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 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 Regulation of protein kinase Cδ Nuclear Import and Apoptosis by Mechanistic Target of Rapamycin Complex-1

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Antonio Layoun ◽  
Alexander A. Goldberg ◽  
Ayesha Baig ◽  
Mikaela Eng ◽  
Ortal Attias ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Nuclear Import ◽  
Target Of Rapamycin ◽  
Interferon Β ◽  
Structural Mechanism ◽  
Mechanistic Target Of Rapamycin ◽  
Kinase C ◽  
Nuclear Content ◽  
In Cells

AbstractInactivation of the protein complex ‘mechanistic target of rapamycin complex 1’ (mTORC1) can increase the nuclear content of transcriptional regulators of metabolism and apoptosis. Previous studies established that nuclear import of signal transducer and activator of transcription-1 (STAT1) requires the mTORC1-associated adaptor karyopherin-α1 (KPNA1) when mTORC1 activity is reduced. However, the role of other mTORC1-interacting proteins in the complex, including ‘protein kinase C delta’ (PKCδ), have not been well characterized. In this study, we demonstrate that PKCδ, a STAT1 kinase, contains a functional ‘target of rapamycin signaling’ (TOS) motif that directs its interaction with mTORC1. Depletion of KPNA1 by RNAi prevented the nuclear import of PKCδ in cells exposed to the mTORC1 inhibitor rapamycin or amino acid restriction. Mutation of the TOS motif in PKCδ led to its loss of regulation by mTORC1 or karyopherin-α1, resulting in increased constitutive nuclear content. In cells expressing wild-type PKCδ, STAT1 activity and apoptosis were increased by rapamycin or interferon-β. Those expressing the PKCδ TOS mutant exhibited increased STAT1 activity and apoptosis; further enhancement by rapamycin or interferon-β, however, was lost. Therefore, the TOS motif in PKCδ is a novel structural mechanism by which mTORC1 prevents PKCδ and STAT1 nuclear import, and apoptosis.

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.

scholarly journals Inhibition of Nuclear Import and Alteration of Nuclear Pore Complex Composition by Rhinovirus

2002 ◽  
Vol 76 (17) ◽  
pp. 8787-8796 ◽  
Author(s):  
Kurt E. Gustin ◽  
Peter Sarnow
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Import ◽  
Nuclear Proteins ◽  
Nuclear Pore ◽  
Nucleocytoplasmic Trafficking ◽  
The Status ◽  
Infected Cells ◽  
Novel Strategy ◽  
In Cells

ABSTRACT Nucleocytoplasmic trafficking pathways and the status of nuclear pore complex (NPC) components were examined in cells infected with rhinovirus type 14. A variety of shuttling and nonshuttling nuclear proteins, using multiple nuclear import pathways, accumulated in the cytoplasm of cells infected with rhinovirus. An in vitro nuclear import assay with semipermeabilized infected cells confirmed that nuclear import was inhibited and that docking of nuclear import receptor-cargo complexes at the cytoplasmic face of the NPC was prevented in rhinovirus-infected cells. The relocation of cellular proteins and inhibition of nuclear import correlated with the degradation of two NPC components, Nup153 and p62. The degradation of Nup153 and p62 was not due to induction of apoptosis, because p62 was not proteolyzed in apoptotic HeLa cells, and Nup153 was cleaved to produce a 130-kDa cleavage product that was not observed in cells infected with poliovirus or rhinovirus. The finding that both poliovirus and rhinovirus cause inhibition of nuclear import and degradation of NPC components suggests that this may be a common feature of the replicative cycle of picornaviruses. Inhibition of nuclear import is predicted to result in the cytoplasmic accumulation of a large number of nuclear proteins that could have functions in viral translation, RNA synthesis, packaging, or assembly. Additionally, inhibition of nuclear import also presents a novel strategy whereby cytoplasmic RNA viruses can evade host immune defenses by preventing signal transduction into the nucleus.

scholarly journals Pleiotropic nuclear defects associated with a conditional allele of the novel nucleoporin Rat9p/Nup85p.

1996 ◽  
Vol 7 (6) ◽  
pp. 917-934 ◽  
Author(s):  
A L Goldstein ◽  
C A Snay ◽  
C V Heath ◽  
C N Cole
Keyword(s):  
Fluorescent Protein ◽  
Nuclear Pore ◽  
Main Body ◽  
The Novel ◽  
Conditional Allele ◽  
Green Fluorescent ◽  
Nucleocytoplasmic Export ◽  
In Cells

In a screen for mutants defective in nucleocytoplasmic export of mRNA, we have identified a new component of the Saccharomyces cerevisiae nuclear pore complex (NPC). The RAT9/NUP85 (ribonucleic acid trafficking) gene encodes an 84.9-kDa protein that we have localized to NPCs by tagging the RAT9/NUP85 gene with the in vivo molecular marker Green Fluorescent Protein. In cells containing either the rat9-1 allele or a complete deletion of the RAT9/NUP85 gene, poly(A)+ RNA accumulates rapidly in nuclei after a shift from 23 degrees C to 37 degrees C. Under these same conditions, rapid fragmentation of the nucleolus was also observed. At the permissive growth temperature in rat9-1 or RAT9 deletion strains, the nuclear envelope (NE) becomes detached from the main body of the nucleus, forming long thin double sheets of NE. NPCs within these sheets are clustered and some appear to be locked together between opposing sheets of NE such that their nucleoplasmic faces are in contact. The Rat9/Nup85 protein could not be detected in cells carrying a mutation of RAT2/NUP120, suggesting that Rat9p/Nup85p cannot be assembled into NPCs in the absence of Rat2p/Nup120p. In contrast,Rat9/ Nup85 protein was readily incorporated into NPCs in strains carrying mutant alleles of other nucleoporin genes. The possible role of Rat9p/Nup85p in NE integrity and the loss of nucleoporins when another nucleoporin is mutant or absent are discussed.

scholarly journals How SARS-CoV-2 and Other Viruses Build an Invasion Route to Hijack the Host Nucleocytoplasmic Trafficking System

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1424
Author(s):  
Elma Sakinatus Sajidah ◽  
Keesiang Lim ◽  
Richard W. Wong
Keyword(s):  
Nuclear Import ◽  
Nuclear Transport ◽  
Antiviral Treatment ◽  
Nuclear Pore ◽  
Nucleocytoplasmic Trafficking ◽  
Nucleocytoplasmic Shuttling ◽  
Transport Receptors ◽  
Import And Export ◽  
Viral Components ◽  
Host Nucleus

The host nucleocytoplasmic trafficking system is often hijacked by viruses to accomplish their replication and to suppress the host immune response. Viruses encode many factors that interact with the host nuclear transport receptors (NTRs) and the nucleoporins of the nuclear pore complex (NPC) to access the host nucleus. In this review, we discuss the viral factors and the host factors involved in the nuclear import and export of viral components. As nucleocytoplasmic shuttling is vital for the replication of many viruses, we also review several drugs that target the host nuclear transport machinery and discuss their feasibility for use in antiviral treatment.

scholarly journals The L1 stalk is required for efficient export of nascent large ribosomal subunits in yeast

2019 ◽  
Author(s):  
Sharmishtha Musalgaonkar ◽  
Joshua J. Black ◽  
Arlen W. Johnson
Keyword(s):  
Ribosomal Protein ◽  
Nuclear Pore Complex ◽  
Nuclear Export ◽  
Nuclear Pore ◽  
Structural Studies ◽  
Ribosomal Subunits

AbstractThe ribosomal protein Rpl1 (uL1 in universal nomenclature) is essential in yeast and constitutes part of the L1 stalk which interacts with E site ligands on the ribosome. Structural studies of nascent pre-60S complexes in yeast have shown that a domain of the Crm1-dependent nuclear export adapter Nmd3, binds in the E site and interacts with Rpl1, inducing closure of the L1 stalk. Based on this observation, we decided to reinvestigate the role of the L1 stalk in nuclear export of pre-60S subunits despite previous work showing that Rpl1-deficient ribosomes are exported from the nucleus and engage in translation. Large cargoes, such as ribosomal subunits, require multiple export factors to facilitate their transport through the nuclear pore complex. Here, we show that pre-60S subunits lacking Rpl1 or truncated for the RNA of the L1 stalk are exported inefficiently. Surprisingly, this is not due to a measurable defect in recruitment of Nmd3 but appears to result from inefficient recruitment of the Mex67-Mtr2 heterodimer.

scholarly journals Hook3 is a scaffold for the opposite-polarity microtubule-based motors cytoplasmic dynein-1 and KIF1C

2019 ◽  
Vol 218 (9) ◽  
pp. 2982-3001 ◽  
Author(s):  
Agnieszka A. Kendrick ◽  
Andrea M. Dickey ◽  
William B. Redwine ◽  
Phuoc Tien Tran ◽  
Laura Pontano Vaites ◽  
...  
Keyword(s):  
Cytoplasmic Dynein ◽  
Opposite Polarity ◽  
Full Length ◽  
Cell Periphery ◽  
Long Distance ◽  
Short Region ◽  
Cargo Transport ◽  
In Cells

The unidirectional and opposite-polarity microtubule-based motors, dynein and kinesin, drive long-distance intracellular cargo transport. Cellular observations suggest that opposite-polarity motors may be coupled. We recently identified an interaction between the cytoplasmic dynein-1 activating adaptor Hook3 and the kinesin-3 KIF1C. Here, using in vitro reconstitutions with purified components, we show that KIF1C and dynein/dynactin can exist in a complex scaffolded by Hook3. Full-length Hook3 binds to and activates dynein/dynactin motility. Hook3 also binds to a short region in the “tail” of KIF1C, but unlike dynein/dynactin, this interaction does not activate KIF1C. Hook3 scaffolding allows dynein to transport KIF1C toward the microtubule minus end, and KIF1C to transport dynein toward the microtubule plus end. In cells, KIF1C can recruit Hook3 to the cell periphery, although the cellular role of the complex containing both motors remains unknown. We propose that Hook3’s ability to scaffold dynein/dynactin and KIF1C may regulate bidirectional motility, promote motor recycling, or sequester the pool of available dynein/dynactin activating adaptors.

scholarly journals Nup358 and Transportin 1 Cooperate in Adenoviral Genome Import

2020 ◽  
Vol 94 (10) ◽  
Author(s):  
Irene Carlon-Andres ◽  
Floriane Lagadec ◽  
Noémie Pied ◽  
Fabienne Rayne ◽  
Marie-Edith Lafon ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Protein Import ◽  
Productive Infection ◽  
Nuclear Pore ◽  
Time Resolved ◽  
Dna Viruses ◽  
Viral Genomes ◽  
Cellular Factors ◽  
Rate Limiting

ABSTRACT Nuclear import of viral genomes is an important step during the life cycle of adenoviruses (AdV), requiring soluble cellular factors as well as proteins of the nuclear pore complex (NPC). We addressed the role of the cytoplasmic nucleoporin Nup358 during adenoviral genome delivery by performing depletion/reconstitution experiments and time-resolved quantification of adenoviral genome import. Nup358-depleted cells displayed reduced efficiencies of nuclear import of adenoviral genomes, and the nuclear import receptor transportin 1 became rate limiting under these conditions. Furthermore, we identified a minimal N-terminal region of Nup358 that was sufficient to compensate for the import defect. Our data support a model where Nup358 functions as an assembly platform that promotes the formation of transport complexes, allowing AdV to exploit a physiological protein import pathway for accelerated transport of its DNA. IMPORTANCE Nuclear import of viral genomes is an essential step to initiate productive infection for several nuclear replicating DNA viruses. On the other hand, DNA is not a physiological nuclear import substrate; consequently, viruses have to exploit existing physiological transport routes. Here, we show that adenoviruses use the nucleoporin Nup358 to increase the efficiency of adenoviral genome import. In its absence, genome import efficiency is reduced and the transport receptor transportin 1 becomes rate limiting. We show that the N-terminal half of Nup358 is sufficient to drive genome import and identify a transportin 1 binding region. In our model, adenovirus genome import exploits an existing protein import pathway and Nup358 serves as an assembly platform for transport complexes.

scholarly journals Kap120 Functions as a Nuclear Import Receptor for Ribosome Assembly Factor Rpf1 in Yeast

2006 ◽  
Vol 26 (8) ◽  
pp. 3170-3180 ◽  
Author(s):  
Stefanie Caesar ◽  
Markus Greiner ◽  
Gabriel Schlenstedt
Keyword(s):  
Nuclear Import ◽  
Nuclear Export ◽  
Nuclear Accumulation ◽  
Nuclear Pore ◽  
Ribosomal Subunits ◽  
Parallel Import ◽  
Β Protein ◽  
Maturation Factor ◽  
Assembly Factor

ABSTRACT The nucleocytoplasmic exchange of macromolecules is mediated by receptors specialized in passage through the nuclear pore complex. The majority of these receptors belong to the importin β protein family, which has 14 members in Saccharomyces cerevisiae. Nine importins carry various cargos from the cytoplasm into the nucleus, whereas four exportins mediate nuclear export. Kap120 is the only receptor whose transport cargo has not been found previously. Here, we characterize Kap120 as an importin for the ribosome maturation factor Rpf1, which was identified in a two-hybrid screen. Kap120 binds directly to Rpf1 in vitro and is released by Ran-GTP. At least three parallel import pathways exist for Rpf1, since nuclear import is defective in strains with the importins Kap120, Kap114, and Nmd5 deleted. Both kap120 and rpf1 mutants accumulate large ribosomal subunits in the nucleus. The nuclear accumulation of 60S ribosomal subunits in kap120 mutants is abolished upon RPF1 overexpression, indicating that Kap120 does not function in the actual ribosomal export step but rather in import of ribosome maturation factors.

Sign in / Sign up

Export Citation Format

Share Document