KARYOPHERIN BETA-2 MEDIATES NUCLEAR IMPORT OF A MESSENGER-RNA BINDING-PROTEIN

We have cloned and sequenced cDNA for human karyopherin beta 2, also k nown as transportin. In a solution binding assay, recombinant beta 2 b ound directly to recombinant nuclear mRNA-binding protein A1. Binding was inhibited by a peptide representing A1's previously characterized M9 nuclear localization sequence (NLS), but not by a peptide represent ing a classical NLS. As previously shown for karyopherin beta 1, karyo pherin beta 2 bound to several nucleoporins containing characteristic peptide repeat motifs, In a solution binding assay, both beta 1 and be ta 2 competed with each other for binding to immobilized repeat nucleo porin Nup98. In digitonin-permeabilized cells, beta 2 was able to dock Al at the nuclear rim and to impart it into the nucleoplasm. At low c oncentrations of beta 2, there was no stimulation of import by the exo genous addition of the GTPase Ran, However, at higher concentrations o f beta 2 there was marked stimulation of import by Ran. Import was inh ibited by the nonhydrolyzable GTP analog guanylyl imidodiphosphate by a Ran mutant that is unable to hydrolyze GTP and also by wheat germ ag glutinin, Consistent with the solution binding results, karyopherin be ta 2 inhibited karyopherin alpha/beta 1-mediated import of a classical NLS containing substrate and, vice versa, beta 1 inhibited beta 2-med iated import of A1 substrate, suggesting that the two import pathways merge at the level of docking of beta 1 and beta 2 to repeat nucleopor ins.