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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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Characterization of the binding of importin β to nucleoporins. Shown are binding isotherms depicting the interaction of selected importin β mutants with Nup153 (895–1475) (A), with full-length Nup62 (B), or with Nup358 (996–1963) (C). Error bars represent the standard deviation of duplicate measurements. The calculated apparent Kd values are indicated next to the binding isotherms. NS, nonsaturated binding at importin β concentrations up to 800 nM.
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fig2: Characterization of the binding of importin β to nucleoporins. Shown are binding isotherms depicting the interaction of selected importin β mutants with Nup153 (895–1475) (A), with full-length Nup62 (B), or with Nup358 (996–1963) (C). Error bars represent the standard deviation of duplicate measurements. The calculated apparent Kd values are indicated next to the binding isotherms. NS, nonsaturated binding at importin β concentrations up to 800 nM.

Mentions: We found that importin β single point mutants containing the substitutions I178D, E214A, F217A, Y255A, and I263R all had a decreased affinity for the Nup153 fragment, but retained wild-type affinity for RanGTP and for importin α (Table I). Weakened nucleoporin binding was most evident for the I178D mutant, which had an ∼40-fold decrease in affinity for Nup153 (895–1475). In contrast, the E214A, F217A, Y255A, and I263R mutants showed a relatively small decrease in affinity for the Nup153 fragment (Kd ≈ 1.8–3.5 nM) (Table I; Fig. 2). All of these mutants had a decreased affinity for Nup62 and Nup358 (996–1963) as well. In several cases, binding of the mutants to Nup62 and Nup358 (996–1963) did not reach saturation in our assay (Table I; Fig. 2), implying a Kd of >>1 μM for the interaction (see Table I legend).


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

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

Characterization of the binding of importin β to nucleoporins. Shown are binding isotherms depicting the interaction of selected importin β mutants with Nup153 (895–1475) (A), with full-length Nup62 (B), or with Nup358 (996–1963) (C). Error bars represent the standard deviation of duplicate measurements. The calculated apparent Kd values are indicated next to the binding isotherms. NS, nonsaturated binding at importin β concentrations up to 800 nM.
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Related In: Results  -  Collection

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fig2: Characterization of the binding of importin β to nucleoporins. Shown are binding isotherms depicting the interaction of selected importin β mutants with Nup153 (895–1475) (A), with full-length Nup62 (B), or with Nup358 (996–1963) (C). Error bars represent the standard deviation of duplicate measurements. The calculated apparent Kd values are indicated next to the binding isotherms. NS, nonsaturated binding at importin β concentrations up to 800 nM.
Mentions: We found that importin β single point mutants containing the substitutions I178D, E214A, F217A, Y255A, and I263R all had a decreased affinity for the Nup153 fragment, but retained wild-type affinity for RanGTP and for importin α (Table I). Weakened nucleoporin binding was most evident for the I178D mutant, which had an ∼40-fold decrease in affinity for Nup153 (895–1475). In contrast, the E214A, F217A, Y255A, and I263R mutants showed a relatively small decrease in affinity for the Nup153 fragment (Kd ≈ 1.8–3.5 nM) (Table I; Fig. 2). All of these mutants had a decreased affinity for Nup62 and Nup358 (996–1963) as well. In several cases, binding of the mutants to Nup62 and Nup358 (996–1963) did not reach saturation in our assay (Table I; Fig. 2), implying a Kd of >>1 μM for the interaction (see Table I legend).

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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