Targeting of most nuclear proteins to the cell nucleus is initiated by interaction between the classical nuclear localization signals (NLSs) contained within them and the importin NLS receptor complex. We have recently delineated a novel 38 amino acid transport signal in the hnRNP A1 protein, termed M9, which confers bidirectional transport across the nuclear envelope. We show here that M9-mediated nuclear import occurs by a novel pathway that is independent of the well-characterized, importin-mediated classical NLS pathway. Additionally, we have identified a specific M9-interacting protein, termed transportin, which binds to wild-type M9 but not to transport-defective M9 mutants. Transportin is a 90 kDa protein, distantly related to importin beta, and we show that it mediates the nuclear import of M9-containing proteins. These findings demonstrate that there are at least two receptor-mediated nuclear protein import pathways. Furthermore, as hnRNP A1 likely participates in mRNA export, it raises the possibility that transportin is a mediator of this process as well.

demonstrated recently using immunoelectron microscopy Philadelphia, PA 19104-6148, USA that mRNAs in transit through the nuclear pore complex 1 Corresponding author (NPC) to the cytoplasm are associated directly with shuttling hnRNPs (Visa et al., 1996). Third, an active, Protein import into the nucleus and export from the transferable nuclear export signal (NES) within the nucleus are signal-mediated processes that require shuttling hnRNP A1 protein has been identified (Michael energy. The nuclear transport process about which the et al., 1995a). Fourth, a direct role for the A1 NES in most information is currently available is classical mRNA export has been demonstrated recently. In these nuclear localization signal (NLS)-mediated nuclear experiments, it was shown, using Xenopus oocytes, that import. However, details concerning the signal-mediinjection of saturating amounts of A1 into the nucleus ated export of proteins and RNAs as well as alternative competitively inhibits export of mRNA while a deletion nuclear import pathways are beginning to emerge. An mutant of A1 which lacks a functional NES does not example of this is the heterogeneous nuclear ribo- (Izaurralde et al., 1997), indicating that the A1 NES plays nucleoprotein (hnRNP) A1 protein which, by virtue of a central role in the export of mRNP particles. its M9 domain, is actively exported from the nucleus A1 is the most extensively characterized of the shuttling and imported into the nucleus via a novel pathway hnRNPs. It is composed of two RNP motif RNA-binding mediated by the recently characterized transportin domains (RBDs) as well as a third RNA-binding domain, protein. Here we report that the shuttling hnRNP K the RGG box (Burd and Dreyfus, 1994). A1 typifies a protein contains a novel shuttling domain (termed class of shuttling hnRNPs which require continuous pol KNS) which has many of the characteristics of M9, inII transcription for complete nuclear localization (Piñol-that it confers bi-directional transport across the Roma and Dreyfuss, 1991;Michael et al., 1995b). Transnuclear envelope. KNS-mediated nuclear import is port of A1 is determined by the M9 domain, a 38 amino dependent on RNA polymerase II transcription, and acid sequence located at the carboxy-terminus. M9 was we show that a classical NLS can override this effect.identified initially as the A1 nuclear localization signal Furthermore, KNS accesses a separate import pathway (NLS) as placement of M9 on normally cytoplasmic from either classical NLSs or M9. This demonstrates reporter proteins results in nuclear localization (Siomi and the existence of a third protein import pathway into Dreyfuss, 1995;Weighardt et al., 1995). Interestingly, M9 the nucleus and thereby defines a new type of nuclear also supplies the A1 nuclear export activity. When fused import/export signal.to a protein which is otherwise retained within the nucleus, Keywords: hnRNP K protein/nuclear export/nuclear such as the pentameric core domain of nucleoplasmin, import/shuttling domain/signal M9 can activ...

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