scholarly journals Molecular determinants of large cargo transport into the nucleus

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Giulia Paci ◽  
Tiantian Zheng ◽  
Joana Caria ◽  
Anton Zilman ◽  
Edward A Lemke
Keyword(s):  
Nuclear Import ◽  
Nucleocytoplasmic Transport ◽  
Biophysical Model ◽  
Nuclear Pore ◽  
Cargo Transport ◽  
Transport Receptors ◽  
Molecular Determinants ◽  
Quantitative Understanding ◽  
Kinetics Of

Nucleocytoplasmic transport is tightly regulated by the nuclear pore complex (NPC). Among the thousands of molecules that cross the NPC, even very large (>15 nm) cargoes such as pathogens, mRNAs and pre-ribosomes can pass the NPC intact. For these cargoes, there is little quantitative understanding of the requirements for their nuclear import, especially the role of multivalent binding to transport receptors via nuclear localisation sequences (NLSs) and the effect of size on import efficiency. Here, we assayed nuclear import kinetics of 30 large cargo models based on four capsid-like particles in the size range of 17–36 nm, with tuneable numbers of up to 240 NLSs. We show that the requirements for nuclear transport can be recapitulated by a simple two-parameter biophysical model that correlates the import flux with the energetics of large cargo transport through the NPC. Together, our results reveal key molecular determinants of large cargo import in cells.

scholarly journals Molecular determinants of large cargo transport into the nucleus

2019 ◽  
Author(s):  
Giulia Paci ◽  
Edward A Lemke
Keyword(s):  
Nuclear Import ◽  
Size Range ◽  
Nuclear Pore ◽  
Ribosomal Subunits ◽  
Cargo Transport ◽  
Transport Receptors ◽  
Molecular Determinants ◽  
Quantitative Understanding ◽  
In Cells

AbstractTransport of molecules between the nucleus and the cytoplasm is tightly regulated by the nuclear pore complex (NPC). Even very large cargoes such as many pathogens, mRNAs and pre-ribosomal subunits can pass the NPC intact. Compared to small import complexes, for such large cargoes >15 nm there is very little quantitative understanding of the mechanism for efficient transport, the role of multivalent binding to nuclear transport receptors via nuclear localisation sequences (NLSs) and effects of size differences. Here, we assayed nuclear import kinetics in cells for a total of 30 large cargo models based on four capsid-like particles in the size range of 17-36 nm, with tuneable numbers of up to 240 NLSs. We show that the requirements for transport scale non-linearly with size and obey a minimal cut off of functional import requiring more than 10 NLS in the lowest case. Together, our results reveal the key molecular determinants on large cargo import kinetics in cells.

scholarly journals Cargo transport through the nuclear pore complex at a glance

2021 ◽  
Vol 134 (2) ◽  
pp. jcs247874
Author(s):  
Giulia Paci ◽  
Joana Caria ◽  
Edward A. Lemke
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Transport ◽  
Cell Cycle Control ◽  
Genetic Material ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Cargo Transport ◽  
Transport Receptors ◽  
Bidirectional Transport ◽  
Transport Of Macromolecules

ABSTRACTBidirectional transport of macromolecules across the nuclear envelope is a hallmark of eukaryotic cells, in which the genetic material is compartmentalized inside the nucleus. The nuclear pore complex (NPC) is the major gateway to the nucleus and it regulates nucleocytoplasmic transport, which is key to processes including transcriptional regulation and cell cycle control. Accordingly, components of the nuclear transport machinery are often found to be dysregulated or hijacked in diseases. In this Cell Science at a Glance article and accompanying poster, we provide an overview of our current understanding of cargo transport through the NPC, from the basic transport signals and machinery to more emerging aspects, all from a ‘cargo perspective’. Among these, we discuss the transport of large cargoes (>15 nm), as well as the roles of different cargo properties to nuclear transport, from size and number of bound nuclear transport receptors (NTRs), to surface and mechanical properties.

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 Simple biophysics underpins collective conformations of the intrinsically disordered proteins of the Nuclear Pore Complex

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Andrei Vovk ◽  
Chad Gu ◽  
Michael G Opferman ◽  
Larisa E Kapinos ◽  
Roderick YH Lim ◽  
...  
Keyword(s):  
Spatial Organization ◽  
Intrinsically Disordered Proteins ◽  
Nucleocytoplasmic Transport ◽  
Transport Proteins ◽  
Disordered Proteins ◽  
Biophysical Model ◽  
Nuclear Pore ◽  
Intrinsically Disordered

Nuclear Pore Complexes (NPCs) are key cellular transporter that control nucleocytoplasmic transport in eukaryotic cells, but its transport mechanism is still not understood. The centerpiece of NPC transport is the assembly of intrinsically disordered polypeptides, known as FG nucleoporins, lining its passageway. Their conformations and collective dynamics during transport are difficult to assess in vivo. In vitro investigations provide partially conflicting results, lending support to different models of transport, which invoke various conformational transitions of the FG nucleoporins induced by the cargo-carrying transport proteins. We show that the spatial organization of FG nucleoporin assemblies with the transport proteins can be understood within a first principles biophysical model with a minimal number of key physical variables, such as the average protein interaction strengths and spatial densities. These results address some of the outstanding controversies and suggest how molecularly divergent NPCs in different species can perform essentially the same function.

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

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.

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.

Nucleocytoplasmic transport in yeast: a few roles for many actors

2010 ◽  
Vol 38 (1) ◽  
pp. 273-277 ◽  
Author(s):  
Jindriska Fiserova ◽  
Martin W. Goldberg
Keyword(s):  
Nuclear Pore Complex ◽  
Model Organism ◽  
Nucleocytoplasmic Transport ◽  
Present Review ◽  
Eukaryotic Cells ◽  
Nuclear Pore ◽  
Controlled Processes ◽  
Bidirectional Transport

Eukaryotic cells have developed a series of highly controlled processes of transport between the nucleus and cytoplasm. The present review focuses on the latest advances in our understanding of nucleocytoplasmic exchange of molecules in yeast, a widely studied model organism in the field. It concentrates on the role of individual proteins such as nucleoporins and karyopherins in the translocation process and relates this to how the organization of the nuclear pore complex effectively facilitates the bidirectional transport between the two compartments.

scholarly journals C9orf72 arginine-rich dipeptide repeat proteins disrupt karyopherin-mediated nuclear import

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Lindsey R Hayes ◽  
Lauren Duan ◽  
Kelly Bowen ◽  
Petr Kalab ◽  
Jeffrey D Rothstein
Keyword(s):  
Nuclear Import ◽  
Genetic Modifier ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Dipeptide Repeat Proteins ◽  
Hexanucleotide Repeat ◽  
Repeat Proteins ◽  
Importin Α ◽  
Dipeptide Repeat

Disruption of nucleocytoplasmic transport is increasingly implicated in the pathogenesis of neurodegenerative diseases, including ALS caused by a C9orf72 hexanucleotide repeat expansion. However, the mechanism(s) remain unclear. Karyopherins, including importin β and its cargo adaptors, have been shown to co-precipitate with the C9orf72 arginine-containing dipeptide repeat proteins (R-DPRs), poly-glycine arginine (GR) and poly-proline arginine (PR), and are protective in genetic modifier screens. Here, we show that R-DPRs interact with importin β, disrupt its cargo loading, and inhibit nuclear import of importin β, importin α/β, and transportin cargoes in permeabilized mouse neurons and HeLa cells, in a manner that can be rescued by RNA. Although R-DPRs induce widespread protein aggregation in this in vitro system, transport disruption is not due to nucleocytoplasmic transport protein sequestration, nor blockade of the phenylalanine-glycine (FG)-rich nuclear pore complex. Our results support a model in which R-DPRs interfere with cargo loading on karyopherins.

scholarly journals Kinetics of the multitasking high-affinity Win binding site of WDR5 in restricted and unrestricted conditions

2021 ◽  
Author(s):  
Ali Imran ◽  
Brandon S. Moyer ◽  
Ashley J. Canning ◽  
Dan Kalina ◽  
Thomas M Duncan ◽  
...  
Keyword(s):  
Binding Site ◽  
Primary Role ◽  
Dissociation Kinetics ◽  
High Affinity ◽  
Histone 3 ◽  
Quantitative Understanding ◽  
Slow Dissociation ◽  
Kinetics Of ◽  
Association Kinetics

Recent advances in quantitative proteomics show that WD40 proteins play a pivotal role in numerous cellular networks. Yet, they have been fairly unexplored and their physical associations with other proteins are ambiguous. A quantitative understanding of these interactions has wide-ranging significance. WD40 repeat protein 5 (WDR5) interacts with all members of human SET1/MLL methyltransferases, which regulate methylation of the histone 3 lysine 4 (H3K4). Here, using real-time binding measurements in a high-throughput setting, we identified the kinetic fingerprint of  transient associations between WDR5 and 14-residue WDR5 interaction (Win) motif peptides of each SET1 protein (SET1Win). Our results reveal that the high-affinity WDR5-SET1Win interactions feature slow association kinetics. This finding is likely due to the requirement of SET1Win to insert into the narrow WDR5 cavity, also named the Win binding site. Furthermore, our explorations indicate fairly slow dissociation kinetics. This conclusion is in accordance with the primary role of WDR5 in maintaining the functional integrity of a large multisubunit complex, which regulates the histone methylation. Because the Win binding site is considered a key therapeutic target, the immediate outcomes of this study could form the basis for accelerated developments in medical biotechnology.

The molecular mechanism of translocation through the nuclear pore complex is highly conserved

2002 ◽  
Vol 115 (14) ◽  
pp. 2997-3005
Author(s):  
Carl Feldherr ◽  
Debra Akin ◽  
Trevor Littlewood ◽  
Murray Stewart
Keyword(s):  
Nuclear Import ◽  
Amoeba Proteus ◽  
Nuclear Pore ◽  
Hnrnp A1 ◽  
Transport Mechanisms ◽  
Evolutionary Changes ◽  
Transport Receptors ◽  
Importin Α ◽  
Transport Factors

In this report we investigated the activity of vertebrate nuclear transport factors in a primitive organism, Amoeba proteus, to better understand evolutionary changes in the transport mechanisms of organisms expected to have different requirements for nucleocytoplasmic exchange. It was initially determined that FxFG-containing nucleoporins and Ran, both of which are essential for nuclear import in vertebrates, as well as yeast, are also present and functional in amoebae. This suggests that there are fundamental similarities in the transport process; however, there are also significant differences. Transport substrates containing either the hnRNP A1 M9 shuttling signal (a GST/GFP/M9 fusion protein) or the classical bipartite NLS (colloidal gold coated with BSA-bipartite NLS conjugates), both of which are effectively transported in vertebrate cells, are excluded from the nucleus when microinjected into amoebae. However, when these substrates are injected along with transportin or importin α/β, respectively, the vertebrate receptors for these signals, they readily accumulate in the nucleoplasm. These results indicate that although the molecular recognition of substrates is not well conserved between vertebrates and amoebae, vertebrate transport receptors are functional in A. proteus, showing that the translocation machinery is highly conserved. Since selected nuclear import pathways can be investigated in the absence of competing endogenous transport, A. proteus might provide a useful in vivo system for investigating specific molecular interactions involved in trafficking.

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