Importin beta can mediate the nuclear import of an arginine-rich nuclear localization signal in the absence of importin alpha

The import of proteins into the nucleus is dependent on cis-acting targeting sequences, nuclear localization signals (NLSs), and members of the nuclear transport receptor (importin-beta-like) superfamily. The most extensively characterized import pathway, often termed the classical pathway, is utilized by many basic-type (lysine-rich) NLSs and requires an additional component, importin alpha, to serve as a bridge between the NLS and the import receptor importin beta. More recently, it has become clear that a variety of proteins enter the nucleus via alternative import receptors and that their NLSs bind directly to those receptors. By using the digitonin-permeabilized cell system for protein import in vitro, we have defined the import pathway for the Rex protein of human T-cell leukemia virus type 1. Interestingly, the arginine-rich NLS of Rex uses importin beta for import but does so by a mechanism that is importin alpha independent. Based on the ability of the Rex NLS to inhibit the import of the lysine-rich NLS of T antigen and of both NLSs to be inhibited by the domain of importin alpha that binds importin beta (the IBB domain), we infer that the Rex NLS interacts with importin beta directly. In addition, and in keeping with other receptor-mediated nuclear import pathways, Rex import is dependent on the integrity of the Ran GTPase cycle. Based on these results, we suggest that importin beta can mediate the nuclear import of arginine-rich NLSs directly, or lysine-rich NLSs through the action of importin alpha.     

Prenatal diagnosis of Glanzmann thrombasthenia using the polymorphic markers BRCA1 and THRA1 on chromosome 17

Nuclear import of proteins containing a nuclear localization signal (NLS) is dependent on the presence of a cytoplasmic NLS receptor, the GTPase Ran, and p10/ NTF2. The NLS receptor is a heterodimeric proteins consisting of subunits of approximately 60 and 97 kDa, which have been termed importin alpha/beta, karyopherin alpha/beta, or PTAC 58/ 97. Members of the 60-kDa/importin alpha subunit family directly bind to the NLS motif and have been shown to function as adaptors that tether NLS-containing proteins to the p97/ importin beta subunit and to the downstream transport machinery. Herein we report the identification and characterization of hSRP1 gamma, a human importin alpha homologue. The hSRP1 gamma protein is around 45% identical to the previously identified human importin alpha homologues hSRP1 alpha/Rch1 and NPI/ hSRP1. hSRP1 gamma can form a complex with importin beta and is able to mediate import of a BSA-NLS substrate in an in vitro nuclear import system. Interestingly, hSRP1 gamma shows a very selective expression pattern and is most abundantly expressed in skeletal muscle, representing more than 1% of the total protein in this tissue. A potential role for hSRP1 gamma in tissue-specific transport events is discussed.     

Loss of IkappaB alpha-mediated control over nuclear import and DNA binding enables oncogenic activation of c-Rel

The IkappaB alpha protein is able both to inhibit nuclear import of Rel/NF-kappaB proteins and to mediate the export of Rel/NF-kappaB proteins from the nucleus. We now demonstrate that the c-Rel-IkappaB alpha complex is stably retained in the cytoplasm in the presence of leptomycin B, a specific inhibitor of Crm1-mediated nuclear export. In contrast, leptomycin B treatment results in the rapid and complete relocalization of the v-Rel-IkappaB alpha complex from the cytoplasm to the nucleus. IkappaB alpha also mediates the rapid nuclear shuttling of v-Rel in an interspecies heterokaryon assay. Thus, continuous nuclear export is required for cytoplasmic retention of the v-Rel-IkappaB alpha complex. Furthermore, although IkappaB alpha is able to mask the c-Rel-derived nuclear localization sequence (NLS), IkappaB alpha is unable to mask the v-Rel-derived NLS in the context of the v-Rel-IkappaB alpha complex. Taken together, our results demonstrate that IkappaB alpha is unable to inhibit nuclear import of v-Rel. We have identified two amino acid differences between c-Rel and v-Rel (Y286S and L302P) which link the failure of IkappaB alpha to inhibit nuclear import and DNA binding of a mutant c-Rel protein to oncogenesis. Our results support a model in which loss of IkappaB alpha-mediated control over c-Rel leads to oncogenic activation of c-Rel.     

Multiple protein domains determine the cell type-specific nuclear distribution of the catalytic subunit required for apolipoprotein B mRNA editing

Apolipoprotein B (apoB) mRNA editing catalyzed by apoB mRNA editing catalytic subunit 1 (APOBEC-1) has been proposed to be a nuclear process. To test this hypothesis, the subcellular distribution of hemagglutinin- (HA) tagged APOBEC-1 expressed in transiently transfected hepatoma cells was determined by indirect immunofluorescence microscopy. HA-APOBEC-1 was detected in both the nucleus and cytoplasm of rat and human hepatoma cells. Mutagenesis of APOBEC-1 demonstrated that the N-terminal 56 amino acids (1-56) were necessary for the nuclear distribution of APOBEC-1, but this region did not contain a functional nuclear localization signal (NLS). However, we identified a 24-amino acid domain in the C terminus of APOBEC-1 with characteristics of a cytoplasmic retention signal (CRS) or a nuclear export signal (NES). These data suggest, therefore, that the nuclear import of APOBEC-1 may not be mediated by a positive NLS; rather, it may be achieved by overcoming the effect of a CRS/NES. We also demonstrated that the nuclear distribution of APOBEC-1 occurred only in cell lines that were capable of editing apoB RNA. We propose that the cellular distribution of APOBEC-1 is determined by multiple domains within this protein, and a nuclear localization of the enzyme may be regulated by cell type-specific factors that render these cells uniquely editing competent.     

Nucleocytoplasmic recycling of the nuclear localization signal receptor alpha subunit in vivo is dependent on a nuclear export signal, energy, and RCC1

Nuclear protein import requires a nuclear localization signal (NLS) receptor and at least three other cytoplasmic factors. The alpha subunit of the NLS receptor, Rag cohort 1 (Rch1), enters the nucleus, probably in a complex with the beta subunit of the receptor, as well as other import factors and the import substrate. To learn more about which factors and/or events end the import reaction and how the import factors return to the cytoplasm, we have studied nucleocytoplasmic shuttling of Rch1 in vivo. Recombinant Rch1 microinjected into Vero or tsBN2 cells was found primarily in the cytoplasm. Rch1 injected into the nucleus was rapidly exported in a temperature-dependent manner. In contrast, a mutant of Rch1 lacking the first 243 residues accumulated in the nuclei of Vero cells after cytoplasmic injection. After nuclear injection, the truncated Rch1 was retained in the nucleus, but either Rch1 residues 207-217 or a heterologous nuclear export signal, but not a mutant form of residues 207-217, restored nuclear export. Loss of the nuclear transport factor RCC1 (regulator of chromosome condensation) at the nonpermissive temperature in the thermosensitive mutant cell line tsBN2 caused nuclear accumulation of wild-type Rch1 injected into the cytoplasm. However, free Rch1 injected into nuclei of tsBN2 cells at the nonpermissive temperature was exported. These results suggested that RCC1 acts at an earlier step in Rch1 recycling, possibly the disassembly of an import complex that contains Rch1 and the import substrate. Consistent with this possibility, incubation of purified RanGTP and RCC1 with NLS receptor and import substrate prevented assembly of receptor/substrate complexes or stimulated their disassembly.     

The asymmetric distribution of the constituents of the Ran system is essential for transport into and out of the nucleus

The GTPase Ran is essential for nuclear import of proteins with a classical nuclear localization signal (NLS). Ran's nucleotide-bound state is determined by the chromatin-bound exchange factor RCC1 generating RanGTP in the nucleus and the cytoplasmic GTPase activating protein RanGAP1 depleting RanGTP from the cytoplasm. This predicts a steep RanGTP concentration gradient across the nuclear envelope. RanGTP binding to importin-beta has previously been shown to release importin-alpha from -beta during NLS import. We show that RanGTP also induces release of the M9 signal from the second identified import receptor, transportin. The role of RanGTP distribution is further studied using three methods to collapse the RanGTP gradient. Nuclear injection of either RanGAP1, the RanGTP binding protein RanBP1 or a Ran mutant that cannot stably bind GTP. These treatments block major export and import pathways across the nuclear envelope. Different export pathways exhibit distinct sensitivities to RanGTP depletion, but all are more readily inhibited than is import of either NLS or M9 proteins, indicating that the block of export is direct rather than a secondary consequence of import inhibition. Surprisingly, nuclear export of several substrates including importin-alpha and -beta, transportin, HIV Rev and tRNA appears to require nuclear RanGTP but may not require GTP hydrolysis by Ran, suggesting that the energy for their nuclear export is supplied by another source.     

A novel receptor-mediated nuclear protein import pathway

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.