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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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Effects of mutations in the COOH-terminal segment of importin β on nucleoporin binding and nuclear import. (A) Binding isotherms showing the interaction of the importin β fragment comprising residues 304–876 (with or without the mutations L612D and/or F688A) to Nup153 (895–1475). Error bars represent the standard deviation of duplicate measurements. The numbers next to the binding curves indicate apparent dissociation constants. NS, nonsaturated binding at importin β fragment concentrations of up to 800 nM. (B) Kinetics of nuclear accumulation of wild-type (WT), m-C, and m-N/m-C full-length importin β proteins. 3.4 pmol of each importin β construct was used in each 20-μl assay. The nuclear fluorescence was measured by immunofluorescence and confocal microscopy after 20–600 s incubation at room temperature, as indicated. 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 as a fraction of the 100% level. (C) Nuclear import assays with the wild-type (WT), m-N, m-C, and m-N/m-C full-length importin β proteins. 40-μl reactions were incubated for 30 min at 30°C, and the level of nuclear import was measured by flow cytometry. White bars designate control reactions performed in the presence of hexokinase and glucose to deplete ATP. Black bars show the level of fluorescence in the presence of ATP. Error bars represent the standard deviation of duplicate measurements.
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fig5: Effects of mutations in the COOH-terminal segment of importin β on nucleoporin binding and nuclear import. (A) Binding isotherms showing the interaction of the importin β fragment comprising residues 304–876 (with or without the mutations L612D and/or F688A) to Nup153 (895–1475). Error bars represent the standard deviation of duplicate measurements. The numbers next to the binding curves indicate apparent dissociation constants. NS, nonsaturated binding at importin β fragment concentrations of up to 800 nM. (B) Kinetics of nuclear accumulation of wild-type (WT), m-C, and m-N/m-C full-length importin β proteins. 3.4 pmol of each importin β construct was used in each 20-μl assay. The nuclear fluorescence was measured by immunofluorescence and confocal microscopy after 20–600 s incubation at room temperature, as indicated. 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 as a fraction of the 100% level. (C) Nuclear import assays with the wild-type (WT), m-N, m-C, and m-N/m-C full-length importin β proteins. 40-μl reactions were incubated for 30 min at 30°C, and the level of nuclear import was measured by flow cytometry. White bars designate control reactions performed in the presence of hexokinase and glucose to deplete ATP. Black bars show the level of fluorescence in the presence of ATP. Error bars represent the standard deviation of duplicate measurements.

Mentions: Using this structural alignment, we identified three amino acid residues in HEAT repeats 14 and 16 (L612, F688, and L695) that matched residues in the NH2-terminal nucleoporin binding site in HEAT repeats 5 and 7 (I178, Y255, and I263, respectively; Fig. 4 C). To test whether L612, F688, and L695 are important for the interaction of the COOH-terminal fragment of importin β with nucleoporins, we performed a mutational analysis of the three residues. The mutation L612D (analogous to I178D) strongly decreased the affinity of the importin β fragment for Nup153 (895–1475), such that the Kd was not measurable, whereas F688A (analogous to Y255A) decreased the affinity by approximately twofold (Fig. 5 A). The mutation L695R (analogous to I263R) had no detectable effect on the binding of the importin β fragment to Nup153 (unpublished data). A mutant importin β fragment containing a combination of L612D and F688A showed an even lower level of nonsaturated binding for Nup153 (895–1475) than did L612D alone (Fig. 5 A). The L612D and F688A mutations did not significantly affect importin β folding, as only an approximately twofold decrease in the affinity of the mutant protein fragments for importin α was observed (Table II).


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

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

Effects of mutations in the COOH-terminal segment of importin β on nucleoporin binding and nuclear import. (A) Binding isotherms showing the interaction of the importin β fragment comprising residues 304–876 (with or without the mutations L612D and/or F688A) to Nup153 (895–1475). Error bars represent the standard deviation of duplicate measurements. The numbers next to the binding curves indicate apparent dissociation constants. NS, nonsaturated binding at importin β fragment concentrations of up to 800 nM. (B) Kinetics of nuclear accumulation of wild-type (WT), m-C, and m-N/m-C full-length importin β proteins. 3.4 pmol of each importin β construct was used in each 20-μl assay. The nuclear fluorescence was measured by immunofluorescence and confocal microscopy after 20–600 s incubation at room temperature, as indicated. 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 as a fraction of the 100% level. (C) Nuclear import assays with the wild-type (WT), m-N, m-C, and m-N/m-C full-length importin β proteins. 40-μl reactions were incubated for 30 min at 30°C, and the level of nuclear import was measured by flow cytometry. White bars designate control reactions performed in the presence of hexokinase and glucose to deplete ATP. Black bars show the level of fluorescence in the presence of ATP. Error bars represent the standard deviation of duplicate measurements.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172684&req=5

fig5: Effects of mutations in the COOH-terminal segment of importin β on nucleoporin binding and nuclear import. (A) Binding isotherms showing the interaction of the importin β fragment comprising residues 304–876 (with or without the mutations L612D and/or F688A) to Nup153 (895–1475). Error bars represent the standard deviation of duplicate measurements. The numbers next to the binding curves indicate apparent dissociation constants. NS, nonsaturated binding at importin β fragment concentrations of up to 800 nM. (B) Kinetics of nuclear accumulation of wild-type (WT), m-C, and m-N/m-C full-length importin β proteins. 3.4 pmol of each importin β construct was used in each 20-μl assay. The nuclear fluorescence was measured by immunofluorescence and confocal microscopy after 20–600 s incubation at room temperature, as indicated. 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 as a fraction of the 100% level. (C) Nuclear import assays with the wild-type (WT), m-N, m-C, and m-N/m-C full-length importin β proteins. 40-μl reactions were incubated for 30 min at 30°C, and the level of nuclear import was measured by flow cytometry. White bars designate control reactions performed in the presence of hexokinase and glucose to deplete ATP. Black bars show the level of fluorescence in the presence of ATP. Error bars represent the standard deviation of duplicate measurements.
Mentions: Using this structural alignment, we identified three amino acid residues in HEAT repeats 14 and 16 (L612, F688, and L695) that matched residues in the NH2-terminal nucleoporin binding site in HEAT repeats 5 and 7 (I178, Y255, and I263, respectively; Fig. 4 C). To test whether L612, F688, and L695 are important for the interaction of the COOH-terminal fragment of importin β with nucleoporins, we performed a mutational analysis of the three residues. The mutation L612D (analogous to I178D) strongly decreased the affinity of the importin β fragment for Nup153 (895–1475), such that the Kd was not measurable, whereas F688A (analogous to Y255A) decreased the affinity by approximately twofold (Fig. 5 A). The mutation L695R (analogous to I263R) had no detectable effect on the binding of the importin β fragment to Nup153 (unpublished data). A mutant importin β fragment containing a combination of L612D and F688A showed an even lower level of nonsaturated binding for Nup153 (895–1475) than did L612D alone (Fig. 5 A). The L612D and F688A mutations did not significantly affect importin β folding, as only an approximately twofold decrease in the affinity of the mutant protein fragments for importin α was observed (Table II).

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