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Karyopherin binding interactions and nuclear import mechanism of nuclear pore complex protein Tpr
Iris Ben-Efraim, Phyllis D Frosst, Larry Gerace
BMC Cell Biology , 2009, DOI: 10.1186/1471-2121-10-74
Abstract: We found that Tpr binds strongly and specifically to importin α, importin β, and a CRM1 containing trimeric export complex, and that the binding sites for importins α and β are distinct. We also determined that the nuclear import of Tpr is dependent on cytosolic factors and energy and is efficiently mediated by the importin α/β import pathway.Based on the binding and nuclear import assays, we propose that Tpr is imported into the nucleus by the importin α/β heterodimer. In addition, we suggest that Tpr can serve as a nucleoporin binding site for importin β during import of importin β cargo complexes and/or importin β recycling. Our finding that Tpr bound preferentially to CRM1 in an export complex strengthens the notion that Tpr is involved in protein export.Molecules are transported between the cytoplasm and the nucleus through nuclear pore complexes (NPCs), massive proteinaceous structures that span the double membrane of the nuclear envelope (NE). Molecules smaller than ~20-40 kDa in size can passively diffuse through the NPC. However most protein, and nucleic acid is transported by receptor and energy dependent mechanisms (reviewed in [5-8]).Nucleocytoplasmic transport is mediated by shuttling transport receptors termed karyopherins or importins/exportins (reviewed in [5,7]). In the extensively studied classical nuclear import pathway, cargoes carrying a basic amino acid-rich nuclear localization sequence (NLS) bind to the adaptor importin a, which in turn associates with the import receptor importin β that mediates transport into the nucleus. A second class of import cargo directly binds to importin β in the absence of an adaptor. In the classical nuclear export pathway, cargoes carrying a leucine-rich nuclear export signal (NES) bind to the exportin CRM1 together with RanGTP to be transported out of the nucleus.The small GTPase Ran, which binds directly to both importins and exportins, plays a key role in determining the directionality of nuclear transport. Th
Silencing Nuclear Pore Protein Tpr Elicits a Senescent-Like Phenotype in Cancer Cells  [PDF]
Brigitte David-Watine
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0022423
Abstract: Background Tpr is a large coiled-coil protein located in the nuclear basket of the nuclear pore complex for which many different functions were proposed from yeast to human. Methodology/Principal Findings Here we show that depletion of Tpr by RNA interference triggers G0–G1 arrest and ultimately induces a senescent-like phenotype dependent on the presence of p53. We also found that Tpr depletion impairs the NES [nuclear export sequence]-dependent nuclear export of proteins and causes partial co-depletion of Nup153. In addition Tpr depletion impacts on level and function of the SUMO-protease SENP2 thus affecting SUMOylation regulation at the nuclear pore and overall SUMOylation in the cell. Conclusions Our data for the first time provide evidence that a nuclear pore component plays a role in controlling cellular senescence. Our findings also point to new roles for Tpr in the regulation of SUMO-1 conjugation at the nuclear pore and directly confirm Tpr involvement in the nuclear export of NES-proteins.
Localization of Nucleoporin Tpr to the Nuclear Pore Complex Is Essential for Tpr Mediated Regulation of the Export of Unspliced RNA  [PDF]
Kalpana Rajanala, Vinay Kumar Nandicoori
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0029921
Abstract: Nucleoporin Tpr is a component of the nuclear pore complex (NPC) that localizes exclusively to intranuclear filaments. Tpr functions as a scaffolding element in the nuclear phase of the NPC and plays a role in mitotic spindle checkpoint signalling. Export of intron-containing mRNA in Mason Pfizer Monkey Virus is regulated by direct interaction of cellular proteins with the cis-acting Constitutive Transport Element (CTE). In mammalian cells, the transport of Gag/Pol-CTE reporter construct is not very efficient, suggesting a regulatory mechanism to retain this unspliced RNA. Here we report that the knockdown of Tpr in mammalian cells leads to a drastic enhancement in the levels of Gag proteins (p24) in the cytoplasm, which is rescued by siRNA resistant Tpr. Tpr's role in the retention of unspliced RNA is independent of the functions of Sam68 and Tap/Nxf1 proteins, which are reported to promote CTE dependent export. Further, we investigated the possible role for nucleoporins that are known to function in nucleocytoplasmic transport in modulating unspliced RNA export. Results show that depletion of Nup153, a nucleoporin required for NPC anchoring of Tpr, plays a role in regulating the export, while depletion of other FG repeat-containing nucleoporins did not alter the unspliced RNA export. Results suggest that Tpr and Nup153 both regulate the export of unspliced RNA and they are most likely functioning through the same pathway. Importantly, we find that localization of Tpr to the NPC is necessary for Tpr mediated regulation of unspliced RNA export. Collectively, the data indicates that perinuclear localization of Tpr at the nucleopore complex is crucial for regulating intron containing mRNA export by directly or indirectly participating in the processing and degradation of aberrant mRNA transcripts.
A CRM1-Mediated Nuclear Export Signal Is Essential for Cytoplasmic Localization of Neurogenin 3 in Neurons  [PDF]
Julia Simon-Areces, Estefania Acaz-Fonseca, Isabel Ruiz-Palmero, Luis-Miguel Garcia-Segura, Maria-Angeles Arevalo
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0055237
Abstract: Neurogenin 3 (Ngn3), a proneural gene, regulates dendritogenesis and synaptogenesis in mouse hippocampal neurons. Ngn3 is transiently exported from the cell nucleus to the cytoplasm when neuronal polarity is initiated, suggesting that the nucleo-cytoplasmic transport of the protein is important for its action on neuronal development. In this study, we identified for the first time a functional nuclear export sequence (NES2; 131YIWALTQTLRIA142) in Ngn3. The green fluorescent protein (EGFP)-NES2 fusion protein was localized in the cytoplasm and its nucleo-cytoplasmic shuttling was blocked by the CRM1 specific export inhibitor leptomycin B. Mutation of a leucine residue to alanine (L135A) in the NES2 motif resulted in both cytoplasmic and nuclear localization of the EGFP-NES2 fusion protein and in the nuclear accumulation of ectopic full-length myc-Ngn3. In addition, point mutation of the leucine 135 counteracted the effects of Ngn3 on neuronal morphology and synaptic inputs indicating that the cytoplasmic localization of Ngn3 is important for neuronal development. Pharmacological perturbation of the cytoskeleton revealed that cytoplasmic Ngn3 is associated with microtubules.
Heart Failure Induces Significant Changes in Nuclear Pore Complex of Human Cardiomyocytes  [PDF]
Estefanía Tarazón, Miguel Rivera, Esther Roselló-Lletí, Maria Micaela Molina-Navarro, Ignacio José Sánchez-Lázaro, Francisco Espa?a, José Anastasio Montero, Francisca Lago, José Ramón González-Juanatey, Manuel Portolés
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0048957
Abstract: Aims The objectives of this study were to analyse the effect of heart failure (HF) on several proteins of nuclear pore complex (NPC) and their relationship with the human ventricular function. Methods and Results A total of 88 human heart samples from ischemic (ICM, n = 52) and dilated (DCM, n = 36) patients undergoing heart transplant and control donors (CNT, n = 9) were analyzed by Western blot. Subcellular distribution of nucleoporins was analysed by fluorescence and immunocytochemistry. When we compared protein levels according to etiology, ICM showed significant higher levels of NDC1 (65%, p<0.0001), Nup160 (88%, p<0.0001) and Nup153 (137%, p = 0.004) than those of the CNT levels. Furthermore, DCM group showed significant differences for NDC1 (41%, p<0.0001), Nup160 (65%, p<0.0001), Nup153 (155%, p = 0.006) and Nup93 (88%, p<0.0001) compared with CNT. However, Nup155 and translocated promoter region (TPR) did not show significant differences in their levels in any etiology. Regarding the distribution of these proteins in cell nucleus, only NDC1 showed differences in HF. In addition, in the pathological group we obtained good relationship between the ventricular function parameters (LVEDD and LVESD) and Nup160 (r = ?0382, p = 0.004; r = ?0.290, p = 0.033; respectively). Conclusions This study shows alterations in specific proteins (NDC1, Nup160, Nup153 and Nup93) that compose NPC in ischaemic and dilated human heart. These changes, related to ventricular function, could be accompanied by alterations in the nucleocytoplasmic transport. Therefore, our findings may be the basis for a new approach to HF management.
Translocation Biosensors – Cellular System Integrators to Dissect CRM1-Dependent Nuclear Export by Chemicogenomics  [PDF]
Verena Fetz,Shirley K. Knauer,Carolin Bier,Jens Peter Von Kries,Roland H. Stauber
Sensors , 2009, DOI: 10.3390/s90705423
Abstract: Fluorescent protein biosensors are powerful cellular systems biology tools for dissecting the complexity of cellular processes with high spatial and temporal resolution. As regulated nucleo-cytoplasmic transport is crucial for the modulation of numerous (patho)physiological cellular responses, a detailed understanding of its molecular mechanism would open up novel options for a rational manipulation of the cell. In contrast to genetic approaches, we here established and employed high-content cellular translocation biosensors applicable for dissecting nuclear export by chemicogenomics. A431 cell lines, stably expressing a translocation biosensor composed of glutathione S-transferase, GFP and a rational combination of nuclear import and export signals, were engineered by antibiotic selection and flow cytometry sorting. Using an optimized nuclear translocation algorithm, the translocation response could be robustly quantified on the Cellomics Arrayscan? VTI platform. Subsequent to assay optimization, the assay was developed into a higher density 384-well format high-content assay and employed for the screening of the 17K ChemBioNet compound collection. This library was selected on the basis of a genetic algorithm used to identify maximum common chemical substructures in a database of annotated bioactive molecules and hence, is well-placed in the chemical space covered by bioactive compounds. Automated multiparameter data analysis combined with visual inspection allowed us to identify and to rationally discriminate true export inhibitors from false positives, which included fluorescent compounds or cytotoxic substances that dramatically affected the cellular morphology. A total of 120 potential hit compounds were selected for Cellomics Arrayscan? VTI based rescreening. The export inhibitory activity of 20 compounds effective at concentrations < 25 μM were confirmed by fluorescence microscopy in several cell lines. Interestingly, kinetic analysis allowed the identification of inhibitors capable to interfere with the export receptor CRM1-mediated nuclear export not only in an irreversible, but also in a reversible fashion. In sum, exploitation of biosensor based screening allows the identification of chemicogenomic tools applicable for dissecting nucleo-cytoplasmic transport in living cells.
Intermolecular masking of the HIV-1 Rev NLS by the cellular protein HIC: Novel insights into the regulation of Rev nuclear import
Lili Gu, Takahiro Tsuji, Mohamed Jarboui, Geok P Yeo, Noreen Sheehy, William W Hall, Virginie W Gautier
Retrovirology , 2011, DOI: 10.1186/1742-4690-8-17
Abstract: In our study, we have identified the cellular protein HIC (Human I-mfa domain-Containing protein) as a novel interactor of HIV-1 Rev. We demonstrate that HIC selectively interferes with Rev NLS interaction with importin β and impedes its nuclear import and function, but does not affect Rev nuclear import mediated by transportin. Hence, the molecular determinants mediating Rev-NLS recognition by importin β and transportin appear to be distinct. Furthermore, we have employed HIC and M9 M, a peptide specifically designed to inhibit the transportin-mediated nuclear import pathway, to characterise Rev nuclear import pathways within different cellular environments. Remarkably, we could show that in 293T, HeLa, COS7, Jurkat, U937, THP-1 and CEM cells, Rev nuclear import is cell type specific and alternatively mediated by transportin or importin β, in a mutually exclusive fashion.Rev cytoplasmic sequestration by HIC may represent a novel mechanism for the control of Rev function. These studies highlight that the multivalent nature of the Rev NLS for different import receptors enables Rev to adapt its nuclear trafficking strategy.The HIV-1 regulatory protein Rev (18 kDa) is essential for HIV-1 replication [1,2]. Rev is predominantly localised in the nucleus/nucleolus [3], and its primary function is to mediate the nuclear export of partially spliced and unspliced viral transcripts. Rev has also been shown to modulate splicing and translation of viral transcripts, and their subsequent packaging, and to interfere with integration of the HIV-1 genome [4-7]. Rev nuclear export of unspliced viral transcripts requires active shuttling of the protein between the nucleus and cytoplasm via nuclear pore complexes (NPCs) which is mediated by two major functional domains, the Nuclear Localisation Signal (NLS) and the Nuclear Export Signal (NES) [8,9]. The leucine-rich Rev NES binds directly to CRM1, which in concert with DDX3, a DEAD box RNA helicase, facilitates Rev nuclear export of u
The nuclear pore complex
Stephen A Adam
Genome Biology , 2001, DOI: 10.1186/gb-2001-2-9-reviews0007
Abstract: The nuclear pore complex (NPC) forms the conduit for the exchange of information between the nucleus and cytoplasm. NPC structures of amazingly similar appearance have been identified in all eukaryotes from yeast to human, yet differences in NPCs between species are likely to provide important clues to NPC function. As the exclusive site of macromolecular traffic between the nucleus and cytoplasm, the NPC provides an important control point for the regulation of gene expression. Precisely how the NPC regulates the traffic of ions and macromolecules remains a topic of speculation and has become the primary research focus of a number of laboratories in recent years. Recent advances towards the identification and characterization of all NPC components by proteomics and genomics, as well as studies of NPCs in vitro, have propelled the field rapidly forward, and are discussed in this article.Nuclear pore complexes are proteinaceous structures embedded in the double membrane of the nuclear envelope. In order to understand how the NPC functions, it is useful to examine the similarities and differences between NPCs from the yeast Saccharomyces cerevisiae and the frog Xenopus laevis. The NPC is a large structure with a molecular weight of approximately 125 MDa in vertebrates and 66 MDa in yeast [1,2]. A vertebrate cell nucleus contains on the order of 2,000 NPCs, whereas the smaller yeast nucleus contains approximately 200. NPCs have eight-fold rotational symmetry through the central axis of the pore and two-fold mirror symmetry through the plane of the nuclear envelope, suggesting assembly as a modular structure, a notion that is supported by structural and biochemical analysis of pore complex assembly in vitro [3,4].In addition to their difference in size, yeast NPCs (yNPCs) and vertebrate NPCs (vNPCs) differ in several fundamental structural features. The main mass of the vNPC is contained in a three-part structure that surrounds and supports a central transporter (see Fi
TNF Stimulates Nuclear Export and Secretion of IL-15 by Acting on CRM1 and ARF6  [PDF]
Suidong Ouyang, Hung Hsuchou, Abba J. Kastin, Weihong Pan
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0069356
Abstract: Interleukin (IL)-15 is a ubiquitously expressed cytokine that in the basal state is mainly localized intracellularly, including the nucleus. Unexpectedly, tumor necrosis factor-α (TNF) time-dependently induced nuclear export of IL-15Rα and IL15. This process was inhibited by leptomycine B (LMB), a specific inhibitor of nuclear export receptor chromosomal region maintenance 1 (CRM1). In the presence of TNF, LMB co-treatment led to accumulation of both IL-15Rα and IL-15 in the nucleus of HeLa cells, suggesting that CRM1 facilitates nuclear export and that TNF enhances CRM1 activity. Once in the cytoplasm, IL-15 showed partial co-localization with late endosomes but very little with other organelles tested 4 h after TNF treatment. IL-15Rα showed co-localization with both early and late endosomes, and to a lesser extent with endoplasmic reticulum and Golgi. This indicates different kinetics and possibly different trafficking routes of IL-15 from its specific receptor. The TNF-induced secretion of IL-15 was attenuated by pretreatment of cells by brefeldin A that inhibits ER-to-Golgi transport, or by use of domain negative ADP-ribosylation factor 6 (ARF6) that interferes with exocytotic sorting. We conclude that TNF abolishes nuclear localization of IL-15 and IL-15Rα by acting on CRM1, and it facilitates exocytosis of IL-15 with the involvement of ARF6.
Insights into the Function of the CRM1 Cofactor RanBP3 from the Structure of Its Ran-Binding Domain  [PDF]
Karla Langer,Cyril Dian,Vladimir Rybin,Christoph W. Müller,Carlo Petosa
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0017011
Abstract: Proteins bearing a leucine-rich nuclear export signal (NES) are exported from the nucleus by the transport factor CRM1, which forms a cooperative ternary complex with the NES-bearing cargo and with the small GTPase Ran. CRM1-mediated export is regulated by RanBP3, a Ran-interacting nuclear protein. Unlike the related proteins RanBP1 and RanBP2, which promote disassembly of the export complex in the cytosol, RanBP3 acts as a CRM1 cofactor, enhancing NES export by stabilizing the export complex in the nucleus. RanBP3 also alters the cargo selectivity of CRM1, promoting recognition of the NES of HIV-1 Rev and of other cargos while deterring recognition of the import adaptor protein Snurportin1. Here we report the crystal structure of the Ran-binding domain (RBD) from RanBP3 and compare it to RBD structures from RanBP1 and RanBP2 in complex with Ran and CRM1. Differences among these structures suggest why RanBP3 binds Ran with unusually low affinity, how RanBP3 modulates the cargo selectivity of CRM1, and why RanBP3 promotes assembly rather than disassembly of the export complex. The comparison of RBD structures thus provides an insight into the functional diversity of Ran-binding proteins.
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