(cDNA-deduced sequence / recombinant2 / Ran / GTP hydrolysis / repeat nucleoporins)

Neris Bonifaci, Junona Moroianu, Aurelian Radu, and Günter Blobel

Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10021

Contributed by Günter Blobel, March 6, 1997

We have cloned and sequenced cDNA for human karyopherin2, also known as transportin. In a solution binding assay, recombinant2 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 karyopherin1, karyopherin2 bound to several nucleoporins containing characteristic peptide repeat motifs. In a solution binding assay, both1 and 2 competed with each other for binding to immobilized repeat nucleoporin Nup98. In digitonin-permeabilized cells, 2 was able to dock A1 at the nuclear rim and to import it into the nucleoplasm. At low concentrations of2, there was no stimulation ofimport by the exogenous addition of the GTPase Ran. However, at higher concentrations of2 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, karyopherin2 inhibited karyopherin/1-mediated import of a classical NLS containing substrate and, vice versa, 1 inhibited 2-mediated import of A1 substrate, suggesting that the two import pathways merge at the level of docking of1 and 2 to repeat nucleoporins.

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