Showing papers on "Importin published in 2016"
TL;DR: Importin α performs the indispensable role of ferrying proteins from the cytoplasm into the nucleus with a transport carrier, importin β1 as mentioned in this paper, and has been shown to serve non-transport functions.
Abstract: Importin α performs the indispensable role of ferrying proteins from the cytoplasm into the nucleus with a transport carrier, importin β1. Mammalian cells from mouse or human contain either six or seven importin α subtypes, respectively, each with a tightly regulated expression. Therefore, the combination of subtype expression in a cell defines distinct signaling pathways to achieve progressive changes in gene expression essential for cellular events, such as differentiation. Recent studies reveal that, in addition to nucleocytoplasmic transport, importin αs also serve non-transport functions. In this review, we first discuss the physiological significance of importin α as a nuclear transport regulator, and then focus on the functional diversities of importin αs based on their specific subcellular and cellular localizations, such as the nucleus and plasma membrane. These findings enrich our knowledge of how importin αs actively contribute to various cellular events.
107 citations
TL;DR: Targeting the key mediators of nucleocytoplasmic transport in cancer cells represents a novel strategy in cancer intervention with the potential to significantly affect outcomes.
105 citations
TL;DR: The RanGTP system has moonlighting functions exerting a spatial control over protein interactions that drive specific functions depending on the cellular context, and the recent advances made through the characterization of its mitotic targets are focused on.
Abstract: The small GTPase Ran regulates the interaction of transport receptors with a number of cellular cargo proteins. The high affinity binding of the GTP-bound form of Ran to import receptors promotes cargo release, whereas its binding to export receptors stabilizes their interaction with the cargo. This basic mechanism linked to the asymmetric distribution of the two nucleotide-bound forms of Ran between the nucleus and the cytoplasm generates a switch like mechanism controlling nucleo-cytoplasmic transport. Since 1999, we have known that after nuclear envelope breakdown (NEBD) Ran and the above transport receptors also provide a local control over the activity of factors driving spindle assembly and regulating other aspects of cell division. The identification and functional characterization of RanGTP mitotic targets is providing novel insights into mechanisms essential for cell division. Here we review our current knowledge on the RanGTP system and its regulation and we focus on the recent advances made through the characterization of its mitotic targets. We then briefly review the novel functions of the pathway that were recently described. Altogether, the RanGTP system has moonlighting functions exerting a spatial control over protein interactions that drive specific functions depending on the cellular context.
103 citations
TL;DR: It is shown that motor-dependent mRNA localization regulates neuronal growth and cycling cell size and subcellular sequestration of nucleolin or importin β1 enhances axonal growth and causes a sub cellular shift in protein synthesis.
57 citations
TL;DR: It is shown that O-GlcNAcylation of Ser389, which resides in the tetratricopeptide repeats, plays an important role in the nuclear localisation of OGT and may explain how OGT, which possesses a NLS, exists in the nucleus and cytosol simultaneously.
Abstract: Nucleocytoplasmic O-GlcNAc transferase (OGT) attaches a single GlcNAc to hydroxyl groups of serine and threonine residues. Although the cellular localisation of OGT is important to regulate a variety of cellular processes, the molecular mechanisms regulating the nuclear localisation of OGT is unclear. Here, we characterised three amino acids (DFP; residues 451-453) as the nuclear localisation signal of OGT and demonstrated that this motif mediated the nuclear import of non-diffusible β-galactosidase. OGT bound the importin α5 protein, and this association was abolished when the DFP motif of OGT was mutated or deleted. We also revealed that O-GlcNAcylation of Ser389, which resides in the tetratricopeptide repeats, plays an important role in the nuclear localisation of OGT. Our findings may explain how OGT, which possesses a NLS, exists in the nucleus and cytosol simultaneously.
47 citations
TL;DR: It is suggested that nuclear entry is important for the prooncogenic activity of eIF4E, at least in this context, and importin 8 is highly elevated in untreated patients with AML, leading to eif4E nuclear accumulation.
Abstract: Regulation of nuclear-cytoplasmic trafficking of oncoproteins is critical for growth homeostasis. Dysregulated trafficking contributes to malignancy, whereas understanding the process can reveal unique therapeutic opportunities. Here, we focus on eukaryotic translation initiation factor 4E (eIF4E), a prooncogenic protein highly elevated in many cancers, including acute myeloid leukemia (AML). Typically, eIF4E is localized to both the nucleus and cytoplasm, where it acts in export and translation of specific methyl 7-guanosine (m7G)–capped mRNAs, respectively. Nuclear accumulation of eIF4E in patients who have AML is correlated with increased eIF4E-dependent export of transcripts encoding oncoproteins. The subcellular localization of eIF4E closely correlates with patients’ responses. During clinical responses to the m7G-cap competitor ribavirin, eIF4E is mainly cytoplasmic. At relapse, eIF4E reaccumulates in the nucleus, leading to elevated eIF4E-dependent mRNA export. We have identified importin 8 as a factor that directly imports eIF4E into the nucleus. We found that importin 8 is highly elevated in untreated patients with AML, leading to eIF4E nuclear accumulation. Importin 8 only imports cap-free eIF4E. Cap-dependent changes to the structure of eIF4E underpin this selectivity. Indeed, m7G cap analogs or ribavirin prevents nuclear entry of eIF4E, which mirrors the trafficking phenotypes observed in patients with AML. Our studies also suggest that nuclear entry is important for the prooncogenic activity of eIF4E, at least in this context. These findings position nuclear trafficking of eIF4E as a critical step in its regulation and position the importin 8–eIF4E complex as a novel therapeutic target.
37 citations
TL;DR: It is indicated that in BCS, nuclear disassembly of the import complex and release of EMG1D86G lead to its nuclear aggregation and degradation, resulting in the reduced nucleolar recruitment of the RNA methyltransferase and defects in the biogenesis of the small ribosomal subunit.
Abstract: Bowen-Conradi syndrome (BCS) is a severe genetic disorder that is characterised by various developmental abnormalities, bone marrow failure and early infant death. This disease is caused by a single mutation leading to the aspartate 86 to glycine (D86G) exchange in the essential nucleolar RNA methyltransferase EMG1. EMG1 is required for the synthesis of the small ribosomal subunit and is involved in modification of the 18S ribosomal RNA. Here, we identify the pre-ribosomal factors NOP14, NOC4L and UTP14A as members of a nucleolar subcomplex that contains EMG1 and is required for its recruitment to nucleoli. The BCS mutation in EMG1 leads to reduced nucleolar localisation, accumulation of EMG1D86G in nuclear foci and its proteasome-dependent degradation. We further show that EMG1 can be imported into the nucleus by the importins (Imp) Impα/β or Impβ/7. Interestingly, in addition to its role in nuclear import, binding of the Impβ/7 heterodimer can prevent unspecific aggregation of both EMG1 and EMG1D86G on RNAs in vitro, indicating that the importins act as chaperones by binding to basic regions of the RNA methyltransferase. Our findings further indicate that in BCS, nuclear disassembly of the import complex and release of EMG1D86G lead to its nuclear aggregation and degradation, resulting in the reduced nucleolar recruitment of the RNA methyltransferase and defects in the biogenesis of the small ribosomal subunit.
37 citations
TL;DR: This study provides novel evidence that functional importin α1 is located at the cell surface, where it accelerates the proliferation of cancer cells.
Abstract: Importin α1 is involved in nuclear import as a receptor for proteins with a classical nuclear localization signal (cNLS). Here, we report that importin α1 is localized to the cell surface in several cancer cell lines and detected in their cultured medium. We also found that exogenously added importin α1 is associated with the cell membrane via interaction with heparan sulfate. Furthermore, we revealed that the cell surface importin α1 recognizes cNLS-containing substrates. More particularly, importin α1 bound directly to FGF1 and FGF2, secreted cNLS-containing growth factors, and addition of exogenous importin α1 enhanced the activation of ERK1/2, downstream targets of FGF1 signalling, in FGF1-stimulated cancer cells. Additionally, anti-importin α1 antibody treatment suppressed the importin α1-FGF1 complex formation and ERK1/2 activation, resulting in decreased cell growth. This study provides novel evidence that functional importin α1 is located at the cell surface, where it accelerates the proliferation of cancer cells.
34 citations
TL;DR: 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.
Abstract: 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.
34 citations
TL;DR: Evidence is provided that the N-terminal 1–55 amino acids of Yorkie, especially Arg-15, were essential for its nuclear localization, and that the upstream component Hippo can inhibit Importin α1-mediated Yorkie nuclear import.
33 citations
TL;DR: It is shown that heregulin-stimulation induces trafficking of phosphorylated ErbB3 from the plasma membrane to the nucleus via a clathrin-independent mechanism, and it is demonstrated that ErBB3 nuclear localization is transient as it is exported out of the nucleus by the nuclear receptor protein crm-1.
TL;DR: The complex structure of SART3 nuclear localization signal (NLS) and importin-α reveals bipartite binding, and removal of Sart3 NLS prevents the entry of USP4 (and USP15) into the nucleus and abrogates the subsequent deubiquitinase activity ofUSP4.
Abstract: Squamous cell carcinoma antigen recognized by T-cells 3 (SART3) is a U4/U6 recycling factor as well as a targeting factor of USP4 and USP15. However, the details of how SART3 recognizes these deubiquitinases and how they get subsequently translocated into the nucleus are not known. Here, we present the crystal structures of the SART3 half-a-tetratricopeptide (HAT) repeat domain alone and in complex with the domain present in ubiquitin-specific protease (DUSP)-ubiquitin-like (UBL) domains of ubiquitin specific protease 4 (USP4). The 12 HAT repeats of SART3 are in two sub-domains (HAT-N and HAT-C) forming a dimer through HAT-C. USP4 binds SART3 at the opposite surface of the HAT-C dimer interface utilizing the β-structured linker between the DUSP and the UBL domains. The binding affinities of USP4 and USP15 to SART3 are 0.9 μM and 0.2 μM, respectively. The complex structure of SART3 nuclear localization signal (NLS) and importin-α reveals bipartite binding, and removal of SART3 NLS prevents the entry of USP4 (and USP15) into the nucleus and abrogates the subsequent deubiquitinase activity of USP4.
TL;DR: In vitro nuclear import assays showed that TRα1 nuclear localization is temperature and energy-dependent and can be reconstituted by the addition of cytosol, suggesting that TR α1 trafficking into the nucleus is mediated by two distinct pathways.
TL;DR: Several lines of evidence now suggest that nuclear trafficking of structural proteins such as nucleocapsid and matrix proteins may be critical to pathogenesis and thus could provide new targets for vaccine development and antiviral therapies.
Abstract: Genome replication and virion production by most negative-sense RNA viruses (NSVs) occurs exclusively in the cytoplasm, but many NSV-expressed proteins undergo active nucleocytoplasmic trafficking via signals that exploit cellular nuclear transport pathways. Nuclear trafficking has been reported both for NSV accessory proteins (including isoforms of the rabies virus phosphoprotein, and V, W and C proteins of paramyxoviruses) and for structural proteins. Trafficking of the former is thought to enable accessory functions in viral modulation of antiviral responses including the type I IFN system, but the intranuclear roles of structural proteins such as nucleocapsid and matrix proteins, which have critical roles in extranuclear replication and viral assembly, are less clear. Nevertheless, nuclear trafficking of matrix protein has been reported to be critical for efficient production of Nipah virus and Respiratory syncytial virus, and nuclear localization of nucleocapsid protein of several morbilliviruses has been linked to mechanisms of immune evasion. Together, these data point to the nucleus as a significant host interface for viral proteins during infection by NSVs with otherwise cytoplasmic life cycles. Importantly, several lines of evidence now suggest that nuclear trafficking of these proteins may be critical to pathogenesis and thus could provide new targets for vaccine development and antiviral therapies.
TL;DR: It is reported for the first time a transport factor that mediates YB-1 nuclear import - transportin-1 - and transport of Yb-1 and its truncated form (P201A/Y202A, 1-219) bearing inactivating mutations in the transportIn-1-dependent nuclear localization signal was practically abolished.
TL;DR: It is reported that redundant pathways contribute to the nuclear import of Rps3, with the classical importin α/β pathway (Kap60/Kap95 in yeast) constituting a main import route.
Abstract: After their cytoplasmic synthesis, ribosomal proteins need to be transported into the nucleus, where they assemble with ribosomal RNA into pre-ribosomal particles. Due to their physicochemical properties, they need protection from aggregation on this path. Newly synthesized ribosomal protein Rps3 forms a dimer that is associated with one molecule of its specific chaperone Yar1. Here we report that redundant pathways contribute to the nuclear import of Rps3, with the classical importin α/β pathway (Kap60/Kap95 in yeast) constituting a main import route. The Kap60/Kap95 heterodimer mediates efficient nuclear import of Rps3 by recognition of an N-terminal monopartite nuclear localization signal (NLS). This Rps3-NLS is located directly adjacent to the Yar1-binding site and, upon binding of Kap60 to Rps3, Yar1 is displaced from the ribosomal protein in vitro. While Yar1 does not directly interact with Kap60 in vitro, affinity purifications of Yar1 and Rps3, however, revealed that Kap60 is present in the Rps3/Yar1 complex in vivo. Indeed we could reconstitute such a protein complex containing Rps3 and both Yar1 and Kap60 in vitro. Our data suggest that binding of Yar1 to one N-domain and binding of Kap60 to the second N-domain of dimerized Rps3 orchestrates import and protection of the ribosomal protein.
TL;DR: It is proposed that actin polymerization drives not only migration of baculovirus through the cytoplasm but also pushes the nucleocapsid through the nuclear pore complex to enter the cell nucleus.
Abstract: The transport of macromolecules into the nucleus is mediated by soluble cellular receptors of the importin β superfamily and requires the Ran-GTPase cycle. Several studies have provided evidence that there are exceptions to this canonical nuclear import pathway. Here, we report a new unconventional nuclear import mechanism exploited by the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). We found that AcMNPV nucleocapsids entered the nucleus of digitonin-permeabilized cells in the absence of exogenous cytosol or under conditions that blocked the Ran-GTPase cycle. AcMNPV contains a protein that activates the Arp2/3 complex and induces actin polymerization at one end of the rod-shaped nucleocapsid. We show that inhibitors of Arp2/3 blocked nuclear import of nucleocapsids in semi-permeabilized cells. Nuclear import of nucleocapsids was also reconstituted in purified nuclei supplemented with G-actin and Arp2/3 under actin polymerization conditions. Thus, we propose that actin polymerization drives not only migration of baculovirus through the cytoplasm but also pushes the nucleocapsid through the nuclear pore complex to enter the cell nucleus. Our findings point to a very distinct role of actin-based motility during the baculovirus infection cycle.
TL;DR: An X-ray crystallographic study of Vpr complexes containing importin-α, a known Vpr binding partner present in host cells, demonstrates the multifunctional aspects that enable Vpr interaction with a variety of cellular proteins.
TL;DR: The data shows that the nuclear-cytoplasmic shuttling machinery plays a critical role mediating the subcellular distribution of Gli2 and the activation of the pathway, but distinct importins likely play a differential role mediates its ciliary and nuclear translocation.
Abstract: Gli2 is the primary transcriptional activator of Hedgehog signalling in mammals. Upon stimulation of the pathway, Gli2 moves into the cilium before reaching the nucleus. However, the mechanisms underlying its entry into the cilium are not completely understood. Since several similarities have been reported between nuclear and ciliary import, we investigated if the nuclear import machinery participates in Gli2 ciliary entry. Here we show that while two conserved classical nuclear localization signals mediate Gli2 nuclear localization via importin (Imp)-α/β1, these sequences are not required for Gli2 ciliary import. However, blocking Imp-mediated transport through overexpression of GTP-locked Ran reduced the percentage of Gli2 positive cilia, an effect that was not explained by increased CRM1-dependent export of Gli2 from the cilium. We explored the participation of Imp-β2 in Gli2 ciliary traffic and observed that this transporter is involved in moving Gli2 into the cilium, as has been described for other ciliary proteins. In addition, our data indicate that Imp-β2 might also collaborate in Gli2 nuclear entry. How does Imp-β2 determine the final destination of a protein that can localize to two distinct subcellular compartments remains an open question. Therefore, our data shows that the nuclear-cytoplasmic shuttling machinery plays a critical role mediating the subcellular distribution of Gli2 and the activation of the pathway, but distinct importins likely play a differential role mediating its ciliary and nuclear translocation.
TL;DR: The findings reported herein disclose the NLS and molecular mechanism for nuclear transport of P-ICP22, these results will uncover new avenues for depicting the biological roles of the P- ICP22 during PRV infection.
Abstract: Herpes simplex virus 1 (HSV-1) ICP22 is a multifunctional protein and important for HSV-1 replication. Pseudorabies virus (PRV) ICP22 (P-ICP22) is a homologue of HSV-1 ICP22 and is reported to be able to selectively modify the transcription of different kinetic classes of PRV genes, however, the subcellular localization, localization signal and molecular determinants for its transport to execute this function is less well understood. In this study, by utilizing live cells fluorescent microscopy, P-ICP22 fused to enhanced yellow fluorescent protein (EYFP) gene was transient expressed in live cells and shown to exhibit a predominantly nucleus localization in the absence of other viral proteins. By transfection of a series of P-ICP22 deletion mutants fused to EYFP, a bona fide nuclear localization signal (NLS) and its key amino acids (aa) of P-ICP22 was, for the first time, determined and mapped to aa 41–60 (PASTPTPPKRGRYVVEHPEY) and aa 49–50 (KR), respectively. Besides, the P-ICP22 was demonstrated to be targeted to the nucleus via Ran-, importin α1-, and α7-mediated pathway. Our findings reported herein disclose the NLS and molecular mechanism for nuclear transport of P-ICP22, these results will uncover new avenues for depicting the biological roles of P-ICP22 during PRV infection.
TL;DR: It is reported for the first time that additional sites modulate nucleocytoplasmic localisation of W and that W undergoes exportin-1 mediated nuclear export, dependent on a leucine at position 174.
TL;DR: By utilizing live cells fluorescent microscopy and co-immunoprecipitation assays, the nuclear import of UL31 was characterized to be dependent on Ran-, importin α1- and transportin-1-mediated pathway, which will promote the understanding of UL 31-mediated biological functions in HSV-1 infection cycle.
Abstract: As an important protein, UL31 has been demonstrated to play multiple roles in herpes simplex virus 1 (HSV-1) replication. Previous studies showed that UL31 predominantly locates in the nucleus in chemical fixed cells and live cells, however, the determining mechanisms for its nuclear translocation is not clear. In the present study, by utilizing live cells fluorescent microscopy and co-immunoprecipitation assays, the nuclear import of UL31 was characterized to be dependent on Ran-, importin α1- and transportin-1-mediated pathway. Therefore, these results will promote the understanding of UL31-mediated biological functions in HSV-1 infection cycle.
TL;DR: The characterized nuclear import pathways of the PRV DNA polymerase catalytic and accessory subunits suggest that UL42 contains an importin α/β-mediated bipartite NLS that transports the viralDNA polymerase holoenzyme into the nucleus in an in vitro expression system.
Abstract: Pseudorabies virus (PRV) DNA replication occurs in the nuclei of infected cells and requires the viral DNA polymerase. The PRV DNA polymerase comprises a catalytic subunit, UL30, and an accessory subunit, UL42, that confers processivity to the enzyme. Its nuclear localization is a prerequisite for its enzymatic function in the initiation of viral DNA replication. However, the mechanisms by which the PRV DNA polymerase holoenzyme enters the nucleus have not been determined. In this study, we characterized the nuclear import pathways of the PRV DNA polymerase catalytic and accessory subunits. Immunofluorescence analysis showed that UL42 localizes independently in the nucleus, whereas UL30 alone predominantly localizes in the cytoplasm. Intriguingly, the localization of UL30 was completely shifted to the nucleus when it was coexpressed with UL42, demonstrating that nuclear transport of UL30 occurs in an UL42-dependent manner. Deletion analysis and site-directed mutagenesis of the two proteins showed that UL42 contains a functional and transferable bipartite nuclear localization signal (NLS) at amino acids 354–370 and that K354, R355, and K367 are important for the NLS function, whereas UL30 has no NLS. Coimmunoprecipitation assays verified that UL42 interacts with importins α3 and α4 through its NLS. In vitro nuclear import assays demonstrated that nuclear accumulation of UL42 is a temperature- and energy-dependent process and requires both importins α and β, confirming that UL42 utilizes the importin α/β-mediated pathway for nuclear entry. In an UL42 NLS-null mutant, the UL42/UL30 heterodimer was completely confined to the cytoplasm when UL42 was coexpressed with UL30, indicating that UL30 utilizes the NLS function of UL42 for its translocation into the nucleus. Collectively, these findings suggest that UL42 contains an importin α/β-mediated bipartite NLS that transports the viral DNA polymerase holoenzyme into the nucleus in an in vitro expression system.
TL;DR: A dynamic scenario emerges, in which RAN is pivotal in linking spatiotemporal control of centrosome regulators to the cell cycle machinery.
Abstract: Growing lines of evidence implicate the small GTPase RAN, its regulators and effectors--predominantly, nuclear transport receptors--in practically all aspects of centrosome biology in mammalian cells. These include duplication licensing, cohesion, positioning, and microtubule-nucleation capacity. RAN cooperates with the protein nuclear export vector exportin 1/CRM1 to recruit scaffolding proteins containing nuclear export sequences that play roles in the structural organization of centrosomes. Together, they also limit centrosome reduplication by regulating the localization of key "licensing" proteins during the centrosome duplication cycle. In parallel, RAN also regulates the capacity of centrosomes to nucleate and organize functional microtubules, and this predominanlty involves importin vectors: many factors regulating microtubule nucleation or function harbor nuclear localization sequences that interact with importin molecules and such interaction inhibits their activity. Active RANGTP binding to importin molecules removes the inhibition and releases microtubule regulatory factors in the free productive form. A dynamic scenario emerges, in which RAN is pivotal in linking spatiotemporal control of centrosome regulators to the cell cycle machinery.
TL;DR: The results establish a mechanistic connection between the calcium regulation of the motor function of myosin IC in the cytoplasm and the induction of its import into the nucleus, consistent with a mechanism whereby calmodulin binding prevents recognition of the nuclear localization sequence by importin β.
Abstract: Myosin IC is a molecular motor involved in intracellular transport, cell motility, and transcription. Its mechanical properties are regulated by calcium via calmodulin binding, and its functions in the nucleus depend on import from the cytoplasm. The import has recently been shown to be mediated by the nuclear localization signal located within the calmodulin-binding domain. In the present paper, it is demonstrated that mutations in the calmodulin-binding sequence shift the intracellular distribution of myosin IC to the nucleus. The redistribution is displayed by isoform B, described originally as the "nuclear myosin," but is particularly pronounced with isoform C, the normally cytoplasmic isoform. Furthermore, experimental elevation of the intracellular calcium concentration induces a rapid import of myosin into the nucleus. The import is blocked by the importin β inhibitor importazole. These findings are consistent with a mechanism whereby calmodulin binding prevents recognition of the nuclear localization sequence by importin β, and the steric inhibition of import is released by cell signaling leading to the intracellular calcium elevation. The results establish a mechanistic connection between the calcium regulation of the motor function of myosin IC in the cytoplasm and the induction of its import into the nucleus. © 2016 Wiley Periodicals, Inc.
TL;DR: By including interactions involving importin-α and exportin they bring the modelling of spatiotemporal kinetics of transcription factors to a more mechanistic level and reduce the effective relaxation time of the NF-κB regulatory pathway.
Abstract: Importins and exportins influence gene expression by enabling nucleocytoplasmic shuttling of transcription factors. A key transcription factor of innate immunity, NF-κB, is sequestered in the cytoplasm by its inhibitor, IκBα, which masks nuclear localization sequence of NF-κB. In response to TNFα or LPS, IκBα is degraded, which allows importins to bind NF-κB and shepherd it across nuclear pores. NF-κB nuclear activity is terminated when newly synthesized IκBα enters the nucleus, binds NF-κB and exportin which directs the complex to the cytoplasm. Although importins/exportins are known to regulate spatiotemporal kinetics of NF-κB and other transcription factors governing innate immunity, the mechanistic details of these interactions have not been elucidated and mathematically modelled. Based on our quantitative experimental data, we pursue NF-κB system modelling by explicitly including NF-κB–importin and IκBα–exportin binding to show that the competition between importins and IκBα enables NF-κB nuclear translocation despite high levels of IκBα. These interactions reduce the effective relaxation time and allow the NF-κB regulatory pathway to respond to recurrent TNFα pulses of 45-min period, which is about twice shorter than the characteristic period of NF-κB oscillations. By stochastic simulations of model dynamics we demonstrate that randomly appearing, short TNFα pulses can be converted to essentially digital pulses of NF-κB activity, provided that intervals between input pulses are not shorter than 1 h. By including interactions involving importin-α and exportin we bring the modelling of spatiotemporal kinetics of transcription factors to a more mechanistic level. Basing on the analysis of the pursued model we estimated the information transmission rate of the NF-κB pathway as 1 bit per hour. This article was reviewed by Marek Kimmel, James Faeder and William Hlavacek.
01 Jun 2016
TL;DR: The importin-β structures obtained exhibit significant conformational changes and suggest an influence of the polarity of the environment, resulting in an extended conformation in the PEG condition.
Abstract: In eukaryotic cells, the exchange of macromolecules between the nucleus and cytoplasm is highly selective and requires specialized soluble transport factors. Many of them belong to the importin-β superfamily, the members of which share an overall superhelical structure owing to the tandem arrangement of a specific motif, the HEAT repeat. This structural organization leads to great intrinsic flexibility, which in turn is a prerequisite for the interaction with a variety of proteins and for its transport function. During the passage from the aqueous cytosol into the nucleus, the receptor passes the gated channel of the nuclear pore complex filled with a protein meshwork of unknown organization, which seems to be highly selective owing to the presence of FG-repeats, which are peptides with hydrophobic patches. Here, the structural changes of free importin-β from a single organism, crystallized in polar (salt) or apolar (PEG) buffer conditions, are reported. This allowed analysis of the structural changes, which are attributable to the surrounding milieu and are not affected by bound interaction partners. The importin-β structures obtained exhibit significant conformational changes and suggest an influence of the polarity of the environment, resulting in an extended conformation in the PEG condition. The significance of this observation is supported by SAXS experiments and the analysis of other crystal structures of importin-β deposited in the Protein Data Bank.
TL;DR: Data demonstrate that DLK exerts distinct functions, depending on its subcellular localization and thus provide a novel level of regulating DLK action, and the prevention of the nuclear localization of DLK as induced by prediabetic signals with consecutive suppression of β-cell apoptosis might constitute a novel target in the therapy of diabetes mellitus.
TL;DR: To gain insight into the structural basis for the nuclear import process for the XPG protein, crystal structures of complexes formed by the Impα and peptides corresponding to both putative nuclear localization signal (NLS) sequences were solved and isothermal titration calorimetry assays demonstrated that XPG1 and XPG2 peptides bind to two separate binding sites, but with high affinity to the major NLS-binding site of the Imp α.
TL;DR: The concept that basal nucleocytoplasmic shuttling of STAT3 is different from active cytokine-induced nuclear import and does not require conserved N- or SH2-terminal domains, preformed dimer formation and GAS-element-specific DNA recognition is supported.