Scaffold nucleoporins Nup188 and Nup192 share structural and functional properties with nuclear transport receptors

Nucleocytoplasmic transport is mediated by nuclear pore complexes (NPCs) embedded in the nuclear envelope. About 30 different proteins (nucleoporins, nups) arrange around a central eightfold rotational axis to build the modular NPC. Nup188 and Nup192 are related and evolutionary conserved, large nucleoporins that are part of the NPC scaffold. Here we determine the structure of Nup188. The protein folds into an extended stack of helices where an N-terminal 130 kDa segment forms an intricate closed ring, while the C-terminal region is a more regular, superhelical structure. Overall, the structure has distant similarity with flexible S-shaped nuclear transport receptors (NTRs). Intriguingly, like NTRs, both Nup188 and Nup192 specifically bind FG-repeats and are able to translocate through NPCs by facilitated diffusion. This blurs the existing dogma of a clear distinction between stationary nups and soluble NTRs and suggests an evolutionary relationship between the NPC and the soluble nuclear transport machinery.

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O-GlcNAc modification of nuclear pore complexes accelerates bi-directional transport

10.1101/2020.10.09.334029 ◽

2020 ◽

Author(s):

Tae Yeon Yoo ◽

Timothy J Mitchison

Keyword(s):

Nuclear Import ◽

Nuclear Transport ◽

Super Resolution ◽

Nucleocytoplasmic Transport ◽

Nuclear Pore ◽

Permeability Barrier ◽

Nuclear Pore Complexes ◽

Facilitated Diffusion ◽

Import And Export ◽

Pore Complexes

AbstractMacromolecular transport across the nuclear envelope depends on facilitated diffusion through nuclear pore complexes (NPCs). The interior of NPCs contains a permeability barrier made of phenylalanine-glycine (FG) repeat domains that selectively facilitates the permeation of cargoes bound to nuclear transport receptors (NTRs). FG repeats in NPC are a major site of O-linked N-acetylglucosamine (O-GlcNAc) modification, but the functional role of this modification in nucleocytoplasmic transport is unclear. We developed high-throughput assays based on optogenetic probes to quantify the kinetics of nuclear import and export in living human cells. We found that increasing O-GlcNAc modification of the NPC accelerated NTR-facilitated nucleocytoplasmic transport of proteins in both directions, and decreasing modification slowed transport. Super-resolution imaging revealed strong enrichment of O-GlcNAc at the FG-repeat barrier. O-GlcNAc modification also accelerated passive permeation of a small, inert protein through NPCs. We conclude that O-GlcNAc modification accelerates nucleocytoplasmic transport by enhancing the non-specific permeability the FG-repeat barrier, perhaps by steric inhibition of interactions between FG repeats.SummaryNuclear pore complexes mediate nuclear transport and are highly modified with O-linked N-acetylglucosamine (O-GlcNAc) on FG repeat domains. Using a new quantitative live-cell imaging assay, Yoo and Mitchison demonstrate acceleration of nuclear import and export by O-GlcNAc modification.

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Binding Site Distribution of Nuclear Transport Receptors and Transport Complexes in Single Nuclear Pore Complexes

Traffic ◽

10.1111/j.1600-0854.2009.00947.x ◽

2009 ◽

Vol 10 (9) ◽

pp. 1228-1242 ◽

Cited By ~ 15

Author(s):

Martin Kahms ◽

Philipp Lehrich ◽

Jana Hüve ◽

Nils Sanetra ◽

Reiner Peters

Keyword(s):

Binding Site ◽

Nuclear Transport ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Site Distribution ◽

Transport Receptors ◽

Pore Complexes

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Transport into and out of the Nucleus

Microbiology and Molecular Biology Reviews ◽

10.1128/mmbr.65.4.570-594.2001 ◽

2001 ◽

Vol 65 (4) ◽

pp. 570-594 ◽

Cited By ~ 608

Author(s):

Ian G. Macara

Keyword(s):

Rna Processing ◽

Nucleocytoplasmic Transport ◽

Small Gtpase ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Facilitated Diffusion ◽

Transport Mechanisms ◽

Pore Complexes ◽

Transport Of Macromolecules ◽

Gtpase Ran

SUMMARY A defining characteristic of eukaryotic cells is the possession of a nuclear envelope. Transport of macromolecules between the nuclear and cytoplasmic compartments occurs through nuclear pore complexes that span the double membrane of this envelope. The molecular basis for transport has been revealed only within the last few years. The transport mechanism lacks motors and pumps and instead operates by a process of facilitated diffusion of soluble carrier proteins, in which vectoriality is provided by compartment-specific assembly and disassembly of cargo-carrier complexes. The carriers recognize localization signals on the cargo and can bind to pore proteins. They also bind a small GTPase, Ran, whose GTP-bound form is predominantly nuclear. Ran-GTP dissociates import carriers from their cargo and promotes the assembly of export carriers with cargo. The ongoing discovery of numerous carriers, Ran-independent transport mechanisms, and cofactors highlights the complexity of the nuclear transport process. Multiple regulatory mechanisms are also being identified that control cargo-carrier interactions. Circadian rhythms, cell cycle, transcription, RNA processing, and signal transduction are all regulated at the level of nucleocytoplasmic transport. This review focuses on recent discoveries in the field, with an emphasis on the carriers and cofactors involved in transport and on possible mechanisms for movement through the nuclear pores.

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Leukemia-Associated Nup214 Fusion Proteins Disturb the XPO1-Mediated Nuclear-Cytoplasmic Transport Pathway and Thereby the NF-κB Signaling Pathway

Molecular and Cellular Biology ◽

10.1128/mcb.00158-16 ◽

2016 ◽

Vol 36 (13) ◽

pp. 1820-1835 ◽

Cited By ~ 18

Author(s):

Shoko Saito ◽

Sadik Cigdem ◽

Mitsuru Okuwaki ◽

Kyosuke Nagata

Keyword(s):

Signaling Pathway ◽

Nuclear Export ◽

Fusion Proteins ◽

Nuclear Transport ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Transport Pathway ◽

Cytoplasmic Transport ◽

Transport Receptors ◽

Pore Complexes

Nuclear-cytoplasmic transport through nuclear pore complexes is mediated by nuclear transport receptors. Previous reports have suggested that aberrant nuclear-cytoplasmic transport due to mutations or overexpression of nuclear pore complexes and nuclear transport receptors is closely linked to diseases. Nup214, a component of nuclear pore complexes, has been found as chimeric fusion proteins in leukemia. Among various Nup214 fusion proteins, SET-Nup214 and DEK-Nup214 have been shown to be engaged in tumorigenesis, but their oncogenic mechanisms remain unclear. In this study, we examined the functions of the Nup214 fusion proteins by focusing on their effects on nuclear-cytoplasmic transport. We found that SET-Nup214 and DEK-Nup214 interact with exportin-1 (XPO1)/CRM1 and nuclear RNA export factor 1 (NXF1)/TAP, which mediate leucine-rich nuclear export signal (NES)-dependent protein export and mRNA export, respectively. SET-Nup214 and DEK-Nup214 decreased the XPO1-mediated nuclear export of NES proteins such as cyclin B and proteins involved in the NF-κB signaling pathway by tethering XPO1 onto nuclear dots where Nup214 fusion proteins are localized. We also demonstrated that SET-Nup214 and DEK-Nup214 expression inhibited NF-κB-mediated transcription by abnormal tethering of the complex containing p65 and its inhibitor, IκB, in the nucleus. These results suggest that SET-Nup214 and DEK-Nup214 perturb the regulation of gene expression through alteration of the nuclear-cytoplasmic transport system.

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Nucleocytoplasmic transport of macromolecules

Microbiology and Molecular Biology Reviews ◽

10.1128/mmbr.61.2.193-211.1997 ◽

1997 ◽

Vol 61 (2) ◽

pp. 193-211

Author(s):

A H Corbett ◽

P A Silver

Keyword(s):

Nuclear Envelope ◽

Nuclear Transport ◽

Bound State ◽

Nucleocytoplasmic Transport ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Yeast Saccharomyces Cerevisiae ◽

Soluble Factors ◽

Pore Complexes ◽

Transport Of Macromolecules

Nucleocytoplasmic transport is a complex process that consists of the movement of numerous macromolecules back and forth across the nuclear envelope. All macromolecules that move in and out of the nucleus do so via nuclear pore complexes that form large proteinaceous channels in the nuclear envelope. In addition to nuclear pores, nuclear transport of macromolecules requires a number of soluble factors that are found both in the cytoplasm and in the nucleus. A combination of biochemical, genetic, and cell biological approaches have been used to identify and characterize the various components of the nuclear transport machinery. Recent studies have shown that both import to and export from the nucleus are mediated by signals found within the transport substrates. Several studies have demonstrated that these signals are recognized by soluble factors that target these substrates to the nuclear pore. Once substrates have been directed to the pore, most transport events depend on a cycle of GTP hydrolysis mediated by the small Ras-like GTPase, Ran, as well as other proteins that regulate the guanine nucleotide-bound state of Ran. Many of the essential factors have been identified, and the challenge that remains is to determine the exact mechanism by which transport occurs. This review attempts to present an integrated view of our current understanding of nuclear transport while highlighting the contributions that have been made through studies with genetic organisms such as the budding yeast, Saccharomyces cerevisiae.

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The dynamics of karyopherin-mediated nuclear transport

Biochemistry and Cell Biology ◽

10.1139/o01-149 ◽

2001 ◽

Vol 79 (5) ◽

pp. 603-612 ◽

Cited By ~ 18

Author(s):

Marcello Marelli ◽

David J Dilworth ◽

Richard W Wozniak ◽

John D Aitchison

Keyword(s):

Biochemical Characterization ◽

Nucleocytoplasmic Transport ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Complex Interplay ◽

Conventional View ◽

Cytoplasmic Transport ◽

Transport Receptors ◽

Pore Complexes

The regulated exchange of proteins and nucleic acids between the nucleus and cytoplasm demands a complex interplay between nuclear pore complexes (NPCs), which provide conduits in the nuclear envelope, and mobile transport receptors (or karyopherins, also known as importins/exportins) that bind and mediate the translocation of cargoes through the NPCs. Biochemical characterization of individual karyopherins has led to the identification of many of their cargoes and to the elucidation of the mechanisms by which they mediate transport. Likewise, the characterization of numerous NPC-associated components, in combination with structural studies of NPCs, have begun to address the possible mechanisms that drive nucleocytoplasmic transport, and the role that different nucleoporins play in the transport process. Some recent studies indicate that several NPC-associated factors, previously thought to be stable components of the NPC, dynamically interact with both nuclear and cytoplasmic aspects of the NPC. The mobility of these components challenges our conventional view of the NPC as the stationary phase of transport. These components and their potiential roles in nucleo-cytoplasmic transport are discussed.Key words: Nucleocytoplasmic transport, nuclear pore complex, nucleoporin, karyopherin, Nup2p.

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Ran-binding protein 5 (RanBP5) is related to the nuclear transport factor importin-beta but interacts differently with RanBP1.

Molecular and Cellular Biology ◽

10.1128/mcb.17.9.5087 ◽

1997 ◽

Vol 17 (9) ◽

pp. 5087-5096 ◽

Cited By ~ 63

Author(s):

R Deane ◽

W Schäfer ◽

H P Zimmermann ◽

L Mueller ◽

D Görlich ◽

...

Keyword(s):

Nuclear Transport ◽

Binding Protein ◽

Nucleocytoplasmic Transport ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Nucleotide Exchange ◽

Transport Pathway ◽

Importin Beta ◽

Pore Complexes

We report the identification and characterization of a novel 124-kDa Ran binding protein, RanBP5. This protein is related to importin-beta, the key mediator of nuclear localization signal (NLS)-dependent nuclear transport. RanBP5 was identified by two independent methods: it was isolated from HeLa cells by using its interaction with RanGTP in an overlay assay to monitor enrichment, and it was also found by the yeast two-hybrid selection method with RanBP1 as bait. RanBP5 binds to RanBP1 as part of a trimeric RanBP1-Ran-RanBP5 complex. Like importin-beta, RanBP5 strongly binds the GTP-bound form of Ran, stabilizing it against both intrinsic and RanGAP1-induced GTP hydrolysis and also against nucleotide exchange. The GAP resistance of the RanBP5-RanGTP complex can be relieved by RanBP1, which might reflect an in vivo role for RanBP1. RanBP5 is a predominantly cytoplasmic protein that can bind to nuclear pore complexes. We propose that RanBP5 is a mediator of a nucleocytoplasmic transport pathway that is distinct from the importin-alpha-dependent import of proteins with a classical NLS.

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O-GlcNAc modification of nuclear pore complexes accelerates bidirectional transport

The Journal of Cell Biology ◽

10.1083/jcb.202010141 ◽

2021 ◽

Vol 220 (7) ◽

Author(s):

Tae Yeon Yoo ◽

Timothy J. Mitchison

Keyword(s):

Nucleocytoplasmic Transport ◽

Nuclear Pore ◽

Permeability Barrier ◽

Nuclear Pore Complexes ◽

Facilitated Diffusion ◽

Macromolecular Transport ◽

Superresolution Imaging ◽

Bidirectional Transport ◽

Import And Export ◽

Pore Complexes

Macromolecular transport across the nuclear envelope depends on facilitated diffusion through nuclear pore complexes (NPCs). The interior of NPCs contains a permeability barrier made of phenylalanine-glycine (FG) repeat domains that selectively facilitates the permeation of cargoes bound to nuclear transport receptors (NTRs). FG-repeat domains in NPCs are a major site of O-linked N-acetylglucosamine (O-GlcNAc) modification, but the functional role of this modification in nucleocytoplasmic transport is unclear. We developed high-throughput assays based on optogenetic probes to quantify the kinetics of nuclear import and export in living human cells. We found that increasing O-GlcNAc modification of the NPC accelerated NTR-facilitated transport of proteins in both directions, and decreasing modification slowed transport. Superresolution imaging revealed strong enrichment of O-GlcNAc at the FG-repeat barrier. O-GlcNAc modification also accelerated passive permeation of a small, inert protein through NPCs. We conclude that O-GlcNAc modification accelerates nucleocytoplasmic transport by enhancing the nonspecific permeability of the FG-repeat barrier, perhaps by steric inhibition of interactions between FG repeats.

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Mechanosensitivity of nucleocytoplasmic transport

10.1101/2021.07.23.453478 ◽

2021 ◽

Author(s):

Ion Andreu ◽

Ignasi Granero ◽

Nimesh Chahare ◽

Kessem Clein ◽

Marc Molina Jordan ◽

...

Keyword(s):

Molecular Weight ◽

Nucleocytoplasmic Transport ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Facilitated Diffusion ◽

Nuclear Forces ◽

General Applicability ◽

Nuclear Localization Signals ◽

Applied Forces ◽

Pore Complexes

Mechanical force controls fundamental cellular processes in health and disease, and increasing evidence shows that the nucleus both experiences and senses applied forces. Here we show that nuclear forces differentially control both passive and facilitated nucleocytoplasmic transport, setting the rules for the mechanosensitivity of shuttling proteins. We demonstrate that nuclear force increases permeability across nuclear pore complexes, with a dependence on molecular weight that is stronger for passive than facilitated diffusion. Due to this differential effect, force leads to the translocation into or out of the nucleus of cargoes within a given range of molecular weight and affinity for nuclear transport receptors. Further, we show that the mechanosensitivity of several transcriptional regulators can be both explained by this mechanism, and engineered exogenously by introducing appropriate nuclear localization signals. Our work sets a novel framework to understand mechanically induced signalling, with potential general applicability across signalling pathways and pathophysiological scenarios.

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Nup98 FG domains from diverse species spontaneously phase-separate into particles with nuclear pore-like permselectivity

eLife ◽

10.7554/elife.04251 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 119

Author(s):

Hermann Broder Schmidt ◽

Dirk Görlich

Keyword(s):

Aqueous Solutions ◽

Nuclear Transport ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Experimental Conditions ◽

Intrinsically Disordered ◽

Diverse Species ◽

Transport Receptors ◽

Pore Complexes

Nuclear pore complexes (NPCs) conduct massive transport mediated by shuttling nuclear transport receptors (NTRs), while keeping nuclear and cytoplasmic contents separated. The NPC barrier in Xenopus relies primarily on the intrinsically disordered FG domain of Nup98. We now observed that Nup98 FG domains of mammals, lancelets, insects, nematodes, fungi, plants, amoebas, ciliates, and excavates spontaneously and rapidly phase-separate from dilute (submicromolar) aqueous solutions into characteristic ‘FG particles’. This required neither sophisticated experimental conditions nor auxiliary eukaryotic factors. Instead, it occurred already during FG domain expression in bacteria. All Nup98 FG phases rejected inert macromolecules and yet allowed far larger NTR cargo complexes to rapidly enter. They even recapitulated the observations that large cargo-domains counteract NPC passage of NTR⋅cargo complexes, while cargo shielding and increased NTR⋅cargo surface-ratios override this inhibition. Their exquisite NPC-typical sorting selectivity and strong intrinsic assembly propensity suggest that Nup98 FG phases can form in authentic NPCs and indeed account for the permeability properties of the pore.

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