eEF1A mediates the nuclear export of SNAG-containing proteins via the Exportin5-aminoacyl-tRNA complex.

Exportin5 mediates the nuclear export of double-stranded RNAs, including pre-microRNAs, adenoviral RNAs, and tRNAs. When tRNAs are aminoacylated, the Exportin5-aminoacyl (aa)-tRNA complex recruits and coexports the translation elongation factor eEF1A. Here, we show that eEF1A binds to Snail transcription factors when bound to their main target, the E-cadherin promoter, facilitating their export to the cytoplasm in association with the aa-tRNA-Exportin5 complex. Snail binds to eEF1A through the SNAG domain, a protein nuclear export signal present in several transcription factor families, and this binding is regulated by phosphorylation. Thus, we describe a nuclear role for eEF1A and provide a mechanism for protein nuclear export that attenuates the activity of SNAG-containing transcription factors.

c-Jun localizes to the nucleus independent of its phosphorylation by and interaction with JNK and vice versa promotes nuclear accumulation of JNK.

In order to activate gene expression, transcription factors such as c-Jun have to reside in the nucleus. The abundance of c-Jun in the nucleus correlates with the activity of its target genes. As a consequence of excessive c-Jun activation, cells undergo apoptosis or changes in differentiation whereas decreased c-Jun function can reduce proliferation. In the present study we addressed how nuclear accumulation of the transcription factor c-Jun is regulated. First, we analyzed which functions of c-Jun are required for efficient nuclear accumulation. Mutants of c-Jun deficient in dimerization or DNA-binding show no defect in nuclear transport. Furthermore, c-Jun import into the nucleus of living cells occurred when the c-Jun phosphorylation sites were mutated as well in cells that lack the major c-Jun kinase, JNK, suggesting that c-Jun transport into the nucleus does not require JNK signaling. Conversely, however, binding of c-Jun seemed to enhance nuclear accumulation of JNK. In order to identify proteins that might be relevant for the nuclear translocation of c-Jun we searched for novel binding partners by a proteomic approach. In addition to the heat shock protein HSP70 and the DNA damage repair factors Ku70 and 80, we isolated human importin 8 as a novel interactor of c-Jun. Interaction of Imp 8 with c-Jun in human cells was confirmed by co-immunoprecipitation experiments. Nuclear accumulation of c-Jun does not require its functions as a transcription factor or the interaction with its kinase JNK. Interestingly, nuclear accumulation of JNK is regulated by interaction with c-Jun. Unraveling the mechanisms of c-Jun and JNK transport to the nucleus and its regulation will improve our understanding of their role in biological and pathophysiological processes.

Transport of hypoxia-inducible factor HIF-1alpha into the nucleus involves importins 4 and 7.

Hypoxia-inducible transcription factor 1 (HIF-1) mediates the cellular response to hypoxia. HIF-1 activity is controlled via the synthesis, degradation or intracellular localization of its alpha subunit. HIF-1alpha contains a C-terminal bipartite basic NLS that interacts with importins alpha. We have recently shown that HIF-1alpha also contains an atypical hydrophobic CRM1- and phosphorylation-dependent NES and can therefore shuttle in and out of the nucleus. We now report that C-terminal NLS mutants of HIF-1alpha can still enter the nucleus when CRM1-dependent nuclear export is inhibited, indicating that HIF-1alpha contains an additional functional nuclear import signal. Using an in vitro nuclear import assay, we further show that importins 4 and 7 accomplish nuclear import of HIF-1alpha more efficiently than the classical importin alpha/beta NLS receptor. Binding assays confirmed the specific physical interaction between HIF-1alpha and importins 4 and 7. Moreover, the interaction of importin 7 with HIF-1alpha is mapped at its N-terminal part encompassing the bHLH-PAS(A) domain. By expressing functional HIF-1 in yeast, we show that Nmd5, the yeast orthologue of importin 7, is required for HIF-1alpha nuclear accumulation and activity. Taken together, our data show that shuttling of HIF-1alpha between cytoplasm and nucleus is a complex process involving several members of the nuclear transport receptor family.

Characterization of Snail nuclear import pathways as representatives of C2H2 zinc finger transcription factors.

Snail proteins are C(2)H(2) class zinc finger transcription factors involved in different processes during embryonic development, as well as in several adult pathologies including cancer and organ fibrosis. The expression of Snail transcription factors is tightly regulated at the transcriptional level and their activity is modulated by their subcellular localization. Given the importance of this gene family in physiology and pathology, it is essential to understand the mechanisms by which Snail proteins are imported into or exported out of the nucleus. Here we show that several importins mediate the nuclear import of the human Snail proteins and we identify a unique nuclear localization signal (NLS), recognized by all the importins, that has been conserved during the evolution of the Snail family. This NLS is characterized by the presence of basic residues at defined positions in at least three consecutive zinc fingers. Interestingly, the consensus residues for importin-binding are also involved in DNA binding, suggesting that importins could prevent non-specific binding of these transcription factors to cytoplasmic polyanions. Importantly, the identified basic residues are also conserved in other families of C(2)H(2) transcription factors whose nuclear localization requires the zinc finger region.

Exportin 7 defines a novel general nuclear export pathway.

Most transport pathways between cell nucleus and cytoplasm are mediated by nuclear transport receptors of the importin beta family. These receptors are in continuous circulation between the two compartments and transfer cargo molecules from one side of the nuclear envelope to the other. RanBP16 is a family member from higher eukaryotes of so far unknown function. We now show that it exports p50RhoGAP from the nucleus and thereby confines this activity to the cytoplasm. It also accounts for nuclear exclusion of 14-3-3sigma, which in turn is known to anchor, for example, cyclin-dependent kinases in the cytoplasm. Our data further suggest that RanBP16 exports several additional cargoes. It thus appears to be a nuclear export mediator with broad substrate specificity and we will therefore refer to it as exportin 7 (Exp7). Finally, we demonstrate that Exp7-dependent nuclear export signals differ fundamentally from the leucine-rich, CRM1-dependent ones: First, they are not just short linear sequences, but instead include folded motifs. Second, basic residues are critical for Exp7 recruitment.

Nuclear import of HIV-1 intracellular reverse transcription complexes is mediated by importin 7.

Human immunodeficiency virus type 1 (HIV-1), like other lentiviruses, can infect non-dividing cells. This property depends on the active nuclear import of its intracellular reverse transcription complex (RTC). We have studied nuclear import of purified HIV-1 RTCs in primary macrophages and found that importin 7, an import receptor for ribosomal proteins and histone H1, is involved in the process. Nuclear import of RTCs requires, in addition, energy and the components of the Ran system. Depletion of importin 7 from cultured cells by small interfering RNA inhibits HIV-1 infection. These results provide a new insight into the molecular mechanism for HIV-1 nuclear import and reveal potential targets for therapeutic intervention.

Importins fulfil a dual function as nuclear import receptors and cytoplasmic chaperones for exposed basic domains.

Many nuclear transport pathways are mediated by importin beta-related transport receptors. Here, we identify human importin (Imp) 4b as well as mouse Imp4a, Imp9a and Imp9b as novel family members. Imp4a mediates import of the ribosomal protein (rp) S3a, while Imp9a and Imp9b import rpS7, rpL18a and apparently numerous other substrates. Ribosomal proteins, histones and many other nuclear import substrates are very basic proteins that aggregate easily with cytoplasmic polyanions such as RNA. Imp9 effectively prevents such precipitation of, for example, rpS7 and rpL18a by covering their basic domains. The same applies to Imp4, Imp5, Imp7 and Impbeta and their respective basic import substrates. The Impbeta-Imp7 heterodimer appears specialized for the most basic proteins, such as rpL4, rpL6 and histone H1, and is necessary and sufficient to keep them soluble in a cytoplasmic environment prior to rRNA or DNA binding, respectively. Thus, just as heat shock proteins function as chaperones for exposed hydrophobic patches, importins act as chaperones for exposed basic domains, and we suggest that this represents a major and general cellular function of importins.