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Importin beta contains a COOH-terminal nucleoporin binding region important for nuclear transport.

Bednenko J, Cingolani G, Gerace L - J. Cell Biol. (2003)

Bottom Line: Although the affinity of the COOH-terminal region for nucleoporins is dramatically weaker than that of the NH2-terminal region, sets of mutations that perturb the nucleoporin binding of either region reduce the nuclear import activity of importin beta to a similar extent ( approximately 50%).An importin beta mutant with a combination of mutations in the NH2- and COOH-terminal regions is completely inactive for nuclear import.Thus, importin beta possesses two nucleoporin binding sites, both of which are important for its nuclear import function.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

ABSTRACT
Proteins containing a classical NLS are transported into the nucleus by the import receptor importin beta, which binds to cargoes via the adaptor importin alpha. The import complex is translocated through the nuclear pore complex by interactions of importin beta with a series of nucleoporins. Previous studies have defined a nucleoporin binding region in the NH2-terminal half of importin beta. Here we report the identification of a second nucleoporin binding region in its COOH-terminal half. Although the affinity of the COOH-terminal region for nucleoporins is dramatically weaker than that of the NH2-terminal region, sets of mutations that perturb the nucleoporin binding of either region reduce the nuclear import activity of importin beta to a similar extent ( approximately 50%). An importin beta mutant with a combination of mutations in the NH2- and COOH-terminal regions is completely inactive for nuclear import. Thus, importin beta possesses two nucleoporin binding sites, both of which are important for its nuclear import function.

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Effect of mutations in HEAT repeats 5–7 on nuclear import activity of importin β. (A) Nuclear import assays with wild-type importin β and with importin β mutants. 40-μl samples were incubated for 30 min at 30°C, and the level of nuclear import of FITC-labeled BSA-NLS was measured by flow cytometry. White bars depict control reactions performed in the presence of hexokinase and glucose to deplete ATP. Black bars show the levels of fluorescence in the presence of ATP. Error bars represent the standard deviation of duplicate measurements. (B) Kinetics of nuclear accumulation of importin β. 3.4 pmol of importin β or importin β (304–876) fragment was used in each 20-μl assay. The level of intranuclear importin β was quantified by immunofluorescence and confocal microscopy in samples fixed at the various time points. Error bars represent the standard deviation of two independent experiments. The level of intranuclear wild-type importin β for the 3-min time point was set at 100%, and all other values were normalized to the 100% level.
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fig3: Effect of mutations in HEAT repeats 5–7 on nuclear import activity of importin β. (A) Nuclear import assays with wild-type importin β and with importin β mutants. 40-μl samples were incubated for 30 min at 30°C, and the level of nuclear import of FITC-labeled BSA-NLS was measured by flow cytometry. White bars depict control reactions performed in the presence of hexokinase and glucose to deplete ATP. Black bars show the levels of fluorescence in the presence of ATP. Error bars represent the standard deviation of duplicate measurements. (B) Kinetics of nuclear accumulation of importin β. 3.4 pmol of importin β or importin β (304–876) fragment was used in each 20-μl assay. The level of intranuclear importin β was quantified by immunofluorescence and confocal microscopy in samples fixed at the various time points. Error bars represent the standard deviation of two independent experiments. The level of intranuclear wild-type importin β for the 3-min time point was set at 100%, and all other values were normalized to the 100% level.

Mentions: To test the functional effects of mutations in the NH2-terminal nucleoporin binding domain of importin β, we examined the mutants in a permeabilized cell nuclear import assay using the fluorescently labeled importin α–dependent import cargo BSA-NLS (Adam et al., 1992). At an optimal concentration of wild-type importin β, we observed an approximately fivefold accumulation of fluorescent cargo in nuclei under standard assay conditions, as compared with control reactions in which ATP was depleted (Fig. 3 A, compare white and black columns). Examination of cells by confocal microscopy revealed that almost all of the cell fluorescence was in the nucleus (see Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200303085/DC1), validating the use of flow cytometry for quantification. No significant stimulation of import was observed in the absence of recombinant importin β (Fig. 3 A). A time course of import revealed linear nuclear accumulation of cargo over the 30-min time frame of our experiment with wild-type importin β and with all the importin β mutants (unpublished data). This indicates that the observed variations in the level of nuclear import with the various importin β constructs are due to import rate differences.


Importin beta contains a COOH-terminal nucleoporin binding region important for nuclear transport.

Bednenko J, Cingolani G, Gerace L - J. Cell Biol. (2003)

Effect of mutations in HEAT repeats 5–7 on nuclear import activity of importin β. (A) Nuclear import assays with wild-type importin β and with importin β mutants. 40-μl samples were incubated for 30 min at 30°C, and the level of nuclear import of FITC-labeled BSA-NLS was measured by flow cytometry. White bars depict control reactions performed in the presence of hexokinase and glucose to deplete ATP. Black bars show the levels of fluorescence in the presence of ATP. Error bars represent the standard deviation of duplicate measurements. (B) Kinetics of nuclear accumulation of importin β. 3.4 pmol of importin β or importin β (304–876) fragment was used in each 20-μl assay. The level of intranuclear importin β was quantified by immunofluorescence and confocal microscopy in samples fixed at the various time points. Error bars represent the standard deviation of two independent experiments. The level of intranuclear wild-type importin β for the 3-min time point was set at 100%, and all other values were normalized to the 100% level.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172684&req=5

fig3: Effect of mutations in HEAT repeats 5–7 on nuclear import activity of importin β. (A) Nuclear import assays with wild-type importin β and with importin β mutants. 40-μl samples were incubated for 30 min at 30°C, and the level of nuclear import of FITC-labeled BSA-NLS was measured by flow cytometry. White bars depict control reactions performed in the presence of hexokinase and glucose to deplete ATP. Black bars show the levels of fluorescence in the presence of ATP. Error bars represent the standard deviation of duplicate measurements. (B) Kinetics of nuclear accumulation of importin β. 3.4 pmol of importin β or importin β (304–876) fragment was used in each 20-μl assay. The level of intranuclear importin β was quantified by immunofluorescence and confocal microscopy in samples fixed at the various time points. Error bars represent the standard deviation of two independent experiments. The level of intranuclear wild-type importin β for the 3-min time point was set at 100%, and all other values were normalized to the 100% level.
Mentions: To test the functional effects of mutations in the NH2-terminal nucleoporin binding domain of importin β, we examined the mutants in a permeabilized cell nuclear import assay using the fluorescently labeled importin α–dependent import cargo BSA-NLS (Adam et al., 1992). At an optimal concentration of wild-type importin β, we observed an approximately fivefold accumulation of fluorescent cargo in nuclei under standard assay conditions, as compared with control reactions in which ATP was depleted (Fig. 3 A, compare white and black columns). Examination of cells by confocal microscopy revealed that almost all of the cell fluorescence was in the nucleus (see Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200303085/DC1), validating the use of flow cytometry for quantification. No significant stimulation of import was observed in the absence of recombinant importin β (Fig. 3 A). A time course of import revealed linear nuclear accumulation of cargo over the 30-min time frame of our experiment with wild-type importin β and with all the importin β mutants (unpublished data). This indicates that the observed variations in the level of nuclear import with the various importin β constructs are due to import rate differences.

Bottom Line: Although the affinity of the COOH-terminal region for nucleoporins is dramatically weaker than that of the NH2-terminal region, sets of mutations that perturb the nucleoporin binding of either region reduce the nuclear import activity of importin beta to a similar extent ( approximately 50%).An importin beta mutant with a combination of mutations in the NH2- and COOH-terminal regions is completely inactive for nuclear import.Thus, importin beta possesses two nucleoporin binding sites, both of which are important for its nuclear import function.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

ABSTRACT
Proteins containing a classical NLS are transported into the nucleus by the import receptor importin beta, which binds to cargoes via the adaptor importin alpha. The import complex is translocated through the nuclear pore complex by interactions of importin beta with a series of nucleoporins. Previous studies have defined a nucleoporin binding region in the NH2-terminal half of importin beta. Here we report the identification of a second nucleoporin binding region in its COOH-terminal half. Although the affinity of the COOH-terminal region for nucleoporins is dramatically weaker than that of the NH2-terminal region, sets of mutations that perturb the nucleoporin binding of either region reduce the nuclear import activity of importin beta to a similar extent ( approximately 50%). An importin beta mutant with a combination of mutations in the NH2- and COOH-terminal regions is completely inactive for nuclear import. Thus, importin beta possesses two nucleoporin binding sites, both of which are important for its nuclear import function.

Show MeSH
Related in: MedlinePlus