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The 70-kD heat shock cognate protein (hsc70) facilitates the nuclear export of the import receptors.

Kose S, Furuta M, Koike M, Yoneda Y, Imamoto N - J. Cell Biol. (2005)

Bottom Line: Cytosol, depleted of ATP-binding proteins, did not support the sufficient nuclear export of importin beta.These effects of hsc70 were observed in the nuclear export of importin beta, but also for other import receptors, transportin and importin alpha.These results suggest that hsc70 broadly modulates nucleocytoplasmic transport systems by regulating the nuclear export of receptor proteins.

View Article: PubMed Central - PubMed

Affiliation: Cellular Dynamics Laboratory, Discovery Research Institute, RIKEN, Wako, Saitama 351-0198, Japan.

ABSTRACT
Transport receptors of the importin beta family continuously shuttle between the nucleus and cytoplasm. We previously reported that the nuclear export of importin beta involves energy-requiring step(s) in living cells. Here, we show that the in vitro nuclear export of importin beta also requires energy input. Cytosol, depleted of ATP-binding proteins, did not support the sufficient nuclear export of importin beta. Further purification revealed that the active component in the absorbed fraction was a 70-kD heat shock cognate protein (hsc70). The addition of recombinant hsc70, but not an ATPase-deficient hsc70 mutant, to the depleted cytosol restored the export activity. In living cells, depletion of hsc70 caused the significant nuclear accumulation of importin beta. These effects of hsc70 were observed in the nuclear export of importin beta, but also for other import receptors, transportin and importin alpha. These results suggest that hsc70 broadly modulates nucleocytoplasmic transport systems by regulating the nuclear export of receptor proteins.

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Hsc70 facilitates nuclear export of transportin and importin α. (A) Digitonin-permeabilized cells were incubated with 0.3 μM GFP–transportin (TRN) and were reincubated with total cytosol (cyt; a, b), or depleted cytosol (c and d) in the absence (a–c) or presence (d) of 0.3 μM hsc70. Export reactions were performed in the presence of apyrase (a) or an ATP regeneration system (b–d). (B) For import reactions, digitonin-permeabilized cells were incubated with 0.6 μM GFP–importin α, 0.6 μM importin β, 0.4 μM RanGDP, and an ATP regeneration system. Export reactions were performed as described above.
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fig4: Hsc70 facilitates nuclear export of transportin and importin α. (A) Digitonin-permeabilized cells were incubated with 0.3 μM GFP–transportin (TRN) and were reincubated with total cytosol (cyt; a, b), or depleted cytosol (c and d) in the absence (a–c) or presence (d) of 0.3 μM hsc70. Export reactions were performed in the presence of apyrase (a) or an ATP regeneration system (b–d). (B) For import reactions, digitonin-permeabilized cells were incubated with 0.6 μM GFP–importin α, 0.6 μM importin β, 0.4 μM RanGDP, and an ATP regeneration system. Export reactions were performed as described above.

Mentions: To determine whether the function of hsc70 is restricted to importin β, we examined the effect of hsc70 in the recycling of another import receptor, transportin (Fig. 4 A). As in the case of nuclear export of importin β, the nuclear export of transportin requires energy input and cytosol, consistent with the previous notion that export of the importin β family generally requires the Ran system (Fig. 4 A, a and b). The export activity of transportin was decreased significantly in the depleted cytosol, but was restored by the addition of hsc70, as is the case for importin β. We found that hsc70 also facilitates the nuclear export of importin α, which is known to be mediated by cellular apoptosis susceptibility gene (CAS), an export receptor of the importin β family (Fig. 4 B). These results show that the function of hsc70 is not restricted to importin β, and suggests that hsc70 may function generally in the recycling of import receptors.


The 70-kD heat shock cognate protein (hsc70) facilitates the nuclear export of the import receptors.

Kose S, Furuta M, Koike M, Yoneda Y, Imamoto N - J. Cell Biol. (2005)

Hsc70 facilitates nuclear export of transportin and importin α. (A) Digitonin-permeabilized cells were incubated with 0.3 μM GFP–transportin (TRN) and were reincubated with total cytosol (cyt; a, b), or depleted cytosol (c and d) in the absence (a–c) or presence (d) of 0.3 μM hsc70. Export reactions were performed in the presence of apyrase (a) or an ATP regeneration system (b–d). (B) For import reactions, digitonin-permeabilized cells were incubated with 0.6 μM GFP–importin α, 0.6 μM importin β, 0.4 μM RanGDP, and an ATP regeneration system. Export reactions were performed as described above.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171223&req=5

fig4: Hsc70 facilitates nuclear export of transportin and importin α. (A) Digitonin-permeabilized cells were incubated with 0.3 μM GFP–transportin (TRN) and were reincubated with total cytosol (cyt; a, b), or depleted cytosol (c and d) in the absence (a–c) or presence (d) of 0.3 μM hsc70. Export reactions were performed in the presence of apyrase (a) or an ATP regeneration system (b–d). (B) For import reactions, digitonin-permeabilized cells were incubated with 0.6 μM GFP–importin α, 0.6 μM importin β, 0.4 μM RanGDP, and an ATP regeneration system. Export reactions were performed as described above.
Mentions: To determine whether the function of hsc70 is restricted to importin β, we examined the effect of hsc70 in the recycling of another import receptor, transportin (Fig. 4 A). As in the case of nuclear export of importin β, the nuclear export of transportin requires energy input and cytosol, consistent with the previous notion that export of the importin β family generally requires the Ran system (Fig. 4 A, a and b). The export activity of transportin was decreased significantly in the depleted cytosol, but was restored by the addition of hsc70, as is the case for importin β. We found that hsc70 also facilitates the nuclear export of importin α, which is known to be mediated by cellular apoptosis susceptibility gene (CAS), an export receptor of the importin β family (Fig. 4 B). These results show that the function of hsc70 is not restricted to importin β, and suggests that hsc70 may function generally in the recycling of import receptors.

Bottom Line: Cytosol, depleted of ATP-binding proteins, did not support the sufficient nuclear export of importin beta.These effects of hsc70 were observed in the nuclear export of importin beta, but also for other import receptors, transportin and importin alpha.These results suggest that hsc70 broadly modulates nucleocytoplasmic transport systems by regulating the nuclear export of receptor proteins.

View Article: PubMed Central - PubMed

Affiliation: Cellular Dynamics Laboratory, Discovery Research Institute, RIKEN, Wako, Saitama 351-0198, Japan.

ABSTRACT
Transport receptors of the importin beta family continuously shuttle between the nucleus and cytoplasm. We previously reported that the nuclear export of importin beta involves energy-requiring step(s) in living cells. Here, we show that the in vitro nuclear export of importin beta also requires energy input. Cytosol, depleted of ATP-binding proteins, did not support the sufficient nuclear export of importin beta. Further purification revealed that the active component in the absorbed fraction was a 70-kD heat shock cognate protein (hsc70). The addition of recombinant hsc70, but not an ATPase-deficient hsc70 mutant, to the depleted cytosol restored the export activity. In living cells, depletion of hsc70 caused the significant nuclear accumulation of importin beta. These effects of hsc70 were observed in the nuclear export of importin beta, but also for other import receptors, transportin and importin alpha. These results suggest that hsc70 broadly modulates nucleocytoplasmic transport systems by regulating the nuclear export of receptor proteins.

Show MeSH
Related in: MedlinePlus