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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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Related in: MedlinePlus

Depletion of hsc70 in living cells causes the accumulation of importin β, transportin, and importin α within the nucleus. (A) HeLa cells were treated for 48 h with (+) or without (−) siRNA targeting hsc70 (left panels) or β-actin (right panels). The expression of the indicated proteins was monitored by immunoblotting. β-Actin or α-tubulin was used as an internal control. (B–D) HeLa cells were treated for 48 h (B) or 60 h (C and D) with (+) or without (−) siRNA targeting hsc70 (B, a–f; C and D) or β-actin (B, g–i). The transfected cells were fixed with 3.7% formaldehyde and stained with specific antibodies and DAPI. “Phase” refers to the phase-contrast view. Bar, 10 μm.
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fig5: Depletion of hsc70 in living cells causes the accumulation of importin β, transportin, and importin α within the nucleus. (A) HeLa cells were treated for 48 h with (+) or without (−) siRNA targeting hsc70 (left panels) or β-actin (right panels). The expression of the indicated proteins was monitored by immunoblotting. β-Actin or α-tubulin was used as an internal control. (B–D) HeLa cells were treated for 48 h (B) or 60 h (C and D) with (+) or without (−) siRNA targeting hsc70 (B, a–f; C and D) or β-actin (B, g–i). The transfected cells were fixed with 3.7% formaldehyde and stained with specific antibodies and DAPI. “Phase” refers to the phase-contrast view. Bar, 10 μm.

Mentions: To know the effect of hsc70 on the nuclear export of receptors in living cells, we depleted endogenous hsc70 by transfecting hsc70-specific small interfering RNA (siRNA) duplexes into HeLa cells. After 48–72 h transfection, the expression levels of hsc70 proteins were reduced to 10–20% of the hsc70 protein levels that were detected in untransfected control cells (Fig. 5 A). At this time point, the morphology of the cells that were depleted of hsc70 changed dramatically; some displayed cellular blebbing.


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)

Depletion of hsc70 in living cells causes the accumulation of importin β, transportin, and importin α within the nucleus. (A) HeLa cells were treated for 48 h with (+) or without (−) siRNA targeting hsc70 (left panels) or β-actin (right panels). The expression of the indicated proteins was monitored by immunoblotting. β-Actin or α-tubulin was used as an internal control. (B–D) HeLa cells were treated for 48 h (B) or 60 h (C and D) with (+) or without (−) siRNA targeting hsc70 (B, a–f; C and D) or β-actin (B, g–i). The transfected cells were fixed with 3.7% formaldehyde and stained with specific antibodies and DAPI. “Phase” refers to the phase-contrast view. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Depletion of hsc70 in living cells causes the accumulation of importin β, transportin, and importin α within the nucleus. (A) HeLa cells were treated for 48 h with (+) or without (−) siRNA targeting hsc70 (left panels) or β-actin (right panels). The expression of the indicated proteins was monitored by immunoblotting. β-Actin or α-tubulin was used as an internal control. (B–D) HeLa cells were treated for 48 h (B) or 60 h (C and D) with (+) or without (−) siRNA targeting hsc70 (B, a–f; C and D) or β-actin (B, g–i). The transfected cells were fixed with 3.7% formaldehyde and stained with specific antibodies and DAPI. “Phase” refers to the phase-contrast view. Bar, 10 μm.
Mentions: To know the effect of hsc70 on the nuclear export of receptors in living cells, we depleted endogenous hsc70 by transfecting hsc70-specific small interfering RNA (siRNA) duplexes into HeLa cells. After 48–72 h transfection, the expression levels of hsc70 proteins were reduced to 10–20% of the hsc70 protein levels that were detected in untransfected control cells (Fig. 5 A). At this time point, the morphology of the cells that were depleted of hsc70 changed dramatically; some displayed cellular blebbing.

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