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 Architecture of the Xenopus nuclear pore complex revealed by three-dimensional cryo-electron microscopy

1993 ◽  
Vol 122 (1) ◽  
pp. 1-19 ◽  
Author(s):  
CW Akey ◽  
M Radermacher
Keyword(s):  
Electron Microscopy ◽  
Nuclear Envelope ◽  
Nuclear Pore Complex ◽  
Three Dimensional ◽  
Nucleocytoplasmic Transport ◽  
Domain Model ◽  
Nuclear Pore ◽  
Transport Channel ◽  
Macromolecular Transport ◽  
Cryo Electron Microscopy

The nuclear pore complex spans the nuclear envelope and functions as a macromolecular transporter in the ATP-dependent process of nucleocytoplasmic transport. In this report, we present three dimensional (3D) structures for both membrane-associated and detergent-extracted Xenopus NPCs, imaged in frozen buffers by cryo-electron microscopy. A comparison of the differing configurations present in the 3D maps suggests that the spokes may possess an intrinsic conformational flexibility. When combined with recent data from a 3D map of negatively stained NPCs (Hinshaw, J. E., B. O. Carragher, and R. A. Milligan. 1992. Cell. 69:1133-1141), these observations suggest a minimal domain model for the spoke-ring complex which may account for the observed plasticity of this assembly. Moreover, lumenal domains in adjacent spokes are interconnected by radial arm dimers, forming a lumenal ring that may be responsible for anchoring the NPC within the nuclear envelope pore. Importantly, the NPC transporter is visualized as a centrally tapered cylinder that spans the entire width of the NPC, in a direction normal to the nuclear envelope. The central positioning, tripartite structure, and hollow nature of the transporter suggests that it may form a macromolecular transport channel, with a globular gating domain at each end. Finally, the packing of the transporter within the spokes creates a set of eight internal channels that may be responsible, in part, for the diffusion of ions and small molecules across the nuclear envelope.

scholarly journals Role of the Ndc1 interaction network in yeast nuclear pore complex assembly and maintenance

2009 ◽  
Vol 185 (3) ◽  
pp. 475-491 ◽  
Author(s):  
Evgeny Onischenko ◽  
Leslie H. Stanton ◽  
Alexis S. Madrid ◽  
Thomas Kieselbach ◽  
Karsten Weis
Keyword(s):  
Nuclear Pore Complex ◽  
Structural Integrity ◽  
Fluorescent Protein ◽  
Interaction Network ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Binding Partners ◽  
Interaction Partners ◽  
Redundant Functions

The nuclear pore complex (NPC) mediates all nucleocytoplasmic transport, yet its structure and biogenesis remain poorly understood. In this study, we have functionally characterized interaction partners of the yeast transmembrane nucleoporin Ndc1. Ndc1 forms a distinct complex with the transmembrane proteins Pom152 and Pom34 and two alternative complexes with the soluble nucleoporins Nup53 and Nup59, which in turn bind to Nup170 and Nup157. The transmembrane and soluble Ndc1-binding partners have redundant functions at the NPC, and disruption of both groups of interactions causes defects in Ndc1 targeting and in NPC structure accompanied by significant pore dilation. Using photoconvertible fluorescent protein fusions, we further show that the depletion of Pom34 in cells that lack NUP53 and NUP59 blocks new NPC assembly and leads to the reversible accumulation of newly made nucleoporins in cytoplasmic foci. Therefore, Ndc1 together with its interaction partners are collectively essential for the biosynthesis and structural integrity of yeast NPCs.

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.

Structure, assembly and interactions of the nuclear lamina and the nuclear pore complex

1992 ◽  
Vol 50 (1) ◽  
pp. 490-491
Author(s):  
N. Panté ◽  
M. Jarnik ◽  
E. Heitlinger ◽  
U. Aebi
Keyword(s):  
Nuclear Pore Complex ◽  
Divalent Cations ◽  
Nuclear Lamina ◽  
Dynamic Structure ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Cytoplasmic Filaments ◽  
Radial Spokes ◽  
Nuclear Ring ◽  
Central Plug

The nuclear pore complex (NPC) is a ∼120 MD supramolecular machine implicated in nucleocytoplasmic transport, that is embedded in the double-membraned nuclear envelope (NE). The basic framework of the ∼120 nm diameter NPC consists of a 32 MD cytoplasmic ring, a 66 MD ‘plug-spoke’ assembly, and a 21 MD nuclear ring. The ‘central plug’ seen in en face views of the NPC reveals a rather variable appearance indicating that it is a dynamic structure. Projecting from the cytoplasmic ring are 8 short, twisted filaments (Fig. 1a), whereas the nuclear ring is topped with a ‘fishtrap’ made of 8 thin filaments that join distally to form a fragile, 30-50 nm distal diameter ring centered above the NPC proper (Fig. 1b). While the cytoplasmic filaments are sensitive to proteases, they as well as the nuclear fishtraps are resistant to RNase treatment. Removal of divalent cations destabilizes the distal rings and thereby opens the fishtraps, addition causes them to reform. Protruding from the tips of the radial spokes into perinuclear space are ‘knobs’ that might represent the large lumenal domain of gp210, a membrane-spanning glycoprotein (Fig. 1c) which, in turn, may play a topogenic role in membrane folding and/or act as a membrane-anchoring site for the NPC. The lectin wheat germ agglutinin (WGA) which is known to recognize the ‘nucleoporins’, a family of glycoproteins having O-linked N-acetyl-glucosamine, is found in two locations on the NPC (Fig. 1. d-f): (i) whereas the cytoplasmic filaments appear unlabelled (Fig. 1d&e), WGA-gold labels sites between the central plug and the cytoplasmic ring (Fig. le; i.e., at a radius of 25-35 nm), and (ii) it decorates the distal ring of the nuclear fishtraps (Fig. 1, d&f; arrowheads).

scholarly journals Monoclonal antibodies identify a group of nuclear pore complex glycoproteins.

1987 ◽  
Vol 104 (5) ◽  
pp. 1143-1156 ◽  
Author(s):  
C M Snow ◽  
A Senior ◽  
L Gerace
Keyword(s):  
Monoclonal Antibodies ◽  
Nuclear Envelope ◽  
Nuclear Pore Complex ◽  
Peptide Mapping ◽  
Polyclonal Antibodies ◽  
Integral Membrane Protein ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Nuclear Surface ◽  
Punctate Pattern

Using monoclonal antibodies we identified a group of eight polypeptides of rat liver nuclear envelopes that have common epitopes. Most or all of these proteins are structurally distinct, as shown by tryptic peptide mapping and analysis with polyclonal antibodies. While these polypeptides are relatively tightly bound to nuclear membranes, only one is an integral membrane protein. The eight antigens cofractionate with the nuclear pore complex under various conditions of ionic strength and detergent. It can be seen by immunofluorescence microscopy that the monoclonal antibodies reacting with these antigens stain the nuclear surface of interphase cells in a finely punctate pattern. When the nuclear envelope is disassembled and subsequently reformed during mitosis, the proteins are reversibly dispersed throughout the cytoplasm in the form of minute foci. By EM immunogold localization on isolated nuclear envelopes, the monoclonal antibodies label exclusively the nuclear pore complex, at both its nucleoplasmic and cytoplasmic margins. Considered together, our biochemical and localization data indicate that the eight nuclear envelope polypeptides are pore complex components. As shown in the accompanying paper (Holt, G. D., C. M. Snow, A. Senior, R. S. Haltiwanger, L. Gerace, and G. W. Hart, J. Cell Biol., 104:1157-1164) these eight polypeptides contain a novel form of glycosylation, O-linked N-acetylglucosamine. The relative abundance and disposition of these O-linked glycoproteins in the pore complex are consistent with their having a role in nucleocytoplasmic transport.

The nuclear pore complex: three-dimensional surface structure revealed by field emission, in-lens scanning electron microscopy, with underlying structure uncovered by proteolysis

1993 ◽  
Vol 106 (1) ◽  
pp. 261-274 ◽  
Author(s):  
M.W. Goldberg ◽  
T.D. Allen
Keyword(s):  
Nuclear Pore Complex ◽  
Three Dimensional ◽  
Underlying Structure ◽  
Nuclear Pore ◽  
Electron Microscopic ◽  
Biologically Relevant ◽  
Fine Grained ◽  
Outer Periphery ◽  
Electron Microscopes ◽  
Scanning Electron

The structure of the nuclear pore complex (NPC) has been previously studied by many different electron microscopic techniques. Recently, scanning electron microscopes have been developed that can visualise biologically relevant structural detail at the same level of resolution as transmission electron microscopes and have been used to study NPC structure. We have used such an instrument to visualise directly the structure of both cytoplasmic and nucleoplasmic surfaces of the NPC of manually isolated amphibian oocyte nuclear envelopes that have been spread, fixed, critical point dried and coated with a thin fine-grained film of chromium or tantalum. We present images that directly show features of the NPC that are visible at each surface, including coaxial rings, cytoplasmic particles, plug/spoke complexes and the nucleoplasmic basket or fishtrap. Some cytoplasmic particles are rod-shaped or possibly “T”-shaped, can be quite long structures extending into the cytoplasm and may be joined to the coaxial ring at a position between each subunit. Both coaxial rings, which are proud of the membranes, can be exposed by light proteolytic digestion, revealing eight equal subunits each of which may be bipartite. We have determined that the nucleoplasmic filaments that make up the baskets are attached to the outer periphery of the coaxial ring at a position between each of its subunits. These filaments extend into the nucleoplasm and insert at the distal end to the smaller basket ring. The space left between adjacent basket filaments would exclude particles bigger than about 25 nm, which is consistent with the exclusion limit previously found for NPC-transported molecules.

scholarly journals A nanobody suite for yeast scaffold nucleoporins provides details of the nuclear pore complex structure

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Sarah A. Nordeen ◽  
Kasper R. Andersen ◽  
Kevin E. Knockenhauer ◽  
Jessica R. Ingram ◽  
Hidde L. Ploegh ◽  
...  
Keyword(s):  
Nuclear Pore Complex ◽  
Binding Sites ◽  
Complex Structure ◽  
Constitutive Expression ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Modular Assembly ◽  
High Affinity ◽  
Ring Complex ◽  
Pore Complexes

AbstractNuclear pore complexes (NPCs) are the main conduits for molecular exchange across the nuclear envelope. The NPC is a modular assembly of ~500 individual proteins, called nucleoporins or nups. Most scaffolding nups are organized in two multimeric subcomplexes, the Nup84 or Y complex and the Nic96 or inner ring complex. Working in S. cerevisiae, and to study the assembly of these two essential subcomplexes, we here develop a set of twelve nanobodies that recognize seven constituent nucleoporins of the Y and Nic96 complexes. These nanobodies all bind specifically and with high affinity. We present structures of several nup-nanobody complexes, revealing their binding sites. Additionally, constitutive expression of the nanobody suite in S. cerevisiae detect accessible and obstructed surfaces of the Y complex and Nic96 within the NPC. Overall, this suite of nanobodies provides a unique and versatile toolkit for the study of the NPC.

scholarly journals Nuclear actin and myosin as control elements in nucleocytoplasmic transport.

1986 ◽  
Vol 102 (3) ◽  
pp. 859-862 ◽  
Author(s):  
M Schindler ◽  
L W Jiang
Keyword(s):  
Nuclear Pore Complex ◽  
Nucleocytoplasmic Transport ◽  
Rabbit Serum ◽  
Nuclear Pore ◽  
Enhancement Effect ◽  
Flux Rate ◽  
Effective Transport ◽  
Liver Nuclei ◽  
Stimulation Of

Fluorescence redistribution after photobleaching (FRAP) was used to examine the role of actin and myosin in the transport of dextrans through the nuclear pore complex. Anti-actin antibodies added to isolated rat liver nuclei significantly reduced the flux rate of fluorescently labeled 64-kD dextrans. The addition of 3 mM ATP to nuclei, which enhances the flux rate in control nuclei by approximately 250%, had no enhancement effect in the presence of either anti-actin or anti-myosin antibody. Phalloidin (10 microM) and cytochalasin D (1 micrograms/ml) individually inhibited the ATP stimulation of transport. Rabbit serum, anti-fibronectin, and anti-lamins A and C antibodies had no effect on transport. These results suggest a model for nuclear transport in which actin/myosin are involved in an ATP-dependent process that alters the effective transport rate across the nuclear pore complex.

scholarly journals The nuclear basket proteins Mlp1p and Mlp2p are part of a dynamic interactome including Esc1p and the proteasome

2013 ◽  
Vol 24 (24) ◽  
pp. 3920-3938 ◽  
Author(s):  
Mario Niepel ◽  
Kelly R. Molloy ◽  
Rosemary Williams ◽  
Julia C. Farr ◽  
Anne C. Meinema ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nuclear Pore Complex ◽  
Nuclear Export ◽  
Regulation Of Gene Expression ◽  
Nuclear Pore ◽  
Docking Site ◽  
Discrete Structure ◽  
Transport Channel ◽  
Macromolecular Complexes ◽  
Nuclear Stability

The basket of the nuclear pore complex (NPC) is generally depicted as a discrete structure of eight protein filaments that protrude into the nucleoplasm and converge in a ring distal to the NPC. We show that the yeast proteins Mlp1p and Mlp2p are necessary components of the nuclear basket and that they also embed the NPC within a dynamic protein network, whose extended interactome includes the spindle organizer, silencing factors, the proteasome, and key components of messenger ribonucleoproteins (mRNPs). Ultrastructural observations indicate that the basket reduces chromatin crowding around the central transporter of the NPC and might function as a docking site for mRNP during nuclear export. In addition, we show that the Mlps contribute to NPC positioning, nuclear stability, and nuclear envelope morphology. Our results suggest that the Mlps are multifunctional proteins linking the nuclear transport channel to multiple macromolecular complexes involved in the regulation of gene expression and chromatin maintenance.

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