RanBP3 contains an unusual nuclear localization signal that is imported preferentially by importin-alpha 3

The full range of sequences that constitute nuclear localization signals (N LSs) remains to be established. Even though the sequence of the classical N LS contains polybasic residues that are recognized by importin-alpha, this import receptor can also bind cargo that contains no recognizable signal, s uch as STAT1. The situation is further complicated bg:the existence of six mammalian importin-alpha family members. We report the identification of an unusual type of NLS in human Ran binding protein 3 (RanBP3) that binds pre ferentially to importin-alpha 3. RanBP3 contains a variant Ran binding doma in most similar to that found in the yeast protein Yrb2p. Anti-RanBP3 immun ofluorescence is predominantly nuclear. Microinjection of glutathione S-tra nsferase-green fluorescent protein-RanBP3 fusions demonstrated that a regio n at the N terminus is essential and sufficient for nuclear localization. D eletion analysis further mapped the signal sequence to residues 40 to 57. T his 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 importi n-alpha or importin-beta or by the absence of importin-alpha. Binding assay s using recombinant importin-alpha 1, -alpha 3, -alpha 4, -alpha 5, and -al pha 7 revealed a preferential interaction of the RanBP3 NLS with importin-a lpha 3 and -alpha 4, in contrast to the simian virus 40 T-antigen NLS, whic h interacted to similar extents with all of the isoforms. Nuclear import of the RanBP3 NLS mas most efficient in the presence of importin-alpha 3. The se results demonstrate that members of the importin-alpha family possess di stinct preferences for certain NLS sequences and that the NLS consensus seq uence is broader than was hitherto suspected.