scholarly journals The cytoplasmic filaments of the nuclear pore complex are dispensable for selective nuclear protein import

2002 ◽  
Vol 158 (1) ◽  
pp. 63-77 ◽  
Author(s):  
Tobias C. Walther ◽  
Helen S. Pickersgill ◽  
Volker C. Cordes ◽  
Martin W. Goldberg ◽  
Terry D. Allen ◽  
...  
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Import ◽  
Protein Import ◽  
Nuclear Localization Sequence ◽  
Nuclear Pore ◽  
Slight Reduction ◽  
Cytoplasmic Filaments ◽  
Localization Sequence ◽  
Importin Α

The nuclear pore complex (NPC) mediates bidirectional macromolecular traffic between the nucleus and cytoplasm in eukaryotic cells. Eight filaments project from the NPC into the cytoplasm and are proposed to function in nuclear import. We investigated the localization and function of two nucleoporins on the cytoplasmic face of the NPC, CAN/Nup214 and RanBP2/Nup358. Consistent with previous data, RanBP2 was localized at the cytoplasmic filaments. In contrast, CAN was localized near the cytoplasmic coaxial ring. Unexpectedly, extensive blocking of RanBP2 with gold-conjugated antibodies failed to inhibit nuclear import. Therefore, RanBP2-deficient NPCs were generated by in vitro nuclear assembly in RanBP2-depleted Xenopus egg extracts. NPCs were formed that lacked cytoplasmic filaments, but that retained CAN. These nuclei efficiently imported nuclear localization sequence (NLS) or M9 substrates. NPCs lacking CAN retained RanBP2 and cytoplasmic filaments, and showed a minor NLS import defect. NPCs deficient in both CAN and RanBP2 displayed no cytoplasmic filaments and had a strikingly immature cytoplasmic appearance. However, they showed only a slight reduction in NLS-mediated import, no change in M9-mediated import, and were normal in growth and DNA replication. We conclude that RanBP2 is the major nucleoporin component of the cytoplasmic filaments of the NPC, and that these filaments do not have an essential role in importin α/β– or transportin-dependent import.

scholarly journals Monoclonal Antibodies to NTF2 Inhibit Nuclear Protein Import by Preventing Nuclear Translocation of the GTPase Ran

2000 ◽  
Vol 11 (2) ◽  
pp. 703-719 ◽  
Author(s):  
Susanne M. Steggerda ◽  
Ben E. Black ◽  
Bryce M. Paschal
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Import ◽  
Protein Import ◽  
Nuclear Translocation ◽  
Living Cells ◽  
Nuclear Accumulation ◽  
Nuclear Pore ◽  
Permeabilized Cells ◽  
Dependent Protein

Nuclear transport factor 2 (NTF2) is a soluble transport protein originally identified by its ability to stimulate nuclear localization signal (NLS)-dependent protein import in digitonin-permeabilized cells. NTF2 has been shown to bind nuclear pore complex proteins and the GDP form of Ran in vitro. Recently, it has been reported that NTF2 can stimulate the accumulation of Ran in digitonin-permeabilized cells. Evidence that NTF2 directly mediates Ran import or that NTF2 is required to maintain the nuclear concentration of Ran in living cells has not been obtained. Here we show that cytoplasmic injection of anti-NTF2 mAbs resulted in a dramatic relocalization of Ran to the cytoplasm. This provides the first evidence that NTF2 regulates the distribution of Ran in vivo. Moreover, anti-NTF2 mAbs inhibited nuclear import of both Ran and NLS-containing protein in vitro, suggesting that NTF2 stimulates NLS-dependent protein import by driving the nuclear accumulation of Ran. We also show that biotinylated NTF2-streptavidin microinjected into the cytoplasm accumulated at the nuclear envelope, indicating that NTF2 can target a binding partner to the nuclear pore complex. Taken together, our data show that NTF2 is an essential regulator of the Ran distribution in living cells and that NTF2-mediated Ran nuclear import is required for NLS-dependent protein import.

scholarly journals Proteins Connecting the Nuclear Pore Complex with the Nuclear Interior

1999 ◽  
Vol 144 (5) ◽  
pp. 839-855 ◽  
Author(s):  
Caterina Strambio-de-Castillia ◽  
Günter Blobel ◽  
Michael P. Rout
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Import ◽  
Nuclear Localization Sequence ◽  
Nuclear Pore ◽  
Reporter Protein ◽  
Double Deletion ◽  
Large Structures ◽  
Associated Proteins ◽  
Localization Sequence ◽  
Transport Factors

While much has been learned in recent years about the movement of soluble transport factors across the nuclear pore complex (NPC), comparatively little is known about intranuclear trafficking. We isolated the previously identified Saccharomyces protein Mlp1p (myosin-like protein) by an assay designed to find nuclear envelope (NE) associated proteins that are not nucleoporins. We localized both Mlp1p and a closely related protein that we termed Mlp2p to filamentous structures stretching from the nucleoplasmic face of the NE into the nucleoplasm, similar to the homologous vertebrate and Drosophila Tpr proteins. Mlp1p can be imported into the nucleus by virtue of a nuclear localization sequence (NLS) within its COOH-terminal domain. Overexpression experiments indicate that Mlp1p can form large structures within the nucleus which exclude chromatin but appear highly permeable to proteins. Remarkably, cells harboring a double deletion of MLP1 and MLP2 were viable, although they showed a slower net rate of active nuclear import and faster passive efflux of a reporter protein. Our data indicate that the Tpr homologues are not merely NPC-associated proteins but that they can be part of NPC-independent, peripheral intranuclear structures. In addition, we suggest that the Tpr filaments could provide chromatin-free conduits or tracks to guide the efficient translocation of macromolecules between the nucleoplasm and the NPC.

scholarly journals Nuclear import ofArabidopsisPOLY(ADP-RIBOSE) POLYMERASE 2 is mediated by importin-α and a nuclear localization sequence located between the predicted SAP domains

2018 ◽  
Author(s):  
Chao Chen ◽  
Ruth Lintermann ◽  
Lennart Wirthmueller
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Dna Lesions ◽  
Site Directed Mutagenesis ◽  
Covalent Attachment ◽  
Nuclear Localization Sequence ◽  
Diffusion Limit ◽  
Nuclear Pore ◽  
Localization Sequence ◽  
Importin Α

AbstractProteins of the poly(ADP-ribose) polymerase (PARP) family modify target proteins by covalent attachment of ADP-ribose moieties onto amino acid side chains. InArabidopsis, PARP proteins contribute to repair of DNA lesions and modulate plant responses to various abiotic and biotic stressors.ArabidopsisPARP1 and PARP2 are nuclear proteins and given that their molecular weights exceed the diffusion limit of nuclear pore complexes, an active import mechanism into the nucleus is likely. Here we use confocal microscopy of fluorescent protein-taggedArabidopsisPARP2 and PARP2 deletion constructs in combination with site-directed mutagenesis to identify a nuclear localization sequence in PARP2 that is required for nuclear import. We report that in co-immunoprecipitation assays PARP2 interacts with several isoforms of the importin-α group of nuclear transport adapters and that PARP2 binding to IMPORTIN-α2 is mediated by the identified nuclear localization sequence. Our results demonstrate that PARP2 is a cargo protein of the canonical importin-α/β nuclear import pathway.

Novel properties of the protein kinase CK2-site-regulated nuclear- localization sequence of the interferon-induced nuclear factor IFI 16

2000 ◽  
Vol 353 (1) ◽  
pp. 69-77 ◽  
Author(s):  
Lyndall J. BRIGGS ◽  
Ricky W. JOHNSTONE ◽  
Rachel M. ELLIOT ◽  
Chong-Yun XIAO ◽  
Michelle DAWSON ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Protein Kinase Ck2 ◽  
Protein Import ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Nuclear Localization Sequence ◽  
Localization Sequence ◽  
Kinase Ck2

Members of the interferon-induced class of nuclear factors possess a putative CcN motif, comparable with that within proteins such as the simian virus 40 large tumour antigen (T-ag), which confers phosphorylation-mediated regulation of nuclear-localization sequence (NLS)-dependent nuclear import. Here we examine the functionality of the interferon-induced factor 16 (IFI 16) CcN motif, demonstrating its ability to target a heterologous protein to the nucleus, and to be phosphorylated specifically by the CcN-motif-phosphorylating protein kinase CK2 (CK2). The IFI 16 NLS, however, has novel properties, conferring ATP-dependent nuclear import completely independent of cytosolic factors, as well as binding to nuclear components. The IFI 16 NLS is not recognized with high affinity by the NLS-binding importin heterodimer, and transport mediated by it is insensitive to non-hydrolysable GTP analogues. The IFI 16 NLS thus mediates nuclear import through a pathway completely distinct from that of conventional NLSs, such as that of T-ag, but intriguingly resembling that of the NLS of the HIV-1 transactivator protein Tat. Since the IFI 16 CK2 site enhances nuclear import through facilitating binding to nuclear components, this represents a novel mechanism by which the site regulates nuclear-protein import, and constitutes a difference between the IFI 16 and Tat NLSs that may be of importance in the immune response.

scholarly journals Two Isoforms of Npap60 (Nup50) Differentially Regulate Nuclear Protein Import

2010 ◽  
Vol 21 (4) ◽  
pp. 630-638 ◽  
Author(s):  
Yutaka Ogawa ◽  
Yoichi Miyamoto ◽  
Munehiro Asally ◽  
Masahiro Oka ◽  
Yoshinari Yasuda ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Protein Import ◽  
Nuclear Protein ◽  
Time Lapse ◽  
Binding Assays ◽  
Vitro Binding ◽  
Importin Α ◽  
Nuclear Protein Import

Npap60 (Nup50) is a nucleoporin that binds directly to importin α. In humans, there are two Npap60 isoforms: the long (Npap60L) and short (Npap60S) forms. In this study, we provide both in vitro and in vivo evidence that Npap60L and Npap60S function differently in nuclear protein import. In vitro binding assays revealed that Npap60S stabilizes the binding of importin α to classical NLS-cargo, whereas Npap60L promotes the release of NLS-cargo from importin α. In vivo time-lapse experiments showed that when the Npap60 protein level is controlled, allowing CAS to efficiently promote the dissociation of the Npap60/importin α complex, Npap60S and Npap60L suppress and accelerate the nuclear import of NLS-cargo, respectively. These results demonstrate that Npap60L and Npap60S have opposing functions and suggest that Npap60L and Npap60S levels must be carefully controlled for efficient nuclear import of classical NLS-cargo in humans. This study provides novel evidence that nucleoporin expression levels regulate nuclear import efficiency.

scholarly journals Functional Analysis of Tpr: Identification of Nuclear Pore Complex Association and Nuclear Localization Domains and a Role in mRNA Export

1998 ◽  
Vol 143 (7) ◽  
pp. 1801-1812 ◽  
Author(s):  
Peter Bangs ◽  
Brian Burke ◽  
Christine Powers ◽  
Roger Craig ◽  
Aruna Purohit ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Nuclear Pore Complex ◽  
Ectopic Expression ◽  
Coiled Coil ◽  
Full Length ◽  
Nuclear Localization Sequence ◽  
Nuclear Pore ◽  
Nuclear Interior ◽  
Mammalian Cell Lines ◽  
Localization Sequence

Tpr is a 270-kD coiled-coil protein localized to intranuclear filaments of the nuclear pore complex (NPC). The mechanism by which Tpr contributes to the structure and function of the nuclear pore is currently unknown. To gain insight into Tpr function, we expressed the full-length protein and several subdomains in mammalian cell lines and examined their effects on nuclear pore function. Through this analysis, we identified an NH2-terminal domain that was sufficient for association with the nucleoplasmic aspect of the NPC. In addition, we unexpectedly found that the acidic COOH terminus was efficiently transported into the nuclear interior, an event that was apparently mediated by a putative nuclear localization sequence. Ectopic expression of the full-length Tpr caused a dramatic accumulation of poly(A)+ RNA within the nucleus. Similar results were observed with domains that localized to the NPC and the nuclear interior. In contrast, expression of these proteins did not appear to affect nuclear import. These data are consistent with a model in which Tpr is tethered to intranuclear filaments of the NPC by its coiled coil domain leaving the acidic COOH terminus free to interact with soluble transport factors and mediate export of macromolecules from the nucleus.

scholarly journals Yeast N1e3p/Nup170p is required for normal stoichiometry of FG nucleoporins within the nuclear pore complex.

1996 ◽  
Vol 16 (5) ◽  
pp. 2025-2036 ◽  
Author(s):  
M A Kenna ◽  
J G Petranka ◽  
J L Reilly ◽  
L I Davis
Keyword(s):  
Nuclear Pore Complex ◽  
Complementation Group ◽  
Nuclear Localization Sequence ◽  
Nuclear Pore ◽  
Glutathione S Transferase ◽  
Amino Terminal ◽  
Localization Sequence ◽  
Complementation Groups ◽  
Amino Terminal Domain ◽  
Yeast Homolog

The FG nucleoporins are a conserved family of proteins, some of which bind to the nuclear localization sequence receptor, karyopherin. Distinct members of this family are found in each region of the nuclear pore complex (NPC), spanning from the cytoplasmically disposed filaments to the distal end of the nuclear basket. Movement of karyopherin from one FG nucleoporin to the next may be required for translocation of substrates across the NPC. So far, nothing is known about how the FG nucleoporins are localized within the NPC. To identify proteins that interact functionally with one member of this family, the Saccharomyces cerevisiae protein Nup1p, we previously identified 16 complementation groups containing mutants that are lethal in the absence of NUP1 These mutants were referred to as nle (Nup-lethal) mutants. Mutants in the nle3/nlel7 complementation group are lethal in combination with amino-terminal nup1 truncation mutants, which we have previously shown to be defective for localization to the NPC. Here we show that NLE3 (which is allelic to NUP170) encodes a protein with similarity to the mammalian nucleoporin Nup155. We show that Nle3p coprecipitates with glutathione S-transferase fusions containing the amino-terminal domain of Nup1p. Furthermore, a deletion of Nle3p leads to changes in the stoichiometry of several of the XFXFG nucleoporins, including the loss of Nup1p and Nup2p. These results suggest that Nle3p plays a role in localizing specific FG nucleoporins within the NPC. The broad spectrum of synthetic phenotypes observed with the nle3delta mutant provides support for this model. We also identify a redundant yeast homolog that can partially substitute for Nle3p and show that together these proteins are required for viability.

scholarly journals Nuclear Localization Signal and Protein Context both Mediate Importin α Specificity of Nuclear Import Substrates

2006 ◽  
Vol 26 (23) ◽  
pp. 8697-8709 ◽  
Author(s):  
Beate Friedrich ◽  
Christina Quensel ◽  
Thomas Sommer ◽  
Enno Hartmann ◽  
Matthias Köhler
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Protein Import ◽  
Binding Studies ◽  
Vitro Binding ◽  
Importin Α ◽  
Localization Signal ◽  
Experimental Approaches

ABSTRACT The “classical” nuclear protein import pathway depends on importin α and importin β. Importin α binds nuclear localization signal (NLS)-bearing proteins and functions as an adapter to access the importin β-dependent import pathway. In humans, only one importin β is known to interact with importin α, while six α importins have been described. Various experimental approaches provided evidence that several substrates are transported specifically by particular α importins. Whether the NLS is sufficient to mediate importin α specificity is unclear. To address this question, we exchanged the NLSs of two well-characterized import substrates, the seven-bladed propeller protein RCC1, preferentially transported into the nucleus by importin α3, and the less specifically imported substrate nucleoplasmin. In vitro binding studies and nuclear import assays revealed that both NLS and protein context contribute to the specificity of importin α binding and transport.

scholarly journals GTP hydrolysis by Ran occurs at the nuclear pore complex in an early step of protein import.

1995 ◽  
Vol 131 (3) ◽  
pp. 571-581 ◽  
Author(s):  
F Melchior ◽  
T Guan ◽  
N Yokoyama ◽  
T Nishimoto ◽  
L Gerace
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Import ◽  
Protein Import ◽  
Small Gtpase ◽  
Nuclear Pore ◽  
Binding Studies ◽  
Early Step ◽  
Permeabilized Cells ◽  
Complex Protein ◽  
Ran Gtp

Mediated import of proteins into the nucleus involves multiple cytosolic factors, including the small GTPase Ran. Whether Ran functions by interacting with other cytosolic proteins or components of the nuclear pore complex has been unclear. Furthermore, the precise transport step where Ran acts has not been determined. To address these questions, we have analyzed the binding interactions of Ran using permeabilized cells and isolated nuclear envelopes. By light and electron microscope immunolocalization, we have found that Ran accumulates specifically at the cytoplasmic surface of the nuclear pore complex when nuclear import in permeabilized cells is inhibited by nonhydrolyzable analogs of GTP. Ran associates with a peripheral pore complex region that is similar to the area where transport ligands accumulate by depletion of ATP, which arrests an early step of transport. Binding studies with isolated nuclear envelopes in the absence of added cytosol indicate that Ran-GTP directly interacts with a pore complex protein. Using blot overlay techniques, we detected a single prominent polypeptide of isolated nuclear envelopes that binds Ran-GTP. This corresponds to the 358-kD protein RanBP2, a Ran binding pore complex protein recently identified by two-hybrid screening. Thus, RanBP2 is likely to constitute the Ran-GTP-binding site detected at the cytoplasmic periphery of the pore complex. These data support a model in which initial ligand binding to the nuclear pore complex occurs at or near RanBP2, and that hydrolysis of GTP by Ran at this site serves to define commitment to the nuclear import pathway.

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.

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