Nuclear Import System Research Articles

Co-translational binding of importins to nascent proteins

Various cellular quality control mechanisms support proteostasis. While, ribosome-associated chaperones prevent the misfolding of nascent chains during translation, importins were shown to prevent the aggregation of specific cargoes in a post-translational mechanism prior the import into the nucleoplasm. Here, we hypothesize that importins may already bind ribosome-associated cargo in a co-translational manner. We systematically measure the nascent chain association of all importins in Saccharomyces cerevisiae by selective ribosome profiling. We identify a subset of importins that bind to a wide range of nascent, often uncharacterized cargoes. This includes ribosomal proteins, chromatin remodelers and RNA binding proteins that are aggregation prone in the cytosol. We show that importins act consecutively with other ribosome-associated chaperones. Thus, the nuclear import system is directly intertwined with nascent chain folding and chaperoning.

Roles of the CSE1L-mediated nuclear import pathway in epigenetic silencing

Epigenetic silencing can be mediated by various mechanisms, and many regulators remain to be identified. Here, we report a genome-wide siRNA screening to identify regulators essential for maintaining gene repression of a CMV promoter silenced by DNA methylation. We identified CSE1L (chromosome segregation 1 like) as an essential factor for the silencing of the reporter gene and many endogenous methylated genes. CSE1L depletion did not cause DNA demethylation. On the other hand, the methylated genes derepressed by CSE1L depletion largely overlapped with methylated genes that were also reactivated by treatment with histone deacetylase inhibitors (HDACi). Gene silencing defects observed upon CSE1L depletion were linked to its nuclear import function for certain protein cargos because depletion of other factors involved in the same nuclear import pathway, including KPNAs and KPNB1 proteins, displayed similar derepression profiles at the genome-wide level. Therefore, CSE1L appears to be critical for the nuclear import of certain key repressive proteins. Indeed, NOVA1, HDAC1, HDAC2, and HDAC8, genes known as silencing factors, became delocalized into cytosol upon CSE1L depletion. This study suggests that the cargo specificity of the protein nuclear import system may impact the selectivity of gene silencing.

Regulation of Gli ciliary localization and Hedgehog signaling by the PY-NLS/karyopherin-β2 nuclear import system.

Hedgehog (Hh) signaling in vertebrates depends on primary cilia. Upon stimulation, Hh pathway components, including Gli transcription factors, accumulate at primary cilia to transduce the Hh signal, but the mechanisms underlying their ciliary targeting remains largely unknown. Here, we show that the PY-type nuclear localization signal (PY-NLS)/karyopherinβ2 (Kapβ2) nuclear import system regulates Gli ciliary localization and Hh pathway activation. Mutating the PY-NLS in Gli or knockdown of Kapβ2 diminished Gli ciliary localization. Kapβ2 is required for the formation of Gli activator (GliA) in wild-type but not in Sufu mutant cells. Knockdown of Kapβ2 affected Hh signaling in zebrafish embryos, as well as in vitro cultured cerebellum granule neuron progenitors (CGNPs) and SmoM2-driven medulloblastoma cells. Furthermore, Kapβ2 depletion impaired the growth of cultured medulloblastoma cells, which was rescued by Gli overexpression. Interestingly, Kapβ2 is a transcriptional target of the Hh pathway, thus forming a positive feedback loop for Gli activation. Our study unravels the molecular mechanism and cellular machinery regulating Gli ciliary localization and identifies Kapβ2 as a critical regulator of the Hh pathway and a potential drug target for Hh-driven cancers.

Human mutations affect the epigenetic/bookmarking function of HNF1B.

Bookmarking factors are transcriptional regulators involved in the mitotic transmission of epigenetic information via their ability to remain associated with mitotic chromatin. The mechanisms through which bookmarking factors bind to mitotic chromatin remain poorly understood. HNF1β is a bookmarking transcription factor that is frequently mutated in patients suffering from renal multicystic dysplasia and diabetes. Here, we show that HNF1β bookmarking activity is impaired by naturally occurring mutations found in patients. Interestingly, this defect in HNF1β mitotic chromatin association is rescued by an abrupt decrease in temperature. The rapid relocalization to mitotic chromatin is reversible and driven by a specific switch in DNA-binding ability of HNF1β mutants. Furthermore, we demonstrate that importin-β is involved in the maintenance of the mitotic retention of HNF1β, suggesting a functional link between the nuclear import system and the mitotic localization/translocation of bookmarking factors. Altogether, our studies have disclosed novel aspects on the mechanisms and the genetic programs that account for the mitotic association of HNF1β, a bookmarking factor that plays crucial roles in the epigenetic transmission of information through the cell cycle.

Cytoplasmic Dynamics of the General Nuclear Import Machinery in Apically Growing Syncytial Cells

Karyopherins are transporters involved in the bidirectional, selective and active transport of macromolecules through nuclear pores. Importin-β1 is the paradigm of karyopherins and, together with its cargo-adapter importin-α, mediates the general nuclear import pathway. Here we show the existence of different cellular pools of both importin-α and -β1 homologues, KapA and KapB, in the coenocytic ascomycete Aspergillus nidulans. Fluorescence analysis of haploid and diploid strains expressing KapB::GFP and/or KapA::mRFP showed patches of both karyopherins concurrently translocating long distances in apically-growing cells. Anterograde and retrograde movements allowed those patches to reach cell tips and distal regions with an average speed in the range of μm/s. This bidirectional traffic required microtubules as well as kinesin and dynein motors, since it is blocked by benomyl and also by the inactivation of the dynein/dynactin complex through nudA1 or nudK317 mutations. Deletion of Kinesin-3 motor UncA, required for the transport through detyrosinated microtubules, strongly inhibited KapA and KapB movement along hyphae. Overall, this is the first report describing the bidirectional dynamics of the main nuclear import system in coenocytic fungi. A functional link is proposed between two key cellular machines of the filamentous fungal cell: nuclear transport and the tip-growth apparatus.

Factors influencing RhoA protein distribution in the nucleus

The aim of the present study was to observe the influence of various factors on the nuclear distribution of the RhoA protein in the SGC-7901 human gastric cancer cell line. Immunofluorescence microscopy was used to detect the localization of the RhoA protein, and Western blotting was used to determine the quantity of RhoA in the nucleus, cytosol and membrane. The results showed that H2O2-mediated damage and a lipopolysaccharide (LPS)-mediated inflammatory reaction caused the translocation of RhoA from the cytosol toward the nucleus. A P38 mitogen-activated protein kinase (MAPK) inhibitor effectively hindered the LPS-triggered translocation of RhoA into the nucleus at the initial stage. Furthermore, the microtubule-targeted anticancer drug Taxol triggered the translocation of RhoA from the nucleus toward the cytosol and membrane, and Lysophosphatidic acid (LPA) enhanced this translocation. A protein modification inhibitor and a nucleus export inhibitor had no obvious effect on RhoA distribution in the nucleus. The results revealed that the distribution of RhoA protein in the nucleus was influenced by factors related to cell activities but was not affected by the modification of the protein. The translocation of RhoA into the nucleus was not dependent on the active nuclear import system.

Genetic analysis of p53 nuclear importation

A key step in activation of the p53 tumor suppressor is its transport into the nucleus; however, despite intensive study of p53, the regulation of its subcellular localization is still poorly understood. Here we examined the p53 nuclear importation using a series of mutant cell lines that were resistant to the growth inhibitory effects of temperature-sensitive murine p53 (tsp53). Examination of the p53 subcellular localization in these cell lines showed that the protein was cytoplasmic in most of them. Using a digitonin-permeabilized cell in vitro nuclear import system, we show that cytosols from these cell lines do not support nuclear translocation of a p53 nuclear localization signal (NLS)-containing substrate protein, but promote nuclear localization of a SV40TAgNLS-containing substrate. Complementation assays and use of the mutant cells themselves in the in vitro assays demonstrate that both soluble and insoluble protein components are involved in p53 nuclear import. Collectively, our results suggest that there is a p53 NLS-selective nuclear import pathway and that both soluble and insoluble proteins are involved in its function.

An improved genetic system for detection and analysis of protein nuclear import signals

BackgroundNuclear import of proteins is typically mediated by their physical interaction with soluble cytosolic receptor proteins via a nuclear localization signal (NLS). A simple genetic assay to detect active NLSs based on their function in the yeast Saccharomyces cerevisiae has been previously described. In that system, a chimera consisting of a modified bacterial LexA DNA binding domain and the transcriptional activation domain of the yeast Gal4 protein is fused to a candidate NLS. A functional NLS will redirect the chimeric fusion to the yeast cell nucleus and activate transcription of a reporter gene.ResultsWe have reengineered this nuclear import system to expand its utility and tested it using known NLS sequences from adenovirus E1A. Firstly, the vector has been reconstructed to reduce the level of chimera expression. Secondly, an irrelevant "stuffer" sequence from the E. coli maltose binding protein was used to increase the size of the chimera above the passive diffusion limit of the nuclear pore complex. The improved vector also contains an expanded multiple cloning site and a hemagglutinin epitope tag to allow confirmation of expression.ConclusionThe alterations in expression level and composition of the fusions used in this nuclear import system greatly reduce background activity in β-galactosidase assays, improving sensitivity and allowing more quantitative analysis of NLS bearing sequences.

14-3-3 Mediates Transcriptional Regulation by Modulating Nucleocytoplasmic Shuttling of Tobacco DNA-binding Protein Phosphatase-1

Tobacco DBP1 is the founding member of a novel class of plant transcription factors featuring sequence-specific DNA binding and protein phosphatase activity. To understand the mechanisms underlying the function of this family of transcriptional regulators, we have identified the tobacco 14-3-3 isoform G as the first protein interacting with a DBP factor. 14-3-3 recognition involves the N-terminal region of DBP1, which also supports the DNA binding activity attributed to DBP1. The relevance of this interaction is reinforced by its conservation in Arabidopsis plants, where the closest relative of DBP1 in this species also interacts with a homologous 14-3-3 protein through its N-terminal region. Furthermore, we show that in planta 14-3-3 G is directly involved in regulating DBP1 function by promoting nuclear export and subsequent cytoplasmic retention of DBP1 under conditions that in turn alleviate DBP1-mediated repression of target gene expression.

The Drosophila semushi mutation blocks nuclear import of Bicoid during embryogenesis

The maternal transcript of the anterior segmentation gene bicoid (bcd) is localized at the anterior pole of the Drosophila egg and translated to form a gradient in the nuclei of the syncytial blastoderm embryo after fertilization [1–3]. The nuclear gradient of Bcd protein – a transcription factor – leads to differential expression of zygotic segmentation genes. The rapid nuclear division during this stage [4] requires that Bcd quickly enters the nuclei after each mitosis using an active nuclear import system. Nuclear transport depends on the asymmetrical distribution of two forms of the small GTPase Ran: Ran–GTP is concentrated in the nucleus and Ran–GDP in the cytoplasm [5–8]. Ran requires RanGTPase-activating protein-1 (RanGAP1) on the cytoplasmic side of nuclear pore complexes to convert Ran–GTP to Ran–GDP. In vitro studies with vertebrate proteins demonstrate that the RanGAP1 associated with the nuclear pore complex is modified with small ubiquitin related modifier-1 (SUMO-1) by a ubiquitin-conjugating enzyme (E2 enzyme) [9–15]. Here, we show that mutation of the Drosophila semushi (semi) gene, which encodes an E2 enzyme, blocks nuclear import of Bcd during early embryogenesis and results in misregulation of the segmentation genes that are Bcd targets. Consequently, semi embryos have multiple defects in anterior segmentation. This study demonstrates that an E2 enzyme is required for nuclear transport during Drosophila embryogenesis.

Hypothesis: ligand/receptor-assisted nuclear translocation of STATs.

The STAT transcription factors are mediators of signal transduction of a variety of factors, including interferons (IFNs), interleukins, growth factors, and peptide hormones. Subsequent to activation, STATs are translocated to the nucleus apparently through the well-described importin/Ran system, where they activate target genes. Molecules utilizing this nuclear import system require specific nuclear localization sequences (NLSs). Paradoxically, such NLSs are not identifiable on STATs, thus raising the question of how they are imported into the nucleus. Of considerable interest is the observation that ligands and/or receptors that signal through STATs contain putative NLSs and, where examined, either ligand or receptor undergoes nuclear translocation. We hypothesize that ligands and/or their receptors serve as vehicles for the nuclear translocation of STATs, and that they may be directly involved in signal transduction. Using IFNgamma as a model system, we provide a possible mechanism for how this direct role is fulfilled. A functional NLS has been identified in a C-terminal domain of IFNgamma. This domain and the NLS contained within are crucial for the biological properties of IFNgamma in that a peptide encompassing this domain is sufficient to induce an antiviral state. Further, this domain binds specifically to a membrane-proximal region internal cytoplasmic domain of the alpha subunit of the receptor complex in a region that is directly involved in the recruitment and activation of the JAK/STAT pathway. We suggest that this novel mode of receptor recognition and activation may be a driving force for nuclear translocation of molecules like STATs that are associated with the ligand-receptor complex.

Cytokine-receptor complexes as chaperones for nuclear translocation of signal transducers.

A variety of ligands that include interleukins, interferons, and growth hormones activate STAT transcriptions factors. When activated, STATs are translocated to the nucleus apparently through the well described importin/Ran system where they activate target genes. Molecules utilizing this nuclear import system require specific nuclear localization sequences (NLSs). Paradoxically, such NLSs are not identifiable on STATs, raising the question of how they are imported into the nucleus. Surprisingly, most ligands and/or receptors that signal through STATs contain putative NLSs, and where examined either ligand or receptor undergo nuclear translocation. We hypothesize that these ligands and/or their receptors serve as chaperones in the nuclear translocation of STATs, and that they may be directly involved in signal transduction. Using IFN gamma as a model system we provide a possible mechanism for how this direct role is fulfilled. A C-terminal domain of IFN gamma has been identified that contains a functional NLS. Besides the fact that this domain, and the NLS in particular, is crucial for the biological properties of IFN gamma, a peptide encompassing this domain is sufficient to induce an antiviral state. Moreover, this domain interacts exclusively with an internal cytoplasmic domain of a subunit of the receptor complex in a region that is directly involved in the recruitment and activation of the elements of the JAK/STAT pathway. We suggest that this novel mode of receptor recognition and activation may be a driving force for nuclear translocation of molecules like STATs that are associated with the ligand-receptor complex.

Nucleolar protein B23 stimulates nuclear import of the HIV-1 Rev protein and NLS-conjugated albumin.

Nucleolar phosphoprotein B23 is a putative ribosome assembly factor with a relatively high affinity for peptides containing sequences of nuclear localization signals (NLSs) of the SV40 T-antigen type [Szebeni, A., Herrera, J. E., & Olson, O. J. (1995) Biochemistry 34, 8037-8042]. The effects of protein B23 on nuclear import were determined by an in vitro assay [Dean, D. A., & Kasamatsu, H. (1994) J. Biol. Chem. 269, 4910-4916] using NLS peptide-conjugated bovine serum albumin (NLS-BSA) or the HIV-1 Rev protein as substrates for import into isolated rat liver nuclei. The import was ATP-dependent and inhibited by wheat germ agglutinin or by an antibody against p97, a component of the nuclear import system. The rate of import of either substrate was increased if protein B23 was added to the incubation medium. Similar enhancements of import were seen with both isoforms (B23.1 and B23.2). The stimulatory effect on Rev protein import was saturable with maximum stimulation (2-3-fold) at a molar ratio of protein B23:Rev of approximately 1:1. Phosphorylation of protein B23.1 by casein kinase II produced an additional doubling of the import rate. This effect was not seen if protein B23.1 was phosphorylated with a cdc2 type protein kinase. Mutant forms of protein B23.1 in which the nuclear localization signal was either deleted or altered did not stimulate import of the substrates. These results suggest that protein B23 plays a role as an accessory factor in the nuclear import of the NLS-containing proteins and that phosphorylation at sites in the highly acidic segments of the protein enhances the stimulatory effect.

Location of the HIV-1 Rev protein during mitosis: inactivation of the nuclear export signal alters the pathway for postmitotic reentry into nucleoli.

The HIV-1 Rev protein localizes predominantly to the nucleolus of HIV-1-infected or Rev-expressing cells. The subcellular location of Rev during mitotic nucleolar disintegration was examined at various stages of mitosis in synchronized Rev-expressing CMT3 cells. During early prophase Rev was predominantly located in disintegrating nucleoli and began to accumulate at the peripheral regions of chromosomes in late prophase, eventually distributing uniformly on all chromosomes in prometaphase. In anaphase Rev remained associated with the perichromosomal regions, but significant amounts of Rev were also seen in numerous nucleolus-derived foci. The movement of Rev from disintegrating nucleoli to perichromosomal regions and foci was similar to that of nonribosomal nucleolar proteins, including fibrillarin, nucleolin, protein B23 and p52 of the granular component. During telophase Rev remained associated with perichromosomal regions and mitotic foci until the nuclear envelope started to reform. When nuclear envelope formation was complete in late telophase, nonribosomal nucleolar proteins were present in prenucleolar bodies (PNBs) which were eventually incorporated into nucleoli; at the same time, Rev was excluded from nuclei. In contrast, a trans-dominant negative Rev protein containing an inactive nuclear export signal reentered nuclei by the nonribosomal nucleolar protein pathway in late telophase, associating with PNBs and reformed nucleoli. Rev protein reentry into postmitotic nuclei was delayed until early G1 phase, but before the arrival of ribosomal protein S6. Thus, Rev behaves like a nonribosomal nucleolar protein through mitosis until early telophase; however, its nuclear reentry seems to require reestablishment of both a nuclear import system and active nucleoli.

The yeast nuclear import receptor is required for mitosis.

The nuclear import system is highly conserved among eukaryotes. Here we report the effects of a conditional mutation in SRP1, which encodes a Saccharomyces cerevisiae homolog of the vertebrate nuclear import receptor importin. Importin was isolated as a factor required for the initial targeting step of a nuclear import substrate to the nuclear envelope in a mammalian in vitro assay. We show that yeast Srp1 is similarly required for protein import. In addition, Srp1 is also required for the execution of mitosis: we demonstrate that cells containing a conditional mutation of SRP1 arrest with a G2/M phenotype in a manner analogous to classic cdc mutants. This defect may be due to the failure of the mutant to degrade the mitotic cyclin Clb2 and other proteins required for mitosis. The requirement of a nuclear import receptor for cell cycle-regulated proteolysis implies that import of cell cycle regulators into the nucleus is critical for cell cycle progression.

In vitro nuclear import of snRNPs: cytosolic factors mediate m3G-cap dependence of U1 and U2 snRNP transport.

We have established an in vitro snRNP nuclear import system using digitonin permeabilized somatic cells supplemented with cytosolic extracts. As model karyophiles we used digoxygenin labelled U1 snRNPs or fluorescein labelled U2 snRNPs. In vitro nuclear import of snRNPs is inhibited by anti-pore component antibodies, consistent with transport occurring through nuclear pores. This import requires ATP, cytosolic factors and a nuclear localization signal (NLS). SnRNP nuclear accumulation is saturable and distinct from protein transport. Nuclear import of snRNPs, in permeabilized NRK cells supplemented with somatic cell cytosol, requires the same NLS structures as those identified in micro-injected mammalian cells. In contrast to the situation in Xenopus oocytes, the m3G-cap is not required for in vitro nuclear import of U1 and U2 snRNPs in somatic cells. Instead, assembly of the Sm-core domain is both necessary and sufficient to mediate snRNP nuclear targeting. Interestingly, when the in vitro system was provided with cytosol from Xenopus oocytes instead of somatic cells, U1 and U2 snRNP nuclear import was provided with cytosol from Xenopus oocytes instead of somatic cells, U1 and U2 snRNP nuclear import was m3G-cap dependent. These results indicate that soluble cytosolic factors mediate the differential m3G-cap dependence of U1 and U2 snRNP nuclear import in somatic cells and oocytes. We also demonstrate the existence of a soluble cytosolic factor whose interaction with the U2 snRNP m3G-cap is both saturable and essential for U2 snRNP nuclear import in Xenopus oocytes.

11] Nuclear protein import using digitonin-permeabilized cells

The nuclear import system described in this chapter is simple, efficient, and can be tailored to the needs of the individual laboratory. A wide range of different cell types can be used as the source of permeabilized cells or cytosol. The system is particularly useful for purifying and characterizing cytosolic factors that have a role in nuclear import, partly because it does not involve the complications of import assays that rely on nuclear assembly. In addition to being useful for studying nucleocytoplasmic transport, digitonin-permeabilized cells are also useful for studying a number of transport phenomena related to the secretory pathway. As digitonin-permeabilized cells retain a largely intact cytoarchitecture, morphological visualization of the movement of transported proteins between different membrane compartments can be greatly facilitated. Transport of a viral membrane protein from the endoplasmic reticulum (ER) to the Golgi has been successfully visualized by this approach.