without 4 μM Q69L Ran-GTP. Note that the recombinant proteins were present in excess compared with the mitotic chromosomes and mitotic spindles contained in the permeabilized cells, indicating that the reduction observed in the spindle signal of hKid upon addition of importin-α/β was caused by the inhibition of spindle binding. (B) Digitonin-permeabilized mitotic HeLa cells were incubated with 0.4 μM FLAG-hKid in the presence of either 0.8 μM importin-α or -β. All reactions were performed in the presence of apyrase, which depleted the energy source. The same results were obtained without the addition of the energy source to the reaction mixture (not depicted). After incubation, the cells were fixed and processed for indirect immunofluorescence staining to detect FLAG-hKids and the spindle microtubules, as described in Materials and methods. DNA was counterstained with DAPI. The images are projections of five deconvolved sections taken at 0.2-μm focus intervals. Bars, 10 μm.
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fig3: Importin-α/β inhibits spindle binding but facilitates chromosome loading of hKid in digitonin-permeabilized cells. (A) Digitonin-permeabilized mitotic HeLa cells were incubated with 0.4 μM FLAG-hKid or FLAG–ΔNLSs hKid in the absence or presence of 0.8 μM each importin-α and -β, with or without 4 μM Q69L Ran-GTP. Note that the recombinant proteins were present in excess compared with the mitotic chromosomes and mitotic spindles contained in the permeabilized cells, indicating that the reduction observed in the spindle signal of hKid upon addition of importin-α/β was caused by the inhibition of spindle binding. (B) Digitonin-permeabilized mitotic HeLa cells were incubated with 0.4 μM FLAG-hKid in the presence of either 0.8 μM importin-α or -β. All reactions were performed in the presence of apyrase, which depleted the energy source. The same results were obtained without the addition of the energy source to the reaction mixture (not depicted). After incubation, the cells were fixed and processed for indirect immunofluorescence staining to detect FLAG-hKids and the spindle microtubules, as described in Materials and methods. DNA was counterstained with DAPI. The images are projections of five deconvolved sections taken at 0.2-μm focus intervals. Bars, 10 μm.

Mentions: To make sense of the observation in the previous section, we examined the mitotic behavior of hKid in digitonin-permeabilized mitotic cells using purified recombinant proteins. As shown in Fig. 3 A (first column), in permeabilized mitotic cells incubated with purified recombinant hKid, hKid was bound only to the mitotic spindles, and not to the mitotic chromosomes, despite its DNA binding domain. Surprisingly, when hKid was incubated together with importin-α and -β, it was efficiently targeted to the mitotic chromosomes (Fig. 3 A, second column), whereas targeting to the spindle was reduced (Fig. 3 A, compare the first and second columns). The addition of Q69L Ran-GTP, a mutant Ran defective in GTP hydrolysis (Klebe et al., 1995) that disrupts the binding of cargo to importin-β, reversed the effects of importin-α and -β (Fig. 3 A, third column). Accordingly, ΔNLSs hKid was able to bind the spindle microtubules regardless of whether importin-α and -β were present (Fig. 3 A, fourth column). An excess amount of each recombinant protein was incubated with the permeabilized cells; therefore, the reduction in spindle or chromosome binding observed under each condition should represent the inability of hKid to bind either structures, respectively, rather than an indirect effect, such as the consequence of sequestering hKid. We thus concluded that the binding of importin-α and -β to hKid targets hKid to chromosomes and disrupts its ability to bind the mitotic spindle. A previous study showed that importin-α and -β inhibit the microtubule binding of hKid in vitro (Trieselmann et al., 2003), which supports our results. It must be noted that neither phenomena was observed in the presence of importin-α or -β alone (Fig. 3 B), demonstrating the necessity of binding with importin-β and -α in determining the subcellular localization of hKid.


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-α/β inhibits spindle binding but facilitates chromosome loading of hKid in digitonin-permeabilized cells. (A) Digitonin-permeabilized mitotic HeLa cells were incubated with 0.4 μM FLAG-hKid or FLAG–ΔNLSs hKid in the absence or presence of 0.8 μM each importin-α and -β, with or without 4 μM Q69L Ran-GTP. Note that the recombinant proteins were present in excess compared with the mitotic chromosomes and mitotic spindles contained in the permeabilized cells, indicating that the reduction observed in the spindle signal of hKid upon addition of importin-α/β was caused by the inhibition of spindle binding. (B) Digitonin-permeabilized mitotic HeLa cells were incubated with 0.4 μM FLAG-hKid in the presence of either 0.8 μM importin-α or -β. All reactions were performed in the presence of apyrase, which depleted the energy source. The same results were obtained without the addition of the energy source to the reaction mixture (not depicted). After incubation, the cells were fixed and processed for indirect immunofluorescence staining to detect FLAG-hKids and the spindle microtubules, as described in Materials and methods. DNA was counterstained with DAPI. The images are projections of five deconvolved sections taken at 0.2-μm focus intervals. Bars, 10 μm.
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getmorefigures.php?uid=PMC2234231&req=5

fig3: Importin-α/β inhibits spindle binding but facilitates chromosome loading of hKid in digitonin-permeabilized cells. (A) Digitonin-permeabilized mitotic HeLa cells were incubated with 0.4 μM FLAG-hKid or FLAG–ΔNLSs hKid in the absence or presence of 0.8 μM each importin-α and -β, with or without 4 μM Q69L Ran-GTP. Note that the recombinant proteins were present in excess compared with the mitotic chromosomes and mitotic spindles contained in the permeabilized cells, indicating that the reduction observed in the spindle signal of hKid upon addition of importin-α/β was caused by the inhibition of spindle binding. (B) Digitonin-permeabilized mitotic HeLa cells were incubated with 0.4 μM FLAG-hKid in the presence of either 0.8 μM importin-α or -β. All reactions were performed in the presence of apyrase, which depleted the energy source. The same results were obtained without the addition of the energy source to the reaction mixture (not depicted). After incubation, the cells were fixed and processed for indirect immunofluorescence staining to detect FLAG-hKids and the spindle microtubules, as described in Materials and methods. DNA was counterstained with DAPI. The images are projections of five deconvolved sections taken at 0.2-μm focus intervals. Bars, 10 μm.
Mentions: To make sense of the observation in the previous section, we examined the mitotic behavior of hKid in digitonin-permeabilized mitotic cells using purified recombinant proteins. As shown in Fig. 3 A (first column), in permeabilized mitotic cells incubated with purified recombinant hKid, hKid was bound only to the mitotic spindles, and not to the mitotic chromosomes, despite its DNA binding domain. Surprisingly, when hKid was incubated together with importin-α and -β, it was efficiently targeted to the mitotic chromosomes (Fig. 3 A, second column), whereas targeting to the spindle was reduced (Fig. 3 A, compare the first and second columns). The addition of Q69L Ran-GTP, a mutant Ran defective in GTP hydrolysis (Klebe et al., 1995) that disrupts the binding of cargo to importin-β, reversed the effects of importin-α and -β (Fig. 3 A, third column). Accordingly, ΔNLSs hKid was able to bind the spindle microtubules regardless of whether importin-α and -β were present (Fig. 3 A, fourth column). An excess amount of each recombinant protein was incubated with the permeabilized cells; therefore, the reduction in spindle or chromosome binding observed under each condition should represent the inability of hKid to bind either structures, respectively, rather than an indirect effect, such as the consequence of sequestering hKid. We thus concluded that the binding of importin-α and -β to hKid targets hKid to chromosomes and disrupts its ability to bind the mitotic spindle. A previous study showed that importin-α and -β inhibit the microtubule binding of hKid in vitro (Trieselmann et al., 2003), which supports our results. It must be noted that neither phenomena was observed in the presence of importin-α or -β alone (Fig. 3 B), demonstrating the necessity of binding with importin-β and -α in determining the subcellular localization of hKid.

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