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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 dependent on an energy source, and facilitated by ATP-binding proteins. Digitonin-permeabilized cells were incubated with 0.3 μM GFP–importin β and reincubated with cytosol (cyt; a, b), the depleted cytosol (c), the eluate (e) from an ATP–agarose column, or the depleted cytosol and the eluate (e) in the presence of apyrase (a) or an ATP regeneration system (b–e). The addition of ATP-binding proteins to the depleted cytosol restored the nuclear export activity of importin β (e). Bar, 10 μm.
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fig1: Nuclear export of importin β is dependent on an energy source, and facilitated by ATP-binding proteins. Digitonin-permeabilized cells were incubated with 0.3 μM GFP–importin β and reincubated with cytosol (cyt; a, b), the depleted cytosol (c), the eluate (e) from an ATP–agarose column, or the depleted cytosol and the eluate (e) in the presence of apyrase (a) or an ATP regeneration system (b–e). The addition of ATP-binding proteins to the depleted cytosol restored the nuclear export activity of importin β (e). Bar, 10 μm.

Mentions: To determine the energy requirement for the nuclear export of importin β, the nuclear export of GFP–importin β was monitored in the presence of cytosol with or without ATP in an in vitro transport assay (see Materials and methods). In the presence of ATP, importin β exits efficiently from the nucleus (Fig. 1 b), whereas the nuclear export of importin β was inhibited in the presence of apyrase (Fig. 1 a). A nonhydrolyzable ATP analogue, AMPPCP, did not support the nuclear export of importin β (unpublished data). Therefore, consistent with in vivo evidence that was reported previously (Kose et al., 1999), these results show that nuclear export of importin β requires energy input in an in vitro transport assay.


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 dependent on an energy source, and facilitated by ATP-binding proteins. Digitonin-permeabilized cells were incubated with 0.3 μM GFP–importin β and reincubated with cytosol (cyt; a, b), the depleted cytosol (c), the eluate (e) from an ATP–agarose column, or the depleted cytosol and the eluate (e) in the presence of apyrase (a) or an ATP regeneration system (b–e). The addition of ATP-binding proteins to the depleted cytosol restored the nuclear export activity of importin β (e). Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Nuclear export of importin β is dependent on an energy source, and facilitated by ATP-binding proteins. Digitonin-permeabilized cells were incubated with 0.3 μM GFP–importin β and reincubated with cytosol (cyt; a, b), the depleted cytosol (c), the eluate (e) from an ATP–agarose column, or the depleted cytosol and the eluate (e) in the presence of apyrase (a) or an ATP regeneration system (b–e). The addition of ATP-binding proteins to the depleted cytosol restored the nuclear export activity of importin β (e). Bar, 10 μm.
Mentions: To determine the energy requirement for the nuclear export of importin β, the nuclear export of GFP–importin β was monitored in the presence of cytosol with or without ATP in an in vitro transport assay (see Materials and methods). In the presence of ATP, importin β exits efficiently from the nucleus (Fig. 1 b), whereas the nuclear export of importin β was inhibited in the presence of apyrase (Fig. 1 a). A nonhydrolyzable ATP analogue, AMPPCP, did not support the nuclear export of importin β (unpublished data). Therefore, consistent with in vivo evidence that was reported previously (Kose et al., 1999), these results show that nuclear export of importin β requires energy input in an in vitro transport assay.

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