Karyopherin β 2 Mediates Nuclear Import of a mRNA Binding Protein

We have cloned and sequenced cDNA for human karyopherin β 2, also known as transportin. In a solution binding assay, recombinant β 2 bound 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 representing a classical NLS. As previously shown for karyopherin β 1, karyopherin β 2 bound to several nucleoporins containing characteristic peptide repeat motifs. In a solution binding assay, both β 1 and β 2 competed with each other for binding to immobilized repeat nucleoporin Nup98. In digitonin-permeabilized cells, β 2 was able to dock A1 at the nuclear rim and to import it into the nucleoplasm. At low concentrations of β 2, there was no stimulation of import by the exogenous addition of the GTPase Ran. However, at higher concentrations of β 2 there was marked stimulation of import by Ran. Import was inhibited by the nonhydrolyzable GTP analog guanylyl imidodiphosphate by a Ran mutant that is unable to hydrolyze GTP and also by wheat germ agglutinin. Consistent with the solution binding results, karyopherin β 2 inhibited karyopherin α /β 1-mediated import of a classical NLS containing substrate and, vice versa, β 1 inhibited β 2-mediated import of A1 substrate, suggesting that the two import pathways merge at the level of docking of β 1 and β 2 to repeat nucleoporins.