RanBP3 Contains an Unusual Nuclear Localization Signal That Is Imported Preferentially by Importin-α3

Abstract

The full range of sequences that constitute nuclear localization signals (NLSs) remains to be established. Even though the sequence of the classical NLS contains polybasic residues that are recognized by importin-α, this import receptor can also bind cargo that contains no recognizable signal, such as STAT1. The situation is further complicated by the existence of six mammalian importin-α family members. We report the identification of an unusual type of NLS in human Ran binding protein 3 (RanBP3) that binds preferentially to importin-α3. RanBP3 contains a variant Ran binding domain most similar to that found in the yeast protein Yrb2p. Anti-RanBP3 immunofluorescence is predominantly nuclear. Microinjection of glutathione S-transferase–green fluorescent protein–RanBP3 fusions demonstrated that a region at the N terminus is essential and sufficient for nuclear localization. Deletion analysis further mapped the signal sequence to residues 40 to 57. This signal resembles the NLSs of c-Myc and Pho4p. However, several residues essential for import via the c-Myc NLS are unnecessary in the RanBP3 NLS. RanBP3 NLS-mediated import was blocked by competitive inhibitors of importin-α or importin-β or by the absence of importin-α. Binding assays using recombinant importin-α1, -α3, -α4, -α5, and -α7 revealed a preferential interaction of the RanBP3 NLS with importin-α3 and -α4, in contrast to the simian virus 40 T-antigen NLS, which interacted to similar extents with all of the isoforms. Nuclear import of the RanBP3 NLS was most efficient in the presence of importin-α3. These results demonstrate that members of the importin-α family possess distinct preferences for certain NLS sequences and that the NLS consensus sequence is broader than was hitherto suspected.

ACKNOWLEDGMENTS

We thank Dirk Gorlich, Steve Adam, Mark Rush, and Bryce Paschal for the kind gifts of expression clones and proteins necessary for performing in vitro import assays (nucleoplasmin, nucleoplasmin core, human Ran, Rna1p, RanBP1, NTF2, importin-α1, importin-β, and the IBB and BIB domains). We also thank Amy Brownawell, Mike Nemergut, Clark Peterson, Kendra Plafker, and Alicia Smith from the Macara laboratory for reagents and helpful suggestions.

This work was supported by a grant from the National Institutes of Health, DHHS (GM-50526).