scholarly journals Specific Nucleoporin Requirement for Smad Nuclear Translocation

2010 ◽  
Vol 30 (16) ◽  
pp. 4022-4034 ◽  
Author(s):  
Xiaochu Chen ◽  
Lan Xu
Keyword(s):  
Nuclear Localization ◽  
Nuclear Pore Complex ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Transforming Growth Factor ◽  
Nuclear Translocation ◽  
Nuclear Periphery ◽  
Transforming Growth Factor Β ◽  
Nuclear Pore ◽  
Localization Signal

ABSTRACT Cytoplasm-to-nucleus translocation of Smad is a fundamental step in transforming growth factor β (TGF-β) signal transduction. Here we identify a subset of nucleoporins that, in conjunction with Msk (Drosophila Imp7/8), specifically mediate activation-induced nuclear translocation of MAD (Drosophila Smad1) but not the constitutive import of proteins harboring a classic nuclear localization signal (cNLS) or the spontaneous nuclear import of Medea (Drosophila Smad4). Surprisingly, many of these nucleoporins, including Sec13, Nup75, Nup93, and Nup205, are scaffold nucleoporins considered important for the overall integrity of the nuclear pore complex (NPC) but not known to have cargo-specific functions. We demonstrate that the roles of these nucleoporins in supporting Smad nuclear import are separate from their previously assigned functions in NPC assembly. Furthermore, we uncovered novel pathway-specific functions of Sec13 and Nup93; both Sec13 and Nup93 are able to preferentially interact with the phosphorylated/activated form of MAD, and Nup93 acts to recruit the importin Msk to the nuclear periphery. These findings, together with the observation that Sec13 and Nup93 could interact directly with Msk, suggest their direct involvement in the nuclear import of MAD. Thus, we have delineated the nucleoporin requirement of MAD nuclear import, reflecting a unique trans-NPC mechanism.

Nuclear import of glycoconjugates is distinct from the classical NLS pathway

1995 ◽  
Vol 108 (4) ◽  
pp. 1325-1332 ◽  
Author(s):  
E. Duverger ◽  
C. Pellerin-Mendes ◽  
R. Mayer ◽  
A.C. Roche ◽  
M. Monsigny
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Peptide Sequence ◽  
Nuclear Pore ◽  
Dependent Manner ◽  
Permeabilized Cells ◽  
Localization Signal ◽  
Energy Dependent ◽  
Cytosolic Factors

The nuclear import of many proteins depends on a short peptide sequence called the nuclear localization signal. However, glycosylated proteins, which lack such a nuclear localization signal, upon their injection into the cytosol by electroporation, enter the nucleus in a sugar-dependent manner. This paper brings new insights on the mechanism of this process, based on a study of neoglycoprotein nuclear uptake by digitonin-permeabilized cells. The nuclear import of neoglycoproteins is energy dependent: it does not occur when cells are maintained at 4 degrees C or when cells are ATP-depleted by treatment with apyrase. The nuclear import of neoglycoproteins occurs through the nuclear pore: it is inhibited by preincubation of cells with wheat germ agglutinin, a lectin which binds the nuclear pore glycoproteins and blocks the translocation step of nuclear localization signal bearing proteins through the nuclear pore. Furthermore, the nuclear import of neoglycoproteins does not use the pathway of nuclear localization signal bearing proteins: nuclear import of nuclear localization signal bearing proteins depends on cytosolic factors and is inhibited by treatment of cells with N-ethylmaleimide, while the nuclear import of neoglycoproteins neither requires added cytosolic factors nor is sensitive to alkylation by N-ethylmaleimide. In addition, upon incubation in the presence of a large excess of nuclear localization signal bearing protein, the nuclear import of neoglycoproteins is not inhibited.

Nuclear import of protein kinase C occurs by a mechanism distinct from the mechanism used by proteins with a classical nuclear localization signal

1998 ◽  
Vol 111 (13) ◽  
pp. 1823-1830 ◽  
Author(s):  
D. Schmalz ◽  
F. Hucho ◽  
K. Buchner
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Nuclear Localization ◽  
Phorbol Ester ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Nuclear Pore ◽  
Kinase C ◽  
Localization Signal ◽  
Protein Kinase C Alpha

Protein kinase C does not have any known nuclear localization signal but, nevertheless, is redistributed from the cytoplasm to the nucleus upon various stimuli. In NIH 3T3 fibroblasts stimulation with phorbol ester leads to a translocation of protein kinase C alpha to the plasma membrane and into the cell nucleus. We compared the mechanism of protein kinase C alpha's transport into the nucleus with the transport mechanism of a protein with a classical nuclear localization signal at several steps. To this end, we co-microinjected fluorescently labeled bovine serum albumin to which a nuclear localization signal peptide was coupled, together with substances interfering with conventional nuclear protein import. Thereafter, the distribution of both the nuclear localization signal-bearing reporter protein and protein kinase C alpha was analyzed in the same cells. We can show that, in contrast to the nuclear localization signal-dependent transport, the phorbol ester-induced transport of protein kinase C alpha is not affected by microinjection of antibodies against the nuclear import factor p97/importin/karyopherin beta or microinjection of non-hydrolyzable GTP-analogs. This suggests that nuclear import of protein kinase C alpha is independent of p97/importin/karyopherin beta and independent of GTP. At the nuclear pore there are differences between the mechanisms too, since nuclear transport of protein kinase C alpha cannot be inhibited by wheat germ agglutinin or an antibody against nuclear pore complex proteins. Together these findings demonstrate that the nuclear import of protein kinase C alpha occurs by a mechanism distinct from the one used by classical nuclear localization signal-bearing proteins at several stages.

scholarly journals Karyopherins regulate nuclear pore complex barrier and transport function

2017 ◽  
Vol 216 (11) ◽  
pp. 3609-3624 ◽  
Author(s):  
Larisa E. Kapinos ◽  
Binlu Huang ◽  
Chantal Rencurel ◽  
Roderick Y.H. Lim
Keyword(s):  
Nuclear Localization ◽  
Nuclear Pore Complex ◽  
Nuclear Localization Signal ◽  
Barrier Function ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Dependent Manner ◽  
Transport Function ◽  
Guanosine Triphosphate ◽  
Localization Signal

Nucleocytoplasmic transport is sustained by karyopherins (Kaps) and a Ran guanosine triphosphate (RanGTP) gradient that imports nuclear localization signal (NLS)–specific cargoes (NLS-cargoes) into the nucleus. However, how nuclear pore complex (NPC) barrier selectivity, Kap traffic, and NLS-cargo release are systematically linked and simultaneously regulated remains incoherent. In this study, we show that Kapα facilitates Kapβ1 turnover and occupancy at the NPC in a RanGTP-dependent manner that is directly coupled to NLS-cargo release and NPC barrier function. This is underpinned by the binding affinity of Kapβ1 to phenylalanine–glycine nucleoporins (FG Nups), which is comparable with RanGTP·Kapβ1, but stronger for Kapα·Kapβ1. On this basis, RanGTP is ineffective at releasing standalone Kapβ1 from NPCs. Depleting Kapα·Kapβ1 by RanGTP further abrogates NPC barrier function, whereas adding back Kapβ1 rescues it while Kapβ1 turnover softens it. Therefore, the FG Nups are necessary but insufficient for NPC barrier function. We conclude that Kaps constitute integral constituents of the NPC whose barrier, transport, and cargo release functionalities establish a continuum under a mechanism of Kap-centric control.

scholarly journals Identification of a putative nuclear localization signal in maspin protein shed light into its nuclear import regulation

2018 ◽  
Author(s):  
Jeffrey Reina ◽  
Lixin Zhou ◽  
Marcos R.M. Fontes ◽  
Nelly Panté ◽  
Nathalie Cella
Keyword(s):  
Subcellular Localization ◽  
Nuclear Localization ◽  
Hela Cells ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Molecular Mechanisms ◽  
Nuclear Translocation ◽  
Biological Activities ◽  
Dependent Manner ◽  
Localization Signal

AbstractMaspin (SERPINB5) is a potential tumor suppressor gene with pleiotropic biological activities, including regulation of cell proliferation, death, adhesion, migration and gene expression. Several studies suggest that subcellular localization plays an essential role on maspin tumor suppression activity. In this study we investigated the molecular mechanisms underlying maspin nucleocytoplasmic shuttling. Anin vitronuclear-import assay using digitonin-permeabilized HeLa cells demonstrated that maspin enters the nucleus by an energy-and carrier-independent mechanism. However, previous studies indicated that maspin subcellular localization is regulated in the cell. Using a nuclear localization signal (NLS) prediction software, we identified a putative NLS in the maspin amino acid sequence. To distinguish between passive and regulated nuclear translocation, maspinNLS or the full-length protein (MaspinFL) were fused to 5GFP, rendering the construct too large to enter the nucleus passively. Unexpectedly, 5GFP-maspinNLS, but not maspinFL-5GFP, entered the nucleus of HeLa cells. Dominant-negative Ran-GTPase mutants RanQ69L or RanT24N, suppressed 5GFP-maspinNLS nuclear localization. In summary, we provide evidence that maspin translocates to the nucleus passively. In addition, we identified a peptide in the maspin protein sequence, which is able to drive a 5GFP construct to the nucleus in an energy-dependent manner.

scholarly journals RanGTP Targets p97 to RanBP2, a Filamentous Protein Localized at the Cytoplasmic Periphery of the Nuclear Pore Complex

1997 ◽  
Vol 8 (12) ◽  
pp. 2379-2390 ◽  
Author(s):  
Christian Delphin ◽  
Tinglu Guan ◽  
Frauke Melchior ◽  
Larry Gerace
Keyword(s):  
Nuclear Localization ◽  
Nuclear Pore Complex ◽  
Nuclear Localization Signal ◽  
Protein Import ◽  
Critical Role ◽  
In Vitro Assays ◽  
Nuclear Pore ◽  
Localization Signal ◽  
Nuclear Localization Signal Receptor

RanBP2, a protein containing FG repeat motifs and four binding sites for the guanosine triphosphatase Ran, is localized at the cytoplasmic periphery of the nuclear pore complex (NPC) and is believed to play a critical role in nuclear protein import. We purified RanBP2 from rat liver nuclear envelopes and examined its structural and biochemical properties. Electron microscopy showed that RanBP2 forms a flexible filamentous molecule with a length of ∼36 nm, suggesting that it comprises a major portion of the cytoplasmic fibrils implicated in initial binding of import substrates to the NPC. Using in vitro assays, we characterized the ability of RanBP2 to bind p97, a cytosolic factor implicated in the association of the nuclear localization signal receptor with the NPC. We found that RanGTP promotes the binding of p97 to RanBP2, whereas it inhibits the binding of p97 to other FG repeat nucleoporins. These data suggest that RanGTP acts to specifically target p97 to RanBP2, where p97 may support the binding of an nuclear localization signal receptor/substrate complex to RanBP2 in an early step of nuclear import.

scholarly journals SUMO-1 Modification and Its Role in Targeting the Ran GTPase-activating Protein, RanGAP1, to the Nuclear Pore Complex

1998 ◽  
Vol 140 (3) ◽  
pp. 499-509 ◽  
Author(s):  
Michael J. Matunis ◽  
Jian Wu ◽  
Günter Blobel
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Gtpase Activating Protein ◽  
Terminal Domain ◽  
Localization Signal

RanGAP1 is the GTPase-activating protein for Ran, a small ras-like GTPase involved in regulating nucleocytoplasmic transport. In vertebrates, RanGAP1 is present in two forms: one that is cytoplasmic, and another that is concentrated at the cytoplasmic fibers of nuclear pore complexes (NPCs). The NPC-associated form of RanGAP1 is covalently modified by the small ubiquitin-like protein, SUMO-1, and we have recently proposed that SUMO-1 modification functions to target RanGAP1 to the NPC. Here, we identify the domain of RanGAP1 that specifies SUMO-1 modification and demonstrate that mutations in this domain that inhibit modification also inhibit targeting to the NPC. Targeting of a heterologous protein to the NPC depended on determinants specifying SUMO-1 modification and also on additional determinants in the COOH-terminal domain of RanGAP1. SUMO-1 modification and these additional determinants were found to specify interaction between the COOH-terminal domain of RanGAP1 and a region of the nucleoporin, Nup358, between Ran-binding domains three and four. Together, these findings indicate that SUMO-1 modification targets RanGAP1 to the NPC by exposing, or creating, a Nup358 binding site in the COOH-terminal domain of RanGAP1. Surprisingly, the COOH-terminal domain of RanGAP1 was also found to harbor a nuclear localization signal. This nuclear localization signal, and the presence of nine leucine-rich nuclear export signal motifs, suggests that RanGAP1 may shuttle between the nucleus and the cytoplasm.

Nuclear transport of the U2 snRNP-specific U2B'' protein is mediated by both direct and indirect signalling mechanisms

1994 ◽  
Vol 107 (7) ◽  
pp. 1807-1816 ◽  
Author(s):  
C. Kambach ◽  
I.W. Mattaj
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Nuclear Transport ◽  
U2 Snrna ◽  
Central Segment ◽  
U2 Snrnp ◽  
U1 Snrnp ◽  
Localization Signal ◽  
Protein Amino Acids

Experiments investigating the nuclear import of the U2 snRNP-specific B'' protein (U2B'') are presented. U2B'' nuclear transport is shown to be able to occur independently of binding to U2 snRNA. The central segment of the protein (amino acids 90–146) encodes an unusual nuclear localization signal (NLS) that is related to that of the U1 snRNP-specific A protein. However, nuclear import of U2B'' does not depend on this NLS. Sequences in the N-terminal RNP motif of the protein are sufficient to direct nuclear transport, and evidence is presented that the interaction of U2B'' with the U2A' protein mediates this effect. This suggests that U2B'' can ‘piggy-back’ to the nucleus in association with U2A’, and thus be imported to the nucleus by two different mechanisms. U2A' nuclear transport, on the other hand, can occur independently of both U2B'' binding and of U2 snRNA.

scholarly journals SARS-CoV-2 Orf6 hijacks Nup98 to block STAT nuclear import and antagonize interferon signaling

2020 ◽  
Vol 117 (45) ◽  
pp. 28344-28354 ◽  
Author(s):  
Lisa Miorin ◽  
Thomas Kehrer ◽  
Maria Teresa Sanchez-Aparicio ◽  
Ke Zhang ◽  
Phillip Cohen ◽  
...  
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Import ◽  
Nuclear Translocation ◽  
Nuclear Pore ◽  
Interferon Signaling ◽  
Antiviral Signaling ◽  
Pathogenic Viruses ◽  
Transcriptional Induction ◽  
Global Health Problem ◽  
Viral Accessory Protein

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic that is a serious global health problem. Evasion of IFN-mediated antiviral signaling is a common defense strategy that pathogenic viruses use to replicate and propagate in their host. In this study, we show that SARS-CoV-2 is able to efficiently block STAT1 and STAT2 nuclear translocation in order to impair transcriptional induction of IFN-stimulated genes (ISGs). Our results demonstrate that the viral accessory protein Orf6 exerts this anti-IFN activity. We found that SARS-CoV-2 Orf6 localizes at the nuclear pore complex (NPC) and directly interacts with Nup98-Rae1 via its C-terminal domain to impair docking of cargo-receptor (karyopherin/importin) complex and disrupt nuclear import. In addition, we show that a methionine-to-arginine substitution at residue 58 impairs Orf6 binding to the Nup98-Rae1 complex and abolishes its IFN antagonistic function. All together our data unravel a mechanism of viral antagonism in which a virus hijacks the Nup98-Rae1 complex to overcome the antiviral action of IFN.

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