Importin 7 and importin alpha/importin beta are nuclear import receptors for the glucocorticoid receptor

The vertebrate glucocorticoid receptor (GR) is cytoplasmic without hormone and localizes to the nucleus after hormone binding. GR has two nuclear localization signals (NLS): NL1 is similar in sequence to the SV40 NLS; NL2 is poorly defined, residing in the ligand-binding domain. We found that GR displayed similar hormone-regulated compartmentalization in Saccharomyces cerevisiae and required the Sxm1 nuclear import receptor for NL2-mediated import. Two metazoan homologues of Sxm1, importin 7 and importin 8, bound both NL1 and NL2, whereas importin alpha selectively bound NL1. In an in vitro nuclear import assay, both importin 7 and the importin alpha-importin beta heterodimer could import a GR NL1 fragment. Under these conditions, full-length GR localized to nuclei in the presence but not absence of an unidentified component in cell extracts. Interestingly, importin 7, importin 8, and importin alpha bound GR even in the absence of hormone; thus, hormonal control of localization is exerted at a step downstream of import receptor binding.     

Protracted nuclear export of glucocorticoid receptor limits its turnover and does not require the exportin 1/CRM1-directed nuclear export pathway

Glucocorticoid receptors (GRs) are shuttling proteins, yet they preferentially accumulate within either the cytoplasmic or nuclear compartment when overall rates of nuclear import or export, respectively, are limiting. Hormone binding releases receptors from stable heteromeric complexes that restrict their interactions with soluble nuclear import factors and contribute to their cytoplasmic retention. Although hormone dissociation leads to the rapid release of GRs from chromatin, unliganded nuclear receptors are delayed in their export. We have used a chimeric GR that contains a heterologous, leucine-rich nuclear export signal sequence (NES) to assess the consequences of accelerated receptor nuclear export. Leucine-rich NESs utilize the exportin 1/CRM1-dependent nuclear export pathway, which can be blocked by leptomycin B (LMB). The fact that rapid nuclear export of the NES-GR chimera, but not the protracted export of wild-type GR, is sensitive to LMB, suggests that GR does not require the exportin 1/CRM1 pathway to exit the nucleus. Despite its more rapid export, the NES-GR chimera appears indistinguishable from wild-type GR in its transactivation activity in transiently transfected cells. However, accelerated nuclear export of the NES-GR chimera is associated with an increased rate of hormone-dependent down-regulation. The increase in NES-GR down-regulation is overcome by LMB treatment, thereby confirming the connection between receptor nuclear export and down-regulation. Given the presence of a nuclear recycling pathway for GR, the protracted rate of receptor nuclear export may increase the efficiency of biological responses to secondary hormone challenges by limiting receptor down-regulation and hormone desensitization.     

Cse1p Is Involved in Export of Yeast Importin α from the Nucleus

Proteins bearing a nuclear localization signal (NLS) are targeted to the nucleus by the heterodimeric transporter importin. Importin α binds to the NLS and to importin β, which carries it through the nuclear pore complex (NPC). Importin disassembles in the nucleus, evidently by binding of RanGTP to importin β. The importin subunits are exported separately. We investigated the role of Cse1p, the Saccharomyces cerevisiae homologue of human CAS, in nuclear export of Srp1p (yeast importin α). Cse1p is located predominantly in the nucleus but also is present in the cytoplasm and at the NPC. We analyzed the in vivo localization of the importin subunits fused to the green fluorescent protein in wild-type and cse1-1 mutant cells. Srp1p but not importin β accumulated in nuclei of cse1-1 mutants, which are defective in NLS import but not defective in NLS-independent import pathways. Purified Cse1p binds with high affinity to Srp1p only in the presence of RanGTP. The complex is dissociated by the cytoplasmic RanGTP-binding protein Yrb1p. Combined with the in vivo results, this suggests that a complex containing Srp1p, Cse1p, and RanGTP is exported from the nucleus and is subsequently disassembled in the cytoplasm by Yrb1p. The formation of the trimeric Srp1p-Cse1p-RanGTP complex is inhibited by NLS peptides, indicating that only NLS-free Srp1p will be exported to the cytoplasm.     

Separate nuclear import pathways converge on the nucleoporin Nup153 and can be dissected with dominant-negative inhibitors

Background: Proteins generally enter or exit the nucleus as cargo of one of a small family of import and export receptors. These receptors bear distant homology to importin β, a subunit of the receptor for proteins with classical nuclear localisation sequences (NLSs). To understand the mechanism of nuclear transport, the next question involves identifying the nuclear pore proteins that interact with the different transport receptors as they dock at the pore and translocate through it.Results: Two pathways of nuclear import were found to intersect at a single nucleoporin, Nup153, localized on the intranuclear side of the nuclear pore. Nup153 contains separate binding sites for importin α/β, which mediates classical NLS import, and for transportin, which mediates import of different nuclear proteins. Strikingly, a Nup153 fragment containing the importin β binding site acted as a dominant-negative inhibitor of NLS import, with no effect on transportin-mediated import. Conversely, a Nup153 fragment containing the transportin binding site acted as a strong dominant-negative inhibitor of transportin import, with no effect on classical NLS import. The interaction of transportin with Nup153 could be disrupted by a non-hydrolyzable form of GTP or by a GTPase-deficient mutant of Ran, and was not observed if transportin carried cargo. Neither Nup153 fragment affected binding of the export receptor Crm1 at the nuclear rim.Conclusions: Two nuclear import pathways, mediated by importin β and transportin, converge on a single nucleoporin, Nup153. Dominant-negative fragments of Nup153 can now be used to distinguish different nuclear import pathways and, potentially, to dissect nuclear export.     

Major Binding Sites for the Nuclear Import Receptor Are the Internal Nucleoporin Nup153 and the Adjacent Nuclear Filament Protein Tpr

A major question in nuclear import concerns the identity of the nucleoporin(s) that interact with the nuclear localization sequences (NLS) receptor and its cargo as they traverse the nuclear pore. Ligand blotting and solution binding studies of isolated proteins have attempted to gain clues to the identities of these nucleoporins, but the studies have from necessity probed binding events far from an in vivo context. Here we have asked what binding events occur in the more physiological context of a Xenopus egg extract, which contains nuclear pore subcomplexes in an assembly competent state. We have then assessed our conclusions in the context of assembled nuclear pores themselves. We have used immunoprecipitation to identify physiologically relevant complexes of nucleoporins and importin subunits. In parallel, we have demonstrated that it is possible to obtain immunofluorescence localization of nucleoporins to subregions of the nuclear pore and its associated structures. By immunoprecipitation, we find the nucleoporin Nup153 and the pore-associated filament protein Tpr, previously shown to reside at distinct sites on the intranuclear side of assembled pores, are each in stable subcomplexes with importin α and β in Xenopus egg extracts. Importin subunits are not in stable complexes with nucleoporins Nup62, Nup93, Nup98, or Nup214/CAN, either in egg extracts or in extracts of assembled nuclear pores. In characterizing the Nup153 complex, we find that Nup153 can bind to a complete import complex containing importin α, β, and an NLS substrate, consistent with an involvement of this nucleoporin in a terminal step of nuclear import. Importin β binds directly to Nup153 and in vitro can do so at multiple sites in the Nup153 FXFG repeat region. Tpr, which has no FXFG repeats, binds to importin β and to importin α/β heterodimers, but only to those that do not carry an NLS substrate. That the complex of Tpr with importin β is fundamentally different from that of Nup153 is additionally demonstrated by the finding that recombinant β or β_45–462 fragment freely exchanges with the endogenous importin β/Nup153 complex, but cannot displace endogenous importin β from a Tpr complex. However, the GTP analogue GMP-PNP is able to disassemble both Nup153– and Tpr–importin β complexes. Importantly, analysis of extracts of isolated nuclei indicates that Nup153– and Tpr–importin β complexes exist in assembled nuclear pores. Thus, Nup153 and Tpr are major physiological binding sites for importin β. Models for the roles of these interactions are discussed.     

Identification of Importin α 7 Specific Transport Cargoes Using a Proteomic Screening Approach

The importin α:β complex is responsible for the nuclear import of proteins bearing classical nuclear localization signals. In mammals, several importin α subtypes are known to exist that are suggested to have individual functions. Importin α 7 was shown to play a crucial role in early embryonic development in mice. Embryos from importin α 7–depleted females stop at the two-cell stage and show disturbed zygotic genome activation. As there is evidence that individual importin α subtypes possess cargo specificities, we hypothesized that importin α 7 binds a unique set of intracellular proteins. With the use of a collection of in vitro and in vivo binding assays, importin α 7 interaction partners were identified that differed from proteins found to bind to importin α 2 and 3. One of the proteins preferentially binding importin α 7 was the maternal effect protein Brg1. However, Brg1 was localized in oocyte nuclei in importin α 7–deficient embryos, albeit in reduced amounts, suggesting additional modes of nuclear translocation of this factor. An additional SILAC-based screening approach identified Ash2l, Chd3, Mcm3, and Smarcc1, whose nuclear import seems to be disturbed in importin α 7–deficient fibroblasts.The nuclear compartment is spatially separated from the cytoplasm by the nuclear envelope. The nuclear pores, which are embedded in the nuclear membrane, are the gateway for intracellular molecules that must traverse the nuclear envelope to enter or exit the nucleus. Small molecules can pass through the nuclear pores via passive diffusion; molecules weighing more than 40 kDa must be transported actively through the nuclear pore (1). According to the transport direction, carrier proteins that mediate these nuclear trafficking events are called importins or exportins, known collectively as karyopherins. Nuclear trafficking mediated by the importin α:importin β heterodimer is perhaps the best characterized nuclear import pathway. Here, importin α (or karyopherin α) serves as an adaptor molecule that binds cargoes containing classical nuclear localization signals (NLSs)¹ in their primary amino acid sequence. Upon cargo binding, importin α binds to importin β (karyopherin β 1), forming a trimeric transport complex that moves through the nuclear pore into the nucleus. In the nucleoplasm, RanGTP binds to importin β, leading to a conformational change in importin β and to the dissociation of the transport complex. The cargo is released to the nucleoplasm and can fulfill its function, whereas importins α and β are recycled back to the cytoplasm, where they can perform the next round of import (for reviews, see Refs. 2–4).There is only one importin α and one importin β protein present in yeast. However, multiple importin α isoforms, each transcribed from a different gene, are found in higher eukaryotes. Three importin α subtypes have been identified in Caenorhabditis elegans and Drosophila melanogaster, and up to seven importin α isoforms have been identified in mammals (5–7). These importin α isoforms can be grouped into three subfamilies based on sequence similarity (8). Little is known as to why multiple importin α isoforms exist in higher eukaryotes, but there is evidence that each importin α subtype has a tissue-specific expression pattern and distinct cargoes containing classical NLSs (9–12).We have recently shown that importin α 7 is required for embryonic development in mice (13). Oocytes from importin α 7 null females ovulate but produce embryos that fail to develop beyond the two-cell stage. To elucidate the molecular mechanisms behind this phenotype, we were especially interested in the identification of importin α 7 binding partners. Therefore, the aim of this study was to combine in vivo and in vitro screens to identify an importin α 7 subtype-specific cargo set. Through GST pull-down and co-immunoprecipitation experiments, we were able to identify a unique set of importin α 7 interaction partners that are involved in RNA processing, chromosome organization, and chromatin modification. Among them we found Brahma-related gene 1 (Brg1), also known as smarca4 or Baf190a, a known maternal effect protein required for early development in the mouse (14). An additional approach utilizing stable isotope labeling by amino acids in cell culture (SILAC) was used to further narrow down the list of potential importin α 7 specific cargoes. Hereby, we identified Ash2l, Chd3, Mcm3, Mcm5, and Smarcc1, whose nuclear levels were clearly decreased in importin α 7–deficient fibroblasts.     

Nuclear Trafficking of the Rabies Virus Interferon Antagonist P-Protein Is Regulated by an Importin-Binding Nuclear Localization Sequence in the C-Terminal Domain

Rabies virus P-protein is expressed as five isoforms (P1-P5) which undergo nucleocytoplasmic trafficking important to roles in immune evasion. Although nuclear import of P3 is known to be mediated by an importin (IMP)-recognised nuclear localization sequence in the N-terminal region (N-NLS), the mechanisms underlying nuclear import of other P isoforms in which the N-NLS is inactive or has been deleted have remained unresolved. Based on the previous observation that mutation of basic residues K214/R260 of the P-protein C-terminal domain (P-CTD) can result in nuclear exclusion of P3, we used live cell imaging, protein interaction analysis and in vitro nuclear transport assays to examine in detail the nuclear trafficking properties of this domain. We find that the effect of mutation of K214/R260 on P3 is largely dependent on nuclear export, suggesting that nuclear exclusion of mutated P3 involves the P-CTD-localized nuclear export sequence (C-NES). However, assays using cells in which nuclear export is pharmacologically inhibited indicate that these mutations significantly inhibit P3 nuclear accumulation and, importantly, prevent nuclear accumulation of P1, suggestive of effects on NLS-mediated import activity in these isoforms. Consistent with this, molecular binding and transport assays indicate that the P-CTD mediates IMPα2/IMPβ1-dependent nuclear import by conferring direct binding to the IMPα2/IMPβ1 heterodimer, as well as to a truncated form of IMPα2 lacking the IMPβ-binding autoinhibitory domain (ΔIBB-IMPα2), and IMPβ1 alone. These properties are all dependent on K214 and R260. This provides the first evidence that P-CTD contains a genuine IMP-binding NLS, and establishes the mechanism by which P-protein isoforms other than P3 can be imported to the nucleus. These data underpin a refined model for P-protein trafficking that involves the concerted action of multiple NESs and IMP-binding NLSs, and highlight the intricate regulation of P-protein subcellular localization, consistent with important roles in infection.     

Plasmodium falciparum signal recognition particle components and anti-parasitic effect of ivermectin in blocking nucleo-cytoplasmic shuttling of SRP

Signal recognition particle (SRP) is a ubiquitous ribonucleoprotein complex that targets proteins to endoplasmic reticulum (ER) in eukaryotes. Here we report that Plasmodium falciparum SRP is composed of six polypeptides; SRP9, SRP14, SRP19, SRP54, SRP68 and SRP72 and a 303nt long SRP RNA. We generated four transgenic parasite lines expressing SRP-GFP chimeric proteins and co-localization studies showed the nucleo-cytoplasmic localization for these proteins. The evaluation of the effect of known SRP and nuclear import/export inhibitors on P. falciparum revealed that ivermectin, an inhibitor of importin α/β mediated nuclear import inhibited the nuclear import of PfSRP polypeptides at submicromolar concentration, thereby killing the parasites. These findings provide insights into dynamic structure of P. falciparum SRP and also raise the possibility that ivermectin could be used in combination with other antimalarial agents to control the disease.     

Glucocorticoid receptor concentration and the ability to dimerize influence nuclear translocation and distribution

Glucocorticoid receptor (GR) concentrations and the ability of the GR to dimerize are factors which influence sensitivity to glucocorticoids. Upon glucocorticoid binding, the GR is actively transported into the nucleus, a crucial step in determining GR function. We examined the effects of GR concentration and the ability to dimerize on GR nuclear import, export and nuclear distribution using both live cell microscopy of GFP-tagged GR and immunofluorescence of untagged GR, with both wild type GR (GRwt) and dimerization deficient GR (GRdim). We found that the observed rate of GR nuclear import increases significantly at higher GR concentrations, at saturating concentrations of dexamethasone (10^?6 M) using GFP-tagged GR, while with untagged GR it is only discernable at sub-saturating ligand concentrations (10^?10–10^?9 M). Loss of dimerization results in a slower observed rate of nuclear import (2.5- to 3.3-fold decrease for GFP-GRdim) as well as a decreased extent of GR nuclear localization (18–27% decrease for untagged GRdim). These results were linked to an increased rate of GR export at low GR concentrations (1.4- to 1.6-fold increase for untagged GR) and where GR dimerization is abrogated (1.5- to 1.7-fold increase for GFP-GRdim). Furthermore, GR dimerization was shown to be required for the appearance of discrete GC-dependent GR nuclear foci, the loss of which may explain the increased rate of GR export for the GRdim. The reduction in the observed rate of nuclear import and increased rate of nuclear export displayed at low GR concentrations and by the GRdim could explain the lowered glucocorticoid response under these conditions.     

Transportin acts to regulate mitotic assembly events by target binding rather than Ran sequestration

The nuclear import receptors importin β and transportin play a different role in mitosis: both act phenotypically as spatial regulators to ensure that mitotic spindle, nuclear membrane, and nuclear pore assembly occur exclusively around chromatin. Importin β is known to act by repressing assembly factors in regions distant from chromatin, whereas RanGTP produced on chromatin frees factors from importin β for localized assembly. The mechanism of transportin regulation was unknown. Diametrically opposed models for transportin action are as follows: 1) indirect action by RanGTP sequestration, thus down-regulating release of assembly factors from importin β, and 2) direct action by transportin binding and inhibiting assembly factors. Experiments in Xenopus assembly extracts with M9M, a superaffinity nuclear localization sequence that displaces cargoes bound by transportin, or TLB, a mutant transportin that can bind cargo and RanGTP simultaneously, support direct inhibition. Consistently, simple addition of M9M to mitotic cytosol induces microtubule aster assembly. ELYS and the nucleoporin 107–160 complex, components of mitotic kinetochores and nuclear pores, are blocked from binding to kinetochores in vitro by transportin, a block reversible by M9M. In vivo, 30% of M9M-transfected cells have spindle/cytokinesis defects. We conclude that the cell contains importin β and transportin “global positioning system”or “GPS” pathways that are mechanistically parallel.     

Nuclear localization of human DNA mismatch repair protein exonuclease 1 (hEXO1)

Human exonuclease 1 (hEXO1) is implicated in DNA mismatch repair (MMR) and mutations in hEXO1 may be associated with hereditary nonpolyposis colorectal cancer (HNPCC). Since the subcellular localization of MMR proteins is essential for proper MMR function, we characterized possible nuclear localization signals (NLSs) in hEXO1. Using fluorescent fusion proteins, we show that the sequence ⁴¹⁸KRPR⁴²¹, which exhibit strong homology to other monopartite NLS sequences, is responsible for correct nuclear localization of hEXO1. This NLS sequence is located in a region that is also required for hEXO1 interaction with hMLH1 and we show that defective nuclear localization of hEXO1 mutant proteins could be rescued by hMLH1 or hMSH2. Both hEXO1 and hMLH1 form complexes with the nuclear import factors importin β/α1,3,7 whereas hMSH2 specifically recognizes importin β/α3. Taken together, we infer that hEXO1, hMLH1 and hMSH2 form complexes and are imported to the nucleus together, and that redundant NLS import signals in the proteins may safeguard nuclear import and thereby MMR activity.     

Overexpression of the Nucleoporin CAN/NUP214 Induces Growth Arrest, Nucleocytoplasmic Transport Defects, and Apoptosis

The human CAN gene was first identified as a target of t(6;9)(p23;q34), associated with acute myeloid leukemia and myelodysplastic syndrome, which results in the expression of a DEK-CAN fusion gene. CAN, also called NUP214, is a nuclear pore complex (NPC) protein that contains multiple FG-peptide sequence motifs. It interacts at the NPC with at least two other proteins, the nucleoporin NUP88 and hCRM1 (exportin 1), which was recently shown to function as a nuclear export receptor. Depletion of CAN in knockout mouse embryonic cells results in cell cycle arrest in G₂, followed by inhibition of nuclear protein import and a block of mRNA export. We overexpressed CAN and DEK-CAN in U937 myeloid precursor cells. DEK-CAN expression did not interfere with terminal myeloid differentiation of U937 cells, whereas CAN-overexpressing cells arrested in G₀, accumulated mRNA in their nuclei, and died in an apoptotic manner. Interestingly, we found that hCRM1 and import factor p97/importin β colocalized with the ectopically expressed CAN protein, resulting in depletion of both factors from the NPC. Overexpression of the C-terminal FG-repeat region of CAN, which contains the binding site for hCRM1, caused sequestering of hCRM1 in the nucleoplasm and was sufficient to inhibit cell growth and to induce apoptosis. These results confirm that CAN plays a crucial role in nucleocytoplasmic transport and imply an essential role for hCRM1 in cell growth and survival.     

Hormonal regulation of the nuclear localization signals of the human glucocorticosteroid receptor.

Nuclear localization of the rat glucocorticosteroid receptor (rGR) transiently expressed in COS-7 cells appears to be mediated by two nuclear localization signals, NL1 and NL2, in a hormone-dependent mechanism. We investigated the intracellular distribution of the human GR (hGR) expressed in COS-7 cells, by a different immunohistochemical technique involving immunostaining of cell pellet sections, thus avoiding the use of cell permeabilizing agents and allowing rigorous comparison between successive experiments. With a large set of hGR mutants, we could define determinants of the hGR nuclear localization and compare them with those previously reported for rGR. Our study demonstrated two hormone-dependent nuclear localization signals. NL1 activity, overlapping the DNA-binding domain (DBD)-hinge boundary, was repressed by the unliganded ligand-binding domain (LBD), even if the repressed NL1 retained a residual potency to target hGR in the nucleus. Structure/function analysis suggested a bipartite structure of NL1, analogous to that of other nuclear targeting signals (the carboxy-terminal part of DBD between amino acids 478 and 487 and the beginning of the hinge region which includes a basic amino acid stretch between 491 and 498). Upon hormone binding, NL2, located in the LBD, was activated, but was unable by itself to sustain full nuclear localization, which required the derepressed NL1 activity. Only two sequences in the LBD, localized between amino acids 600 and 626 and from amino acid 696 up to the carboxyl-terminal amino acid 777, respectively, were found to inhibit NL1 activity. As previously reported, efficient nuclear retention, mandatory for gene expression, did not required DNA-binding activity. The controversial intracellular localization of the unliganded form of hGR and the role of hsp90 in cytoplasmic localization are further discussed.     

The carboxyl terminus of RNA helicase A contains a bidirectional nuclear transport domain

Human RNA helicase A was recently identified to be a shuttle protein which interacts with the constitutive transport element (CTE) of type D retroviruses. Here we show that a domain of 110 amino acids at the carboxyl terminus of helicase A is both necessary and sufficient for nuclear localization as well as rapid nuclear export of glutathione S-transferase fusion proteins. The import and export activities of this domain overlap but are separable by point mutations. This bidirectional nuclear transport domain (NTD) has no obvious sequence homology to previously identified nuclear import or export signals. However, the Ran-dependent nuclear import of NTD was efficiently competed by excess amounts of the nuclear localization signal (NLS) peptide from simian virus 40 large T antigen, suggesting that import is mediated by the classical NLS pathway. The nuclear export pathway accessed by NTD is insensitive to leptomycin B and thus is distinct from the leucine-rich nuclear export signal pathway mediated by CRM1.