scholarly journals Probing the nucleoporin FG repeat network defines structural and functional features of the nuclear pore complex

2011 ◽  
Vol 195 (2) ◽  
pp. 183-192 ◽  
Author(s):  
Philipp Stelter ◽  
Ruth Kunze ◽  
Jessica Fischer ◽  
Ed Hurt
Keyword(s):  
Nuclear Pore Complex ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Binding Motif ◽  
Dynein Light Chain ◽  
Transport Channel ◽  
Functional Features ◽  
And Function ◽  
Insight Into

Unraveling the organization of the FG repeat meshwork that forms the active transport channel of the nuclear pore complex (NPC) is key to understanding the mechanism of nucleocytoplasmic transport. In this paper, we develop a tool to probe the FG repeat network in living cells by modifying FG nucleoporins (Nups) with a binding motif (engineered dynein light chain–interacting domain) that can drag several copies of an interfering protein, Dyn2, into the FG network to plug the pore and stop nucleocytoplasmic transport. Our method allows us to specifically probe FG Nups in vivo, which provides insight into the organization and function of the NPC transport channel.

scholarly journals Targeting and function in mRNA export of nuclear pore complex protein Nup153.

1996 ◽  
Vol 134 (5) ◽  
pp. 1141-1156 ◽  
Author(s):  
R Bastos ◽  
A Lin ◽  
M Enarson ◽  
B Burke
Keyword(s):  
Nuclear Pore Complex ◽  
Protein Import ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Terminal Domain ◽  
General Decline ◽  
Complex Protein ◽  
Pore Complexes ◽  
And Function

Nup153 is a large (153 kD) O-linked glyco-protein which is a component of the basket structure located on the nucleoplasmic face of nuclear pore complexes. This protein exhibits a tripartite structure consisting of a zinc finger domain flanked by large (60-70 kD) NH2- and COOH-terminal domains. When full-length human Nup153 is expressed in BHK cells, it accumulates appropriately at the nucleoplasmic face of the nuclear envelope. Targeting information for Nup153 resides in the NH2-terminal domain since this region of the molecule can direct an ordinarily cytoplasmic protein, pyruvate kinase, to the nuclear face of the nuclear pore complex. Overexpression of Nup153 results in the dramatic accumulation of nuclear poly (A)+ RNA, suggesting an inhibition of RNA export from the nucleus. This is not due to a general decline in nucleocytoplasmic transport or to occlusion or loss of nuclear pore complexes since nuclear protein import is unaffected. While overexpression of certain Nup153 constructs was found to result in the formation of unusual intranuclear membrane arrays, this structural phenotype could not be correlated with the effects on poly (A)+ RNA distribution. The RNA trafficking defect was, however, dependent upon the Nup153 COOH-terminal domain which contains most of the XFXFG repeats. It is proposed that this region of Nup153, lying within the distal ring of the nuclear basket, represents a docking site for mRNA molecules exiting the nucleus.

scholarly journals Mapping the native organization of the yeast nuclear pore complex using nuclear radial intensity measurements

2019 ◽  
Vol 116 (29) ◽  
pp. 14606-14613 ◽  
Author(s):  
Pascal Vallotton ◽  
Sasikumar Rajoo ◽  
Matthias Wojtynek ◽  
Evgeny Onischenko ◽  
Annemarie Kralt ◽  
...  
Keyword(s):  
Nuclear Pore Complex ◽  
Nucleocytoplasmic Transport ◽  
Live Cells ◽  
Nuclear Pore ◽  
Intrinsically Disordered ◽  
Intensity Measurements ◽  
Composition And Structure ◽  
Associated Proteins ◽  
Multiple Copies

Selective transport across the nuclear envelope (NE) is mediated by the nuclear pore complex (NPC), a massive ∼100-MDa assembly composed of multiple copies of ∼30 nuclear pore proteins (Nups). Recent advances have shed light on the composition and structure of NPCs, but approaches that could map their organization in live cells are still lacking. Here, we introduce an in vivo method to perform nuclear radial intensity measurements (NuRIM) using fluorescence microscopy to determine the average position of NE-localized proteins along the nucleocytoplasmic transport axis. We apply NuRIM to study the organization of the NPC and the mobile transport machinery in budding yeast. This reveals a unique snapshot of the intact yeast NPC and identifies distinct steady-state localizations for various NE-associated proteins and nuclear transport factors. We find that the NPC architecture is robust against compositional changes and could also confirm that in contrast to Chlamydomonas reinhardtii, the scaffold Y complex is arranged symmetrically in the yeast NPC. Furthermore, NuRIM was applied to probe the orientation of intrinsically disordered FG-repeat segments, providing insight into their roles in selective NPC permeability and structure.

scholarly journals The FG-repeat asymmetry of the nuclear pore complex is dispensable for bulk nucleocytoplasmic transport in vivo

2004 ◽  
Vol 167 (4) ◽  
pp. 583-590 ◽  
Author(s):  
Bryan Zeitler ◽  
Karsten Weis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Pore Complex ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Nucleocytoplasmic Trafficking ◽  
Transport Pathways ◽  
Pore Complexes ◽  
Biased Distribution

Nucleocytoplasmic transport occurs through gigantic proteinaceous channels called nuclear pore complexes (NPCs). Translocation through the NPC is exquisitely selective and is mediated by interactions between soluble transport carriers and insoluble NPC proteins that contain phenylalanine-glycine (FG) repeats. Although most FG nucleoporins (Nups) are organized symmetrically about the planar axis of the nuclear envelope, very few localize exclusively to one side of the NPC. We constructed Saccharomyces cerevisiae mutants with asymmetric FG repeats either deleted or swapped to generate NPCs with inverted FG asymmetry. The mutant Nups localize properly within the NPC and exhibit exchanged binding specificity for the export factor Xpo1. Surprisingly, we were unable to detect any defects in the Kap95, Kap121, Xpo1, or mRNA transport pathways in cells expressing the mutant FG Nups. These findings suggest that the biased distribution of FG repeats is not required for major nucleocytoplasmic trafficking events across the NPC.

scholarly journals Architecture of the fungal nuclear pore inner ring complex

Science ◽  
2015 ◽  
Vol 350 (6256) ◽  
pp. 56-64 ◽  
Author(s):  
Tobias Stuwe ◽  
Christopher J. Bley ◽  
Karsten Thierbach ◽  
Stefan Petrovic ◽  
Sandra Schilbach ◽  
...  
Keyword(s):  
Nuclear Pore Complex ◽  
Diffusion Barrier ◽  
Three Dimensional ◽  
Interaction Network ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Transport Channel ◽  
Ring Complex ◽  
And Diffusion

The nuclear pore complex (NPC) constitutes the sole gateway for bidirectional nucleocytoplasmic transport. We present the reconstitution and interdisciplinary analyses of the ~425-kilodalton inner ring complex (IRC), which forms the central transport channel and diffusion barrier of the NPC, revealing its interaction network and equimolar stoichiometry. The Nsp1•Nup49•Nup57 channel nucleoporin heterotrimer (CNT) attaches to the IRC solely through the adaptor nucleoporin Nic96. The CNT•Nic96 structure reveals that Nic96 functions as an assembly sensor that recognizes the three-dimensional architecture of the CNT, thereby mediating the incorporation of a defined CNT state into the NPC. We propose that the IRC adopts a relatively rigid scaffold that recruits the CNT to primarily form the diffusion barrier of the NPC, rather than enabling channel dilation.

scholarly journals A monoclonal antibody against the nuclear pore complex inhibits nucleocytoplasmic transport of protein and RNA in vivo.

1988 ◽  
Vol 107 (4) ◽  
pp. 1289-1297 ◽  
Author(s):  
C Featherstone ◽  
M K Darby ◽  
L Gerace
Keyword(s):  
Monoclonal Antibody ◽  
Ribosomal Rna ◽  
Nuclear Pore Complex ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Small Proteins

A monoclonal antibody that reacts with proteins in the nuclear pore complex of rat liver (Snow, C. M., A. Senior, and L. Gerace. 1987. J. Cell Biol. 104:1143-1156) has been shown to cross react with similar components in Xenopus oocytes, as determined by immunofluorescence microscopy and immunoblotting. We have microinjected the antibody into oocytes to study the possible role of these polypeptides in nucleocytoplasmic transport. The antibody inhibits import of a large nuclear protein, nucleoplasmin, in a time- and concentration-dependent manner. It also inhibits export of 5S ribosomal RNA and mature tRNA, but has no effect on transcription or intranuclear tRNA processing. The antibody does not affect the rate of diffusion into the nucleus of two small proteins, myoglobin and ovalbumin, indicating that antibody binding does not result in occlusion of the channel for diffusion. This suggests that inhibition of protein and RNA transport occurs by binding of the antibody at or near components of the pore that participate in mediated transport.

scholarly journals Nup180, a novel nuclear pore complex protein localizing to the cytoplasmic ring and associated fibrils.

1993 ◽  
Vol 123 (6) ◽  
pp. 1345-1354 ◽  
Author(s):  
N Wilken ◽  
U Kossner ◽  
J L Senécal ◽  
U Scheer ◽  
M C Dabauvalle
Keyword(s):  
Nuclear Pore Complex ◽  
Mammalian Cells ◽  
Protein Transport ◽  
Nucleocytoplasmic Transport ◽  
Transport Processes ◽  
Nuclear Pore ◽  
Nuclear Surface ◽  
Apparent Lack

Using an autoimmune serum from a patient with overlap connective tissue disease we have identified by biochemical and immunocytochemical approaches an evolutionarily conserved nuclear pore complex (NPC) protein with an estimated molecular mass of 180 kD and an isoelectric point of approximately 6.2 which we have designated as nup180. Extraction of isolated nuclear envelopes with 2 M urea and chromatography of the solubilized proteins on WGA-Sepharose demonstrated that nup180 is a peripheral membrane protein and does not react with WGA. Affinity-purified antibodies yielded a punctate immunofluorescent pattern of the nuclear surface of mammalian cells and stained brightly the nuclear envelope of cryosectioned Xenopus oocytes. Nuclei reconstituted in vitro in Xenopus egg extract were also stained in the characteristic punctate fashion. Immunogold EM localized nup180 exclusively to the cytoplasmic ring of NPCs and short fibers emanating therefrom into the cytoplasm. Antibodies to nup180 did not inhibit nuclear protein transport in vivo nor in vitro. Despite the apparent lack of involvement in NPC assembly or nucleocytoplasmic transport processes, the conservation of nup180 across species and its exclusive association with the NPC cytoplasmic ring suggests an important, though currently undefined function for this novel NPC protein.

scholarly journals Proteomic analysis of the mammalian nuclear pore complex

2002 ◽  
Vol 158 (5) ◽  
pp. 915-927 ◽  
Author(s):  
Janet M. Cronshaw ◽  
Andrew N. Krutchinsky ◽  
Wenzhu Zhang ◽  
Brian T. Chait ◽  
Michael J. Matunis
Keyword(s):  
Nuclear Pore Complex ◽  
Proteomic Analysis ◽  
Nucleocytoplasmic Transport ◽  
Structure And Function ◽  
Nuclear Pore ◽  
Allgrove Syndrome ◽  
Associated Proteins ◽  
Wd Repeat ◽  
Protein Components ◽  
And Function

As the sole site of nucleocytoplasmic transport, the nuclear pore complex (NPC) has a vital cellular role. Nonetheless, much remains to be learned about many fundamental aspects of NPC function. To further understand the structure and function of the mammalian NPC, we have completed a proteomic analysis to identify and classify all of its protein components. We used mass spectrometry to identify all proteins present in a biochemically purified NPC fraction. Based on previous characterization, sequence homology, and subcellular localization, 29 of these proteins were classified as nucleoporins, and a further 18 were classified as NPC-associated proteins. Among the 29 nucleoporins were six previously undiscovered nucleoporins and a novel family of WD repeat nucleoporins. One of these WD repeat nucleoporins is ALADIN, the gene mutated in triple-A (or Allgrove) syndrome. Our analysis defines the proteome of the mammalian NPC for the first time and paves the way for a more detailed characterization of NPC structure and function.

scholarly journals A coarse-grained computational model of the nuclear pore complex predicts Phe-Gly nucleoporin dynamics

2017 ◽  
Vol 149 (10) ◽  
pp. 951-966 ◽  
Author(s):  
Joan Pulupa ◽  
Manas Rachh ◽  
Michael D. Tomasini ◽  
Joshua S. Mincer ◽  
Sanford M. Simon
Keyword(s):  
Computational Model ◽  
Nuclear Pore Complex ◽  
Coarse Grained ◽  
Nuclear Pore ◽  
Experimental Measurements ◽  
Central Channel ◽  
Computational Simulations ◽  
Cargo Transport ◽  
And Function

The phenylalanine-glycine–repeat nucleoporins (FG-Nups), which occupy the lumen of the nuclear pore complex (NPC), are critical for transport between the nucleus and cytosol. Although NPCs differ in composition across species, they are largely conserved in organization and function. Transport through the pore is on the millisecond timescale. Here, to explore the dynamics of nucleoporins on this timescale, we use coarse-grained computational simulations. These simulations generate predictions that can be experimentally tested to distinguish between proposed mechanisms of transport. Our model reflects the conserved structure of the NPC, in which FG-Nup filaments extend into the lumen and anchor along the interior of the channel. The lengths of the filaments in our model are based on the known characteristics of yeast FG-Nups. The FG-repeat sites also bind to each other, and we vary this association over several orders of magnitude and run 100-ms simulations for each value. The autocorrelation functions of the orientation of the simulated FG-Nups are compared with in vivo anisotropy data. We observe that FG-Nups reptate back and forth through the NPC at timescales commensurate with experimental measurements of the speed of cargo transport through the NPC. Our results are consistent with models of transport where FG-Nup filaments are free to move across the central channel of the NPC, possibly informing how cargo might transverse the NPC.

scholarly journals C9orf72 poly(GR) aggregation induces TDP-43 proteinopathy

2020 ◽  
Vol 12 (559) ◽  
pp. eabb3774 ◽  
Author(s):  
Casey N. Cook ◽  
Yanwei Wu ◽  
Hana M. Odeh ◽  
Tania F. Gendron ◽  
Karen Jansen-West ◽  
...  
Keyword(s):  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Independent Manner ◽  
Neurofilament Light ◽  
Inclusion Formation ◽  
Repeat Expansions ◽  
Transport Factors ◽  
Insight Into ◽  
Lateral Sclerosis

TAR DNA-binding protein 43 (TDP-43) inclusions are a pathological hallmark of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), including cases caused by G4C2 repeat expansions in the C9orf72 gene (c9FTD/ALS). Providing mechanistic insight into the link between C9orf72 mutations and TDP-43 pathology, we demonstrated that a glycine-arginine repeat protein [poly(GR)] translated from expanded G4C2 repeats was sufficient to promote aggregation of endogenous TDP-43. In particular, toxic poly(GR) proteins mediated sequestration of full-length TDP-43 in an RNA-independent manner to induce cytoplasmic TDP-43 inclusion formation. Moreover, in GFP-(GR)200 mice, poly(GR) caused the mislocalization of nucleocytoplasmic transport factors and nuclear pore complex proteins. These mislocalization events resulted in the aberrant accumulation of endogenous TDP-43 in the cytoplasm where it co-aggregated with poly(GR). Last, we demonstrated that treating G4C2 repeat–expressing mice with repeat-targeting antisense oligonucleotides lowered poly(GR) burden, which was accompanied by reduced TDP-43 pathology and neurodegeneration, including lowering of plasma neurofilament light (NFL) concentration. These results contribute to clarification of the mechanism by which poly(GR) drives TDP-43 proteinopathy, confirm that G4C2-targeted therapeutics reduce TDP-43 pathology in vivo, and demonstrate that alterations in plasma NFL provide insight into the therapeutic efficacy of disease-modifying treatments.

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