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Gradient of increasing affinity of importin beta for nucleoporins along the pathway of nuclear import.

Ben-Efraim I, Gerace L - J. Cell Biol. (2001)

Bottom Line: Our results indicate that the importin beta transport complex binds to nucleoporins with progressively increasing affinity as the complex moves from Nup358 to the Nup62 complex and to Nup153.Antibody inhibition studies support the possibility that importin beta moves from Nup358 to Nup153 via the Nup62 complex during import.These results indicate that nucleoporins themselves, as well as the nucleocytoplasmic compartmentalization of the Ran system, are likely to play an important role in conferring directionality to nuclear protein import.

View Article: PubMed Central - PubMed

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

ABSTRACT
Nuclear import and export signals on macromolecules mediate directional, receptor-driven transport through the nuclear pore complex (NPC) by a process that is suggested to involve the sequential binding of transport complexes to different nucleoporins. The directionality of transport appears to be partly determined by the nucleocytoplasmic compartmentalization of components of the Ran GTPase system. We have analyzed whether the asymmetric localization of discrete nucleoporins can also contribute to transport directionality. To this end, we have used quantitative solid phase binding analysis to determine the affinity of an importin beta cargo complex for Nup358, the Nup62 complex, and Nup153, which are in the cytoplasmic, central, and nucleoplasmic regions of the NPC, respectively. These nucleoporins are proposed to provide progressively more distal binding sites for importin beta during import. Our results indicate that the importin beta transport complex binds to nucleoporins with progressively increasing affinity as the complex moves from Nup358 to the Nup62 complex and to Nup153. Antibody inhibition studies support the possibility that importin beta moves from Nup358 to Nup153 via the Nup62 complex during import. These results indicate that nucleoporins themselves, as well as the nucleocytoplasmic compartmentalization of the Ran system, are likely to play an important role in conferring directionality to nuclear protein import.

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Characterization of the binding of importin β to nucleoporins. Binding was analyzed in the absence (A, C, and E) or presence (B, D, and F) of the IBB domain. Increasing concentrations of importin β were incubated with Nup358-4 (A and B), Nup62 (C and D), and Nup153 (E and F) and the bound importin β was measured (see Materials and Methods). The results are from duplicates of a single typical experiment. The standard deviation was <5% of the mean. A Lineweaver-Burke plot, presented as an inset in each binding isotherm, provides the apparent binding constant (Kd apparent; see Table ). Curves E and F were fitted for a logarithmic function and the rest for a polynomial function using Cricket Graph software. The correlation coefficients for the curve fits were always >0.99.
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Figure 1: Characterization of the binding of importin β to nucleoporins. Binding was analyzed in the absence (A, C, and E) or presence (B, D, and F) of the IBB domain. Increasing concentrations of importin β were incubated with Nup358-4 (A and B), Nup62 (C and D), and Nup153 (E and F) and the bound importin β was measured (see Materials and Methods). The results are from duplicates of a single typical experiment. The standard deviation was <5% of the mean. A Lineweaver-Burke plot, presented as an inset in each binding isotherm, provides the apparent binding constant (Kd apparent; see Table ). Curves E and F were fitted for a logarithmic function and the rest for a polynomial function using Cricket Graph software. The correlation coefficients for the curve fits were always >0.99.

Mentions: The binding experiments were conducted both with importin β alone and with importin β bound to the IBB domain of importin α (Fig. 1 and Table ). The IBB domain behaves as an authentic import cargo for importin β and closely resembles certain NLSs that bind to importin β in an importin α–independent fashion (for review see Gorlich and Kutay 1999). The binding isotherms for each of the six proteins tested showed saturable binding of both importin β and the importin β–IBB domain complex, as evidenced by linear double reciprocal plots (Fig. 1; data not shown). The apparent affinity of importin β for the nucleoporins tested is similar in the presence and absence of the IBB domain. This argues that the region of importin β involved in nucleoporin binding is not conformationally altered by cargo binding. Interestingly, the affinity of importin β was lowest for each of the Nup358 fragments (Kd = 210–225 nM), increased ∼2-fold for each of the Nup62 complex proteins (Kd = 100–105 nM), and increased another ∼10-fold for Nup153-C (Kd = 9 nM) (Table ). Thus, there is a progressively increasing affinity of importin β for nucleoporins that occur progressively closer to the nucleoplasmic periphery of the NPC.


Gradient of increasing affinity of importin beta for nucleoporins along the pathway of nuclear import.

Ben-Efraim I, Gerace L - J. Cell Biol. (2001)

Characterization of the binding of importin β to nucleoporins. Binding was analyzed in the absence (A, C, and E) or presence (B, D, and F) of the IBB domain. Increasing concentrations of importin β were incubated with Nup358-4 (A and B), Nup62 (C and D), and Nup153 (E and F) and the bound importin β was measured (see Materials and Methods). The results are from duplicates of a single typical experiment. The standard deviation was <5% of the mean. A Lineweaver-Burke plot, presented as an inset in each binding isotherm, provides the apparent binding constant (Kd apparent; see Table ). Curves E and F were fitted for a logarithmic function and the rest for a polynomial function using Cricket Graph software. The correlation coefficients for the curve fits were always >0.99.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199621&req=5

Figure 1: Characterization of the binding of importin β to nucleoporins. Binding was analyzed in the absence (A, C, and E) or presence (B, D, and F) of the IBB domain. Increasing concentrations of importin β were incubated with Nup358-4 (A and B), Nup62 (C and D), and Nup153 (E and F) and the bound importin β was measured (see Materials and Methods). The results are from duplicates of a single typical experiment. The standard deviation was <5% of the mean. A Lineweaver-Burke plot, presented as an inset in each binding isotherm, provides the apparent binding constant (Kd apparent; see Table ). Curves E and F were fitted for a logarithmic function and the rest for a polynomial function using Cricket Graph software. The correlation coefficients for the curve fits were always >0.99.
Mentions: The binding experiments were conducted both with importin β alone and with importin β bound to the IBB domain of importin α (Fig. 1 and Table ). The IBB domain behaves as an authentic import cargo for importin β and closely resembles certain NLSs that bind to importin β in an importin α–independent fashion (for review see Gorlich and Kutay 1999). The binding isotherms for each of the six proteins tested showed saturable binding of both importin β and the importin β–IBB domain complex, as evidenced by linear double reciprocal plots (Fig. 1; data not shown). The apparent affinity of importin β for the nucleoporins tested is similar in the presence and absence of the IBB domain. This argues that the region of importin β involved in nucleoporin binding is not conformationally altered by cargo binding. Interestingly, the affinity of importin β was lowest for each of the Nup358 fragments (Kd = 210–225 nM), increased ∼2-fold for each of the Nup62 complex proteins (Kd = 100–105 nM), and increased another ∼10-fold for Nup153-C (Kd = 9 nM) (Table ). Thus, there is a progressively increasing affinity of importin β for nucleoporins that occur progressively closer to the nucleoplasmic periphery of the NPC.

Bottom Line: Our results indicate that the importin beta transport complex binds to nucleoporins with progressively increasing affinity as the complex moves from Nup358 to the Nup62 complex and to Nup153.Antibody inhibition studies support the possibility that importin beta moves from Nup358 to Nup153 via the Nup62 complex during import.These results indicate that nucleoporins themselves, as well as the nucleocytoplasmic compartmentalization of the Ran system, are likely to play an important role in conferring directionality to nuclear protein import.

View Article: PubMed Central - PubMed

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

ABSTRACT
Nuclear import and export signals on macromolecules mediate directional, receptor-driven transport through the nuclear pore complex (NPC) by a process that is suggested to involve the sequential binding of transport complexes to different nucleoporins. The directionality of transport appears to be partly determined by the nucleocytoplasmic compartmentalization of components of the Ran GTPase system. We have analyzed whether the asymmetric localization of discrete nucleoporins can also contribute to transport directionality. To this end, we have used quantitative solid phase binding analysis to determine the affinity of an importin beta cargo complex for Nup358, the Nup62 complex, and Nup153, which are in the cytoplasmic, central, and nucleoplasmic regions of the NPC, respectively. These nucleoporins are proposed to provide progressively more distal binding sites for importin beta during import. Our results indicate that the importin beta transport complex binds to nucleoporins with progressively increasing affinity as the complex moves from Nup358 to the Nup62 complex and to Nup153. Antibody inhibition studies support the possibility that importin beta moves from Nup358 to Nup153 via the Nup62 complex during import. These results indicate that nucleoporins themselves, as well as the nucleocytoplasmic compartmentalization of the Ran system, are likely to play an important role in conferring directionality to nuclear protein import.

Show MeSH
Related in: MedlinePlus