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Katharine S. Ullman - One of the best experts on this subject based on the ideXlab platform.
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NUP153 and Nup50 promote recruitment of 53BP1 to DNA repair foci by antagonizing BRCA1-dependent events
Journal of cell science, 2017Co-Authors: Douglas R. Mackay, Amanda C. Howa, Theresa L. Werner, Katharine S. UllmanAbstract:DNA double-strand breaks are typically repaired through either the high-fidelity process of homologous recombination (HR), in which BRCA1 plays a key role, or the more error-prone process of non-homologous end joining (NHEJ), which relies on 53BP1. The balance between NHEJ and HR depends, in part, on whether 53BP1 predominates in binding to damage sites, where it protects the DNA ends from resection. The nucleoporin NUP153 has been implicated in the DNA damage response, attributed to a role in promoting nuclear import of 53BP1. Here, we define a distinct requirement for NUP153 in 53BP1 intranuclear targeting to damage foci and report that NUP153 likely facilitates the role of another nucleoporin, Nup50, in 53BP1 targeting. The requirement for NUP153 and Nup50 in promoting 53BP1 recruitment to damage foci induced by either etoposide or olaparib is abrogated in cells deficient for BRCA1 or its partner BARD1, but not in cells deficient for BRCA2. Together, our results further highlight the antagonistic relationship between 53BP1 and BRCA1, and place NUP153 and Nup50 in a molecular pathway that regulates 53BP1 function by counteracting BRCA1-mediated events.
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Abstract A46: NUP153 and Nup50 promote recruitment of 53BP1 to DNA repair foci by antagonizing BRCA1-dependent events
Other Topics, 2017Co-Authors: Douglas R. Mackay, Katharine S. UllmanAbstract:This study focuses on an unexpected role for particular nuclear pore proteins in the antagonism between the DNA repair factors BRCA1 and 53BP1, and the impact this has on the response to poly(A) ribose polymerase (PARP) inhibition. Repair of DNA double strand breaks, a cornerstone of genomic integrity, typically follows one of two distinct pathways: the high fidelity process of homologous recombination (HR) repair, in which BRCA1 plays a key role, and the more error-prone process of non-homologous end joining (NHEJ), which relies on 53BP1. The balance between NHEJ and HR depends, in part, on whether 53BP1 predominates in binding to damage sites, where it protects the DNA ends from resection and prevents HR from occurring. Conversely, BRCA1 binding counteracts 53BP1-mediated events to promote HR repair. While mutual antagonism between BRCA1 and 53BP1 has been appreciated for some time, factors that modulate this relationship are only beginning to be elucidated. The nuclear pore component NUP153 has previously been implicated in the DNA damage response, specifically in promoting nuclear import of 53BP1. To further explore the role of NUP153 in this context, we engineered an additional strong nuclear localization sequence (NLS) on 53BP1 and found that this overrides the requirement for NUP153 in nuclear import of 53BP1. While 53BP1-NLS is recruited normally to DNA damage foci in control-treated cells, its accumulation at these sites is still severely impaired in NUP153-depleted cells, indicating that NUP153 has an additional and distinct role in 53BP1 intranuclear targeting. These observations are further underscored by the additional finding that a second nuclear pore protein, Nup50, is also required for intranuclear targeting of 53BP1, yet makes no discernable contribution to 53BP1 nuclear import. Of note, depletion of a third nucleoporin (Tpr), which is part of the same structural element of the nuclear pore, did not alter this DNA damage response pathway. To gain insight into the pathway affected by NUP153 and Nup50 and to understand how their role fits into a clinically relevant context, we probed the consequences of reducing NUP153 and Nup50 levels in cells deficient in BRCA1 activity. We found that the requirement for NUP153 and Nup50 in promoting 53BP1 recruitment to damage foci is abrogated under these circumstances. In cells where HR repair is otherwise defective, however, robust recruitment of 53BP1 to DNA damage foci requires NUP153 and Nup50. Together, our results provide novel insight into 53BP1 regulation during the DNA damage response and are consistent with a model where NUP153 and Nup50 normally promote 53BP1 targeting by counteracting BRCA1-dependent events. These findings have important implications with regard to PARP inhibition, a therapeutic strategy thought to capitalize on defective HR repair by forcing inappropriate use of NHEJ, culminating in cellular toxicity. Accordingly, we found that decreased levels of NUP153 and Nup50 do not affect the 53BP1-dependent pathway in BRCA1-deficient cells exposed to PARP inhibitor treatment, but still impair this response in cells deficient for BRCA2 or when the ATM and ATR signaling pathways are inhibited. Our results indicate that NUP153 and Nup50, as factors that modulate 53BP1 activity, belong to a novel category of biomarkers that could be screened to make better predictions of response to PARP inhibitor treatment. Citation Format: Douglas R. Mackay, Katharine S. Ullman. NUP153 and Nup50 promote recruitment of 53BP1 to DNA repair foci by antagonizing BRCA1-dependent events [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr A46.
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The NUP153-Nup50 Protein Interface and Its Role in Nuclear Import
The Journal of biological chemistry, 2012Co-Authors: Masaki Makise, Douglas R. Mackay, Suzanne Elgort, Sunita S. Shankaran, Stephen A. Adam, Katharine S. UllmanAbstract:Interactions between Nup50 and soluble transport factors underlie the efficiency of certain nucleocytoplasmic transport pathways. The platform on which these interactions take place is important to building a complete understanding of nucleocytoplasmic trafficking. NUP153 is the nucleoporin that provides this scaffold for Nup50. Here, we have delineated requirements for the interaction between NUP153 and Nup50, revealing a dual interface. An interaction between Nup50 and a region in the unique N-terminal region of NUP153 is critical for the nuclear pore localization of Nup50. A second site of interaction is at the distal tail of NUP153 and is dependent on importin α. Both of these interactions involve the N-terminal domain of Nup50. The configuration of the NUP153-Nup50 partnership suggests that the NUP153 scaffold provides not just a means of pore targeting for Nup50 but also serves to provide a local environment that facilitates bringing Nup50 and importin α together, as well as other soluble factors involved in transport. Consistent with this, disruption of the NUP153-Nup50 interface decreases efficiency of nuclear import.
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Two distinct sites in NUP153 mediate interaction with the SUMO proteases SENP1 and SENP2
Nucleus (Austin Tex.), 2012Co-Authors: Kin-hoe Chow, Suzanne Elgort, Mary Dasso, Katharine S. UllmanAbstract:Numerous enzymes of the mammalian SUMO modification pathway, including two members of the SUMO protease family, SENP2 and SENP1, localize to the nuclear periphery. The SUMO proteases play roles both in processing SUMO during the biogenesis of this peptide moiety and also in reversing SUMO modification on specific targets to control the activities conferred by this post-translational modification. Although interaction with the C-terminal domain of the nucleoporin NUP153 is thought to contribute to SENP2 localization at the nuclear pore complex, little is known about the binding partners of SENP1 at the nuclear periphery. We have found that NUP153 binds to both SENP1 and SENP2 and does so by interacting with the unique N-terminal domain of NUP153 as well as a specific region within the C-terminal FG-rich region. We have further found that NUP153 is a substrate for sumoylation, with this modification kept in check by these two SUMO proteases. Specifically, either RNAi depletion of SENP1/SENP2 or expression of dominantly interfering mutants of these proteins results in increased sumoylation of endogenous NUP153. While SENP1 and SENP2 share many characteristics, we show here that SENP1 levels are influenced by the presence of NUP153, whereas SENP2 is not sensitive to changes in NUP153 abundance.
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Defects in nuclear pore assembly lead to activation of an Aurora B-mediated abscission checkpoint.
The Journal of cell biology, 2010Co-Authors: Douglas R. Mackay, Masaki Makise, Katharine S. UllmanAbstract:Correct assembly of nuclear pore complexes (NPCs), which directly and indirectly control nuclear environment and architecture, is vital to genomic regulation. We previously found that nucleoporin 153 (NUP153) is required for timely progression through late mitosis. In this study, we report that disruption of NUP153 function by either small interfering RNA–mediated depletion or expression of a dominant-interfering NUP153 fragment results in dramatic mistargeting of the pore basket components Tpr and Nup50 in midbody-stage cells. We find a concomitant appearance of aberrantly localized active Aurora B and an Aurora B–dependent delay in abscission. Depletion of Nup50 is also sufficient to increase the number of midbody-stage cells and, likewise, triggers distinctive mislocalization of Aurora B. Together, our results suggest that defects in nuclear pore assembly, and specifically the basket structure, at this time of the cell cycle activate an Aurora B–mediated abscission checkpoint, thereby ensuring that daughter cells are generated only when fully formed NPCs are present.
Birthe Fahrenkrog - One of the best experts on this subject based on the ideXlab platform.
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Mitotic checkpoint protein Mad1 is required for early NUP153 recruitment to chromatin and nuclear envelope integrity.
Journal of cell science, 2020Co-Authors: Ikram Mossaid, Valérie Martinelli, Guillaume Chatel, Marcela Vaz, Birthe FahrenkrogAbstract:Nucleoporin NUP153 is a multifunctional protein and mitotic checkpoint protein Mad1 a known binding partner. The functional relevance of their interaction has remained elusive. Here, we have further dissected NUP1539s and Mad19s interface and functional interplay. By in situ proximity ligation assays, we found that the presence of a nuclear envelope (NE) is prerequisite for the NUP153-Mad1 association. Time-lapse microscopy revealed that depletion of Mad1 delayed recruitment of NUP153 to anaphase chromatin, which was often accompanied by a prolongation of anaphase. Furthermore, as seen by electron microscopic and three-dimensional structured illumination investigations, NUP153 and Mad1 depletion led to alterations in NE architecture, characterised by a change of membrane curvature at nuclear pore complexes (NPCs) and an expansion of the spacing between inner and outer nuclear membrane. NUP153 depletion, but not of Mad1, caused defects in interphase NPC assembly with partial displacement of cytoplasmic nucleoporins and a reduction in NPC density. Together our results suggest that NUP153 has separable roles in NE and NPC formation: in post-mitotic NE reformation in concert with Mad1 and in interphase NPC assembly, independent of Mad1.
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Mitotic checkpoint protein Mad1 is required for early NUP153 recruitment to chromatin and nuclear envelope integrity
2020Co-Authors: Ikram Mossaid, Valérie Martinelli, Guillaume Chatel, Marcela Vaz, Birthe FahrenkrogAbstract:The nucleoporin NUP153 is a multifunctional protein and the mitotic checkpoint protein Mad1one of its many binding partners. The functional relevance of their interaction has remained elusive. Here, we have further dissected NUP1539s and Mad19s interface and functional interplay. By in situ proximity ligation assays, we found that the presence of a nuclear envelope (NE) is prerequisite for the NUP153-Mad1 interaction. Time-lapse microscopy revealed that depletion of Mad1 delayed recruitment of NUP153 to anaphase chromatin, which was often accompanied by a prolongation of anaphase. Furthermore, as seen by electron microscopic and three-dimensional structured illumination investigations, NUP153 and Mad1 depletion led to alterations in NE architecture, characterised by a change of the membrane curvature at nuclear pore complexes (NPCs) and an expansion of the spacing between the inner and outer nuclear membranes. NUP153 depletion, but not of Mad1, caused defects in interphase NPC assembly with partial displacement of cytoplasmic nucleoporins and a reduction in NPC density. Together our results suggest that NUP153 has separable roles in NE and NPC formation: in post-mitotic NE reformation in concert with Mad1 and in interphase NPC assembly, independent of Mad1.
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Localisation of NUP153 and SENP1 to nuclear pore complexes is required for 53BP1-mediated DNA double-strand break repair.
Journal of cell science, 2017Co-Authors: Vincent Duheron, Nadine Nilles, Sylvia Pecenko, Valérie Martinelli, Birthe FahrenkrogAbstract:The nuclear basket of nuclear pore complexes (NPCs) is composed of three nucleoporins: NUP153, Nup50 and Tpr. NUP153 has a role in DNA double-strand break (DSB) repair by promoting nuclear import of 53BP1 (also known as TP53BP1), a mediator of the DNA damage response. Here, we provide evidence that loss of NUP153 compromises 53BP1 sumoylation, a prerequisite for efficient accumulation of 53BP1 at DSBs. Depletion of NUP153 resulted in reduced SUMO1 modification of 53BP1 and the displacement of the SUMO protease SENP1 from NPCs. Artificial tethering of SENP1 to NPCs restored non-homologous end joining (NHEJ) in the absence of NUP153 and re-established 53BP1 sumoylation. Furthermore, Nup50 and Tpr, the two other nuclear basket nucleoporins, also contribute to proper DSB repair, in a manner distinct from NUP153. Similar to the role of NUP153, Tpr is implicated in NHEJ and homologous recombination (HR), whereas loss of Nup50 only affects NHEJ. Despite the requirement of all three nucleoporins for accurate NHEJ, only NUP153 is needed for proper nuclear import of 53BP1 and SENP1-dependent sumoylation of 53BP1. Our data support the role of NUP153 as an important regulator of 53BP1 activity and efficient NHEJ.
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The nuclear pore protein NUP153: Dissecting its role in nuclear envelope and nuclear pore complex architecture and its interaction with the spindle assembly checkpoint protein Mad1
2016Co-Authors: Ikram Mossaid, Birthe FahrenkrogAbstract:Nuclear pore complexes (NPCs) are embedded in the nuclear envelope (NE) and composed of proteins called nucleoporins. NPCs as such control the bidirectional traffic of proteins and RNAs between the nucleus and the cytoplasm in eukaryotic cells whereas individual nucleoporins were found to be implicated in other cellular processes such as, cell division, kinetochore assembly, gene expression and cell migration. A prime example for nucleoporin functional versatility can be seen in NUP153. NUP153 is since its discovery known to be a central player in nucleocytoplasmic transport, but additionally participates directly or indirectly, for example, in gene expression and cell cycle control. In this context, it was previously shown that altered levels of NUP153 led to mitotic abnormalities, particularly in cytokinesis and in the spindle assembly checkpoint (SAC). The SAC promotes accurate chromosome separation to ensure the faithful segregation of genetic material to daughter cells. NUP153 was found to interact with the SAC protein Mad1. In the present study, we have further dissected the interaction between NUP153 and Mad1 and investigated the function of the NUP153-Mad1 complex in human cells. By using the high resolution imaging technique “in situ proximity ligation assay”, we found that NUP153 and Mad1 interact with each other exclusively in the presence of a NE, from late mitosis to prophase. By in vitro binding assays, we have confirmed the direct interaction between NUP153 and Mad1 and furthermore identified two independent NUP153-binding sites in Mad1. We have also provided some evidence that NUP153 interacts also with SUMO-modified Mad1.It was previously shown that depletion of NUP153 had no obvious effect on Mad1 and SAC activity. In the present study, we have shown by time-lapse imaging microscopy that the depletion of Mad1 led to a delayed recruitment of NUP153 at the reforming NE during anaphase in living cells, which was often accompanied by a prolongation of anaphase. Furthermore, Mad1 depletion led to alterations in the NE architecture, which were characterized by a change of the membrane curvature at the NPC-NE interface. This was followed by an expansion of the spacing between the inner and outer membranes as seen by electron microscopic and three-dimensional structured illumination investigations. This suggests an implication of Mad1 in a mechanism related to the NE reformation and stability independent of the SAC. Mad1 depletion also resulted in redistribution of the ER network and mitochondria throughout the cell as seen by fluorescence microscopy. NUP153 depletion coincided with the NE abnormalities and alteration of these organelles similar to that seen in Mad1-depleted cells. Further, by fluorescence microscopy, we have shown that NUP153 depletion, but not of Mad1, partially affected the localization of the cytoplasmic nucleoporins in human and in mouse cells and thus the NPC integrity. In conclusion, altogether, our results suggest that NUP153 is essential for NE and NPC integrity. NUP153 has likely separable roles in this context: one in post-mitotic NE reformation with Mad1 and one in interphase in NPC assembly. NUP153-Mad1 complex has a function independent of the spindle checkpoint, but important for the establishment of an intact NE architecture.
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Structural characterization of altered nucleoporin NUP153 expression in human cells by thin-section electron microscopy
Nucleus (Austin Tex.), 2014Co-Authors: Vincent Duheron, Ursula Sauder, Guillaume Chatel, Vesna Oliveri, Birthe FahrenkrogAbstract:Nuclear pore complexes (NPCs) span the 2 membranes of the nuclear envelope (NE) and facilitate nucleocytoplasmic exchange of macromolecules. NPCs have a roughly tripartite structural organization with the so-called nuclear basket emanating from the NPC scaffold into the nucleoplasm. The nuclear basket is composed of the 3 nucleoporins NUP153, Nup50, and Tpr, but their specific role for the structural organization of this NPC substructure is, however, not well established. In this study, we have used thin-section transmission electron microscopy to determine the structural consequences of altering the expression of NUP153 in human cells. We show that the assembly and integrity of the nuclear basket is not affected by NUP153 depletion, whereas its integrity is perturbed in cells expressing high concentrations of the zinc-finger domain of NUP153. Moreover, even mild over-expression of NUP153 is coinciding with massive changes in nuclear organization and it is the excess of the zinc-finger domain of NUP153 that is sufficient to induce these rearrangements. Our data indicate a central function of NUP153 in the organization of the nucleus, not only at the periphery, but throughout the entire nuclear interior.
Joan Massague - One of the best experts on this subject based on the ideXlab platform.
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smad2 nucleocytoplasmic shuttling by nucleoporins can nup214 and NUP153 feeds tgfβ signaling complexes in the cytoplasm and nucleus
Molecular Cell, 2002Co-Authors: Lan Xu, Yibin Kang, Joan MassagueAbstract:The transcription factor Smad2 is released from cytoplasmic retention by TGFβ receptor-mediated phosphorylation, accumulating in the nucleus where it associates with cofactors to regulate transcription. We uncovered direct interactions of Smad2 with the nucleoporins CAN/Nup214 and NUP153. These interactions mediate constitutive nucleocytoplasmic shuttling of Smad2. CAN/Nup214 and NUP153 compete with the cytoplasmic retention factor SARA and the nuclear Smad2 partner FAST-1 for binding to a hydrophobic corridor on the MH2 surface of Smad2. TGFβ receptor-mediated phosphorylation stimulates nuclear accumulation of Smad2 by modifying its affinity for SARA and Smad4 but not for CAN/Nup214 or NUP153. Thus, by directly contacting the nuclear pore complex, Smad2 undergoes constant shuttling, providing a dynamic pool that is competitively drawn by cytoplasmic and nuclear signal transduction partners.
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Smad2 Nucleocytoplasmic Shuttling by Nucleoporins CAN/Nup214 and NUP153 Feeds TGFβ Signaling Complexes in the Cytoplasm and Nucleus
Molecular cell, 2002Co-Authors: Yibin Kang, Seda Çöl, Joan MassagueAbstract:The transcription factor Smad2 is released from cytoplasmic retention by TGFβ receptor-mediated phosphorylation, accumulating in the nucleus where it associates with cofactors to regulate transcription. We uncovered direct interactions of Smad2 with the nucleoporins CAN/Nup214 and NUP153. These interactions mediate constitutive nucleocytoplasmic shuttling of Smad2. CAN/Nup214 and NUP153 compete with the cytoplasmic retention factor SARA and the nuclear Smad2 partner FAST-1 for binding to a hydrophobic corridor on the MH2 surface of Smad2. TGFβ receptor-mediated phosphorylation stimulates nuclear accumulation of Smad2 by modifying its affinity for SARA and Smad4 but not for CAN/Nup214 or NUP153. Thus, by directly contacting the nuclear pore complex, Smad2 undergoes constant shuttling, providing a dynamic pool that is competitively drawn by cytoplasmic and nuclear signal transduction partners.
David Balasundaram - One of the best experts on this subject based on the ideXlab platform.
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Multiple conserved domains of the nucleoporin Nup124p and its orthologs Nup1p and NUP153 are critical for nuclear import and activity of the fission yeast Tf1 retrotransposon.
Molecular biology of the cell, 2007Co-Authors: Srivani Sistla, Junxiong Vincent Pang, Cui Xia Wang, David BalasundaramAbstract:The nucleoporin Nup124p is a host protein required for the nuclear import of both, retrotransposon Tf1-Gag as well as the retroviral HIV-1 Vpr in fission yeast. The human nucleoporin NUP153 and the Saccharomyces cerevisiae Nup1p were identified as orthologs of Nup124p. In this study, we show that all three nucleoporins share a large FG/FXFG-repeat domain and a C-terminal peptide sequence, GRKIxxxxxRRKx, that are absolutely essential for Tf1 retrotransposition. Though the FXFG domain was essential, the FXFG repeats themselves could be eliminated without loss of retrotransposon activity, suggesting the existence of a common element unrelated to FG/FXFG motifs. The Nup124p C-terminal peptide, GRKIAVPRSRRKR, was extremely sensitive to certain single amino acid changes within stretches of the basic residues. On the basis of our comparative study of Nup124p, Nup1p, and NUP153 domains, we have developed peptides that specifically knockdown retrotransposon activity by disengaging the Tf1-Gag from its host nuclear transport machinery without any harmful consequence to the host itself. Our results imply that those domains challenged a specific pathway affecting Tf1 transposition. Although full-length Nup1p or NUP153 does not complement Nup124p, the functionality of their conserved domains with reference to Tf1 activity suggests that these three proteins evolved from a common ancestor.
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The Functionally Conserved Nucleoporins Nup124p from Fission Yeast and the Human NUP153 Mediate Nuclear Import and Activity of the Tf1 Retrotransposon and HIV-1 Vpr
Molecular biology of the cell, 2005Co-Authors: Padmapriya Varadarajan, Sundarasamy Mahalingam, Peiyun Liu, Sheetal Gandotra, Desmond Suresh Kumar Dorairajoo, David BalasundaramAbstract:We report that the fission yeast nucleoporin Nup124p is required for the nuclear import of both, retrotransposon Tf1-Gag as well as the retroviral HIV-1 Vpr. Failure to import Tf1-Gag into the nucleus in a nup124 null mutant resulted in complete loss of Tf1 transposition. Similarly, nuclear import of HIV-1 Vpr was impaired in nup124 null mutant strains and cells became resistant to Vpr's cell-killing activity. On the basis of protein domain similarity, the human nucleoporin NUP153 was identified as a putative homolog of Nup124p. We demonstrate that in vitro-translated Nup124p and NUP153 coimmunoprecipitate Tf1-Gag or HIV-1 Vpr. Though full-length NUP153 was unable to complement the Tf1 transposition defect in a nup124 null mutant, we provide evidence that both nucleoporins share a unique N-terminal domain, Nup124p(AA264-454) and NUP153(AA448-634) that is absolutely essential for Tf1 transposition. Epigenetic overexpression of this domain in a wild-type (nup124(+)) background blocked Tf1 activity implying that sequences from Nup124p and the human NUP153 challenged the same pathway affecting Tf1 transposition. Our results establish a unique relationship between two analogous nucleoporins Nup124p and NUP153 wherein the function of a common domain in retrotransposition is conserved.
Douglass J. Forbes - One of the best experts on this subject based on the ideXlab platform.
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Analysis of the initiation of nuclear pore assembly by ectopically targeting nucleoporins to chromatin.
Nucleus (Austin Tex.), 2015Co-Authors: Michal Schwartz, Anna Travesa, Steven W Martell, Douglass J. ForbesAbstract:Nuclear pore complexes (NPCs) form the gateway to the nucleus, mediating virtually all nucleocytoplasmic trafficking. Assembly of a nuclear pore complex requires the organization of many soluble sub-complexes into a final massive structure embedded in the nuclear envelope. By use of a LacI/LacO reporter system, we were able to assess nucleoporin (Nup) interactions, show that they occur with a high level of specificity, and identify nucleoporins sufficient for initiation of the complex process of NPC assembly in vivo. Eleven nucleoporins from different sub-complexes were fused to LacI-CFP and transfected separately into a human cell line containing a stably integrated LacO DNA array. The LacI-Nup fusion proteins, which bound to the array, were examined for their ability to recruit endogenous nucleoporins to the intranuclear LacO site. Many could recruit nucleoporins of the same sub-complex and a number could also recruit other sub-complexes. Strikingly, Nup133 and Nup107 of the Nup107/160 subcomplex and NUP153 and Nup50 of the nuclear pore basket recruited a near full complement of nucleoporins to the LacO array. Furthermore, Nup133 and NUP153 efficiently targeted the LacO array to the nuclear periphery. Our data support a hierarchical, seeded assembly pathway and identify Nup133 and NUP153 as effective “seeds” for NPC assembly. In addition, we show that this system can be applied to functional studies of individual nucleoporin domains as well as to specific nucleoporin disease mutations. We find that the R391H cardiac arrhythmia/sudden death mutation of Nup155 prevents both its subcomplex assembly and nuclear rim targeting of the LacO array.
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Novel vertebrate nucleoporins Nup133 and Nup160 play a role in mRNA export
The Journal of cell biology, 2001Co-Authors: Sanjay K. Vasu, Sundeep Shah, Arturo V. Orjalo, Minkyu Park, Wolfgang H. Fischer, Douglass J. ForbesAbstract:RNA undergoing nuclear export first encounters the basket of the nuclear pore. Two basket proteins, Nup98 and NUP153, are essential for mRNA export, but their molecular partners within the pore are largely unknown. Because the mechanism of RNA export will be in question as long as significant vertebrate pore proteins remain undiscovered, we set out to find their partners. Fragments of Nup98 and NUP153 were used for pulldown experiments from Xenopus egg extracts, which contain abundant disassembled nuclear pores. Strikingly, Nup98 and NUP153 each bound the same four large proteins. Purification and sequence analysis revealed that two are the known vertebrate nucleoporins, Nup96 and Nup107, whereas two mapped to ORFs of unknown function. The genes encoding the novel proteins were cloned, and antibodies were produced. Immunofluorescence reveals them to be new nucleoporins, designated Nup160 and Nup133, which are accessible on the basket side of the pore. Nucleoporins Nup160, Nup133, Nup107, and Nup96 exist as a complex in Xenopus egg extracts and in assembled pores, now termed the Nup160 complex. Sec13 is prominent in Nup98 and NUP153 pulldowns, and we find it to be a member of the Nup160 complex. We have mapped the sites that are required for binding the Nup160 subcomplex, and have found that in Nup98, the binding site is used to tether Nup98 to the nucleus; in NUP153, the binding site targets NUP153 to the nuclear pore. With transfection and in vivo transport assays, we find that specific Nup160 and Nup133 fragments block poly[A]+ RNA export, but not protein import or export. These results demonstrate that two novel vertebrate nucleoporins, Nup160 and Nup133, not only interact with Nup98 and NUP153, but themselves play a role in mRNA export.
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The Nucleoporin NUP153 Plays a Critical Role in Multiple Types of Nuclear Export
Molecular biology of the cell, 1999Co-Authors: Katharine S. Ullman, Sundeep Shah, Maureen A Powers, Douglass J. ForbesAbstract:The fundamental process of nucleocytoplasmic transport takes place through the nuclear pore. Peripheral pore structures are presumably poised to interact with transport receptors and their cargo as these receptor complexes first encounter the pore. One such peripheral structure likely to play an important role in nuclear export is the basket structure located on the nuclear side of the pore. At present, NUP153 is the only nucleoporin known to localize to the surface of this basket, suggesting that NUP153 is potentially one of the first pore components an RNA or protein encounters during export. In this study, anti-NUP153 antibodies were used to probe the role of NUP153 in nuclear export in Xenopus oocytes. We found that NUP153 antibodies block three major classes of RNA export, that of snRNA, mRNA, and 5S rRNA. NUP153 antibodies also block the NES protein export pathway, specifically the export of the HIV Rev protein, as well as Rev-dependent RNA export. Not all export was blocked; NUP153 antibodies did not impede the export of tRNA or the recycling of importin beta to the cytoplasm. The specific antibodies used here also did not affect nuclear import, whether mediated by importin alpha/beta or by transportin. Overall, the results indicate that NUP153 is crucial to multiple classes of RNA and protein export, being involved at a vital juncture point in their export pathways. This juncture point appears to be one that is bypassed by tRNA during its export. We asked whether a physical interaction between RNA and NUP153 could be observed, using homoribopolymers as sequence-independent probes for interaction. NUP153, unlike four other nucleoporins including Nup98, associated strongly with poly(G) and significantly with poly(U). Thus, NUP153 is unique among the nucleoporins tested in its ability to interact with RNA and must do so either directly or indirectly through an adaptor protein. These results suggest a unique mechanistic role for NUP153 in the export of multiple cargos.
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Major Binding Sites for the Nuclear Import Receptor Are the Internal Nucleoporin NUP153 and the Adjacent Nuclear Filament Protein Tpr
The Journal of cell biology, 1998Co-Authors: Sundeep Shah, Stuart Tugendreich, Douglass J. ForbesAbstract: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 β45–462 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.
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Separate nuclear import pathways converge on the nucleoporin NUP153 and can be dissected with dominant-negative inhibitors
Current biology : CB, 1998Co-Authors: Sundeep Shah, Douglass J. ForbesAbstract: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.
