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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

Nuclear accumulation of hsc70 during in vitro importin β export assay. Digitonin-permeabilized cells were incubated with 0.3 μM EYFP–importin β and were reincubated with cytosol (cyt; a), or depleted cytosol (b–m) in the absence (a) or presence of 1 μM ECFP–hsc70 (b–g) or ECFP-hsc70(D10N) (h–m). Export reactions were performed in the presence of an ATP regeneration system.
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fig3: Nuclear accumulation of hsc70 during in vitro importin β export assay. Digitonin-permeabilized cells were incubated with 0.3 μM EYFP–importin β and were reincubated with cytosol (cyt; a), or depleted cytosol (b–m) in the absence (a) or presence of 1 μM ECFP–hsc70 (b–g) or ECFP-hsc70(D10N) (h–m). Export reactions were performed in the presence of an ATP regeneration system.

Mentions: Hsc70 is known to be localized in the cytoplasm and nucleus, and to shuttle between these two compartments (Mandell and Feldherr, 1990). Therefore, we examined the subcellular localization of hsc70 during an in vitro export assay. ECFP-hsc70, as well as unlabeled hsc70, facilitated the nuclear export of importin β (Fig. 3, b–d). During the export assay of importin β, a portion of the ECFP–hsc70 migrated into the nucleus of most of the cells (Fig. 3, e–g); however, the extent of nuclear migration was heterogeneous. Notably, the level of nuclear migration of hsc70 and the export of importin β showed good correlation in this assay (i.e., a nucleus that contained more hsc70 showed lower levels of importin β). This suggests that the nuclear migration of hsc70 is important to support the export of importin β. In similar experiments, the ECFP–hsc70 (D10N) showed much more heterogeneity in its nuclear migrating activity, and the population of nuclei containing hsc70 (D10N) was decreased substantially (Fig. 3, k–m). Regardless of its nuclear migration, hsc70 (D10N) failed to support the nuclear export of importin β (Fig. 3, h–j).


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 accumulation of hsc70 during in vitro importin β export assay. Digitonin-permeabilized cells were incubated with 0.3 μM EYFP–importin β and were reincubated with cytosol (cyt; a), or depleted cytosol (b–m) in the absence (a) or presence of 1 μM ECFP–hsc70 (b–g) or ECFP-hsc70(D10N) (h–m). 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

fig3: Nuclear accumulation of hsc70 during in vitro importin β export assay. Digitonin-permeabilized cells were incubated with 0.3 μM EYFP–importin β and were reincubated with cytosol (cyt; a), or depleted cytosol (b–m) in the absence (a) or presence of 1 μM ECFP–hsc70 (b–g) or ECFP-hsc70(D10N) (h–m). Export reactions were performed in the presence of an ATP regeneration system.
Mentions: Hsc70 is known to be localized in the cytoplasm and nucleus, and to shuttle between these two compartments (Mandell and Feldherr, 1990). Therefore, we examined the subcellular localization of hsc70 during an in vitro export assay. ECFP-hsc70, as well as unlabeled hsc70, facilitated the nuclear export of importin β (Fig. 3, b–d). During the export assay of importin β, a portion of the ECFP–hsc70 migrated into the nucleus of most of the cells (Fig. 3, e–g); however, the extent of nuclear migration was heterogeneous. Notably, the level of nuclear migration of hsc70 and the export of importin β showed good correlation in this assay (i.e., a nucleus that contained more hsc70 showed lower levels of importin β). This suggests that the nuclear migration of hsc70 is important to support the export of importin β. In similar experiments, the ECFP–hsc70 (D10N) showed much more heterogeneity in its nuclear migrating activity, and the population of nuclei containing hsc70 (D10N) was decreased substantially (Fig. 3, k–m). Regardless of its nuclear migration, hsc70 (D10N) failed to support the nuclear export of importin β (Fig. 3, h–j).

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