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

Nuclear import and export signals on macromolecules mediate directional, re ceptor-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 pa rtly determined by the nucleocytoplasmic compartmentalization of components of the Ran GTPase system. We have analyzed whether the asymmetric localiza tion of discrete nucleoporins can also contribute to transport directionali ty,To this end, we have used quantitative solid phase binding analysis to d etermine the affinity of an importin beta cargo complex for Nup358, the Nup 62 complex, and Nup153, which are in the cytoplasmic, central, and nucleopl asmic regions of the NPC, respectively. These nucleoporins are proposed to provide progressively more distal binding sites for importin beta during im port. Our results indicate that the importin beta transport complex binds t o 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 Ra n system, are likely to play an important role in conferring directionality to nuclear protein import.