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

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.

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 (K _d 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.

Figure 3

Role of the Nup62 complex in nuclear import. Shown are the effects of anti-Nup62 Fab fragment on (A) NLS-mediated import in NRK cells; (B) the solubilization of Nup358 and Nup153 (left) and the association of importin β with these Nups (right); and (C) the binding of importin β to GST-Nup62.

Figure 2

The binding of importin β to nucleoporins in the solid phase assay is sensitive to RanGTP. The binding of 10 nM importin β to nucleoporins adsorbed to microtiter wells was analyzed in the absence of RanGTP, or in the presence of 20 or 100 nM RanGTP, as indicated. The bound importin β was quantified as described in the legend to Fig. 1.

Figure 4

Model for the directional movement of an importin β cargo complex through the NPC. See text for details.