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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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Nuclear export of importin β is restored by the addition of hsc70 in an in vitro transport assay. (A and B) Purification of hsc70 protein from total cytosol of Ehrlich ascites tumor cells. Total cytosol (lane 1), the flow-through (lane 2) and eluate (lane 3) from the ATP-agarose, the Phenyl Sepharose HP fraction (lane 4), and the Superdex 200 peak fraction (lane 5) were separated on 10% SDS-PAGE and subjected to Coomassie blue staining (A) or Western blotting probed with specific antibodies to indicated proteins (B). Ran, NTF2, and RCC1 were not depleted by ATP–agarose. (C) Digitonin-permeabilized cells were incubated with 0.3 μM GFP-importin β and were then reincubated with cytosol (cyt; a), or depleted cytosol (b–d) in the absence (a, b) or presence of 1 μM hsc70 (c) or hsc70(D10N) (d). Export reactions were performed in the presence of an ATP regeneration system.
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fig2: Nuclear export of importin β is restored by the addition of hsc70 in an in vitro transport assay. (A and B) Purification of hsc70 protein from total cytosol of Ehrlich ascites tumor cells. Total cytosol (lane 1), the flow-through (lane 2) and eluate (lane 3) from the ATP-agarose, the Phenyl Sepharose HP fraction (lane 4), and the Superdex 200 peak fraction (lane 5) were separated on 10% SDS-PAGE and subjected to Coomassie blue staining (A) or Western blotting probed with specific antibodies to indicated proteins (B). Ran, NTF2, and RCC1 were not depleted by ATP–agarose. (C) Digitonin-permeabilized cells were incubated with 0.3 μM GFP-importin β and were then reincubated with cytosol (cyt; a), or depleted cytosol (b–d) in the absence (a, b) or presence of 1 μM hsc70 (c) or hsc70(D10N) (d). Export reactions were performed in the presence of an ATP regeneration system.

Mentions: Ran regulates the association and disassociation of importin β family receptors with cargo and nucleoporins, and is required for the recycling of importin β. The loss of export activity could be the result of depletion of Ran and its regulators by ATP–agarose. However, as shown in Fig. 2 B, Ran and its regulators (NTF2, RCC1) were present in the depleted cytosol at a level similar to that in the starting cytosol, which indicated that the Ran systems were not depleted by the ATP–agarose column.


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)

Nuclear export of importin β is restored by the addition of hsc70 in an in vitro transport assay. (A and B) Purification of hsc70 protein from total cytosol of Ehrlich ascites tumor cells. Total cytosol (lane 1), the flow-through (lane 2) and eluate (lane 3) from the ATP-agarose, the Phenyl Sepharose HP fraction (lane 4), and the Superdex 200 peak fraction (lane 5) were separated on 10% SDS-PAGE and subjected to Coomassie blue staining (A) or Western blotting probed with specific antibodies to indicated proteins (B). Ran, NTF2, and RCC1 were not depleted by ATP–agarose. (C) Digitonin-permeabilized cells were incubated with 0.3 μM GFP-importin β and were then reincubated with cytosol (cyt; a), or depleted cytosol (b–d) in the absence (a, b) or presence of 1 μM hsc70 (c) or hsc70(D10N) (d). Export reactions were performed in the presence of an ATP regeneration system.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Nuclear export of importin β is restored by the addition of hsc70 in an in vitro transport assay. (A and B) Purification of hsc70 protein from total cytosol of Ehrlich ascites tumor cells. Total cytosol (lane 1), the flow-through (lane 2) and eluate (lane 3) from the ATP-agarose, the Phenyl Sepharose HP fraction (lane 4), and the Superdex 200 peak fraction (lane 5) were separated on 10% SDS-PAGE and subjected to Coomassie blue staining (A) or Western blotting probed with specific antibodies to indicated proteins (B). Ran, NTF2, and RCC1 were not depleted by ATP–agarose. (C) Digitonin-permeabilized cells were incubated with 0.3 μM GFP-importin β and were then reincubated with cytosol (cyt; a), or depleted cytosol (b–d) in the absence (a, b) or presence of 1 μM hsc70 (c) or hsc70(D10N) (d). Export reactions were performed in the presence of an ATP regeneration system.
Mentions: Ran regulates the association and disassociation of importin β family receptors with cargo and nucleoporins, and is required for the recycling of importin β. The loss of export activity could be the result of depletion of Ran and its regulators by ATP–agarose. However, as shown in Fig. 2 B, Ran and its regulators (NTF2, RCC1) were present in the depleted cytosol at a level similar to that in the starting cytosol, which indicated that the Ran systems were not depleted by the ATP–agarose column.

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