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 The Nup358-RanGAP Complex Is Required for Efficient Importin α/β-dependent Nuclear Import

2008 ◽  
Vol 19 (5) ◽  
pp. 2300-2310 ◽  
Author(s):  
Saskia Hutten ◽  
Annette Flotho ◽  
Frauke Melchior ◽  
Ralph H. Kehlenbach
Keyword(s):  
Nuclear Import ◽  
Protein Transport ◽  
Nucleocytoplasmic Transport ◽  
Limiting Factor ◽  
Dual Function ◽  
Nuclear Pore ◽  
Transport Reaction ◽  
Importin Α

In vertebrate cells, the nucleoporin Nup358/RanBP2 is a major component of the filaments that emanate from the nuclear pore complex into the cytoplasm. Nup358 forms a complex with SUMOylated RanGAP1, the GTPase activating protein for Ran. RanGAP1 plays a pivotal role in the establishment of a RanGTP gradient across the nuclear envelope and, hence, in the majority of nucleocytoplasmic transport pathways. Here, we investigate the roles of the Nup358-RanGAP1 complex and of soluble RanGAP1 in nuclear protein transport, combining in vivo and in vitro approaches. Depletion of Nup358 by RNA interference led to a clear reduction of importin α/β-dependent nuclear import of various reporter proteins. In vitro, transport could be partially restored by the addition of importin β, RanBP1, and/or RanGAP1 to the transport reaction. In intact Nup358-depleted cells, overexpression of importin β strongly stimulated nuclear import, demonstrating that the transport receptor is the most rate-limiting factor at reduced Nup358-concentrations. As an alternative approach, we used antibody-inhibition experiments. Antibodies against RanGAP1 inhibited the enzymatic activity of soluble and nuclear pore–associated RanGAP1, as well as nuclear import and export. Although export could be fully restored by soluble RanGAP, import was only partially rescued. Together, these data suggest a dual function of the Nup358-RanGAP1 complex as a coordinator of importin β recycling and reformation of novel import complexes.

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 Charge influences substrate recognition and self-assembly of hydrophobic FG sequences

2016 ◽  
Author(s):  
Wesley G. Chen ◽  
Jacob Witten ◽  
Scott C. Grindy ◽  
Niels Holten-Andersen ◽  
Katharina Ribbeck
Keyword(s):  
Amino Acid ◽  
Self Assembly ◽  
Amino Acid Sequences ◽  
Nuclear Pore ◽  
Selective Binding ◽  
Charged Amino Acids ◽  
Essential Components ◽  
Transport Receptors

AbstractThe nuclear pore complex controls the passage of molecules via hydrophobic phenylalanine-glycine (FG) domains on nucleoporins. Such FG-domains consist of repeating units of FxFG, FG, or GLFG sequences, which can be interspersed with highly charged amino acid sequences. Despite the high density of charge exhibited in certain FG-domains, if and how charge influences FG-domain self-assembly and selective binding of nuclear transport receptors is largely unexplored. Studying how individual charged amino acids contribute to nuclear pore selectivity is challenging with modern in vivo and in vitro techniques due to the complexity of nucleoporin sequences. Here, we present a rationally designed approach to deconstruct essential components of nucleoporins down to 14 amino acid sequences. With these nucleoporin-based peptides, we systematically dissect how charge type and placement of charge influences self-assembly and selective binding of FG-containing gels. Specifically, we find that charge type determines which hydrophobic substrates FG sequences recognize while spatial localization of charge tunes hydrophobic self-assembly and receptor selectivity of FG sequences.

scholarly journals Expression Analysis and Nuclear Import Study of Full-length Isoforms Importin α as 6x Histidin-tagged Fusion Protein on the Intracellular Localization of Recombinant HBV Core Protein

2015 ◽  
Vol 10 (1) ◽  
Author(s):  
Aris Haryanto
Keyword(s):  
Fusion Protein ◽  
Nuclear Import ◽  
Recombinant Dna ◽  
Core Protein ◽  
Intracellular Localization ◽  
Full Length ◽  
Nuclear Pore ◽  
E Coli ◽  
Importin Α ◽  
Localization Signal

Isoform importin α molecules play a central role in the classical nuclear import pathway, that occurs throughthe nuclear pore complex (NPC) and typically requires a specific nuclear localization signal (NLS). In this study,it was investigated the role of isoforms importin α in the nuclear import of wild type recombinant hepatitis B viruscore protein (WT rHBc), phosphorylated recombinant HBV core (rHBc) and recombinant HBV core without NLSby co-immunoprecipitation. Four recombinant full-length isoforms importin α as 6x histidin-tagged fusion proteinwere expressed and analysed from expression plasmid vectors Rch1, pHM 1969, pHM 1967 and pHM 1965. Theresults indicated that importin α-1, importin α-3, importin α-4 and importin α-5 can be expressed and isolatedfrom E. coli transformed recombinant DNA plasmid as protein in size around 58-60 kDa. By the nuclear transportstudy shown that isoforms importin α are involved in the nuclear import of WT rHBc, phosphorylated rHBc andrHBc without NLS. It also indicated that they have an important role for nuclear transport of from cytoplasm intothe nucleus.Keywords: NPC, NLS, importin α, importin β, isoforms importin α as 6x histidin-tagged fusion protein, WTrHBc, SV40 Tag, co-immunoprecipitation, westernblotting.

scholarly journals Nuclear Import of IκBα Is Accomplished by a Ran-Independent Transport Pathway

2000 ◽  
Vol 20 (5) ◽  
pp. 1571-1582 ◽  
Author(s):  
Shrikesh Sachdev ◽  
Sriparna Bagchi ◽  
Donna D. Zhang ◽  
Angela C. Mings ◽  
Mark Hannink
Keyword(s):  
Nuclear Import ◽  
Nuclear Export ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Nuclear Pore ◽  
Transport Pathway ◽  
Repeat Domain ◽  
Nuclear Export Receptor

ABSTRACT The inhibitor of kappa B alpha (IκBα) protein is able to shuttle between the cytoplasm and the nucleus. We have utilized a combination of in vivo and in vitro approaches to provide mechanistic insight into nucleocytoplasmic shuttling by IκBα. IκBα contains multiple functional domains that contribute to shuttling of IκBα between the cytoplasm and the nucleus. Nuclear import of IκBα is mediated by the central ankyrin repeat domain. Similar to previously described nuclear import pathways, nuclear import of IκBα is temperature and ATP dependent and is blocked by a dominant-negative mutant of importin β. However, in contrast to classical nuclear import pathways, nuclear import of IκBα is independent of soluble cytosolic factors and is not blocked by the dominant-negative RanQ69L protein. Nuclear export of IκBα is mediated by an N-terminal nuclear export sequence. Nuclear export of IκBα requires the CRM1 nuclear export receptor and is blocked by the dominant-negative RanQ69L protein. Our results are consistent with a model in which nuclear import of IκBα is mediated through direct interactions with components of the nuclear pore complex, while nuclear export of IκBα is mediated via a CRM1-dependent pathway.

scholarly journals Mutations in Tap Uncouple RNA Export Activity from Translocation through the Nuclear Pore Complex

2006 ◽  
Vol 17 (2) ◽  
pp. 931-943 ◽  
Author(s):  
Lyne Lévesque ◽  
Yeou-Cherng Bor ◽  
Leah H. Matzat ◽  
Li Jin ◽  
Stephen Berberoglu ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Point Mutations ◽  
Nuclear Pore ◽  
Nucleocytoplasmic Shuttling ◽  
Transport Receptors ◽  
Cellular Mrnas ◽  
Rna Export ◽  
Rna Complex ◽  
Transport Receptor

Interactions between transport receptors and phenylalanine-glycine (FG) repeats on nucleoporins drive the translocation of receptor-cargo complexes through nuclear pores. Tap, a transport receptor that mediates nuclear export of cellular mRNAs, contains a UBA-like and NTF2-like folds that can associate directly with FG repeats. In addition, two nuclear export sequences (NESs) within the NTF2-like region can also interact with nucleoporins. The Tap-RNA complex was shown to bind to three nucleoporins, Nup98, p62, and RanBP2, and these interactions were enhanced by Nxt1. Mutations in the Tap-UBA region abolished interactions with all three nucleoporins, whereas the effect of point mutations within the NTF2-like domain of Tap known to disrupt Nxt1 binding or nucleoporin binding were nucleoporin dependent. A mutation in any of these Tap domains was sufficient to reduce RNA export but was not sufficient to disrupt Tap interaction with the NPC in vivo or its nucleocytoplasmic shuttling. However, shuttling activity was reduced or abolished by combined mutations within the UBA and either the Nxt1-binding domain or NESs. These data suggest that Tap requires both the UBA- and NTF2-like domains to mediate the export of RNA cargo, but can move through the pores independently of these domains when free of RNA cargo.

scholarly journals Cse1p Is Involved in Export of Yeast Importin α from the Nucleus

1998 ◽  
Vol 18 (11) ◽  
pp. 6805-6815 ◽  
Author(s):  
Jens Solsbacher ◽  
Patrick Maurer ◽  
F. Ralf Bischoff ◽  
Gabriel Schlenstedt
Keyword(s):  
Nuclear Export ◽  
Fluorescent Protein ◽  
Nuclear Pore ◽  
Wild Type ◽  
Importin Α ◽  
Localization Signal ◽  
Green Fluorescent ◽  
Mutant Cells

ABSTRACT Proteins bearing a nuclear localization signal (NLS) are targeted to the nucleus by the heterodimeric transporter importin. Importin α binds to the NLS and to importin β, which carries it through the nuclear pore complex (NPC). Importin disassembles in the nucleus, evidently by binding of RanGTP to importin β. The importin subunits are exported separately. We investigated the role of Cse1p, theSaccharomyces cerevisiae homologue of human CAS, in nuclear export of Srp1p (yeast importin α). Cse1p is located predominantly in the nucleus but also is present in the cytoplasm and at the NPC. We analyzed the in vivo localization of the importin subunits fused to the green fluorescent protein in wild-type and cse1-1 mutant cells. Srp1p but not importin β accumulated in nuclei ofcse1-1 mutants, which are defective in NLS import but not defective in NLS-independent import pathways. Purified Cse1p binds with high affinity to Srp1p only in the presence of RanGTP. The complex is dissociated by the cytoplasmic RanGTP-binding protein Yrb1p. Combined with the in vivo results, this suggests that a complex containing Srp1p, Cse1p, and RanGTP is exported from the nucleus and is subsequently disassembled in the cytoplasm by Yrb1p. The formation of the trimeric Srp1p-Cse1p-RanGTP complex is inhibited by NLS peptides, indicating that only NLS-free Srp1p will be exported to the cytoplasm.

scholarly journals Simple kinetic relationships and nonspecific competition govern nuclear import rates in vivo

2006 ◽  
Vol 175 (4) ◽  
pp. 579-593 ◽  
Author(s):  
Benjamin L. Timney ◽  
Jaclyn Tetenbaum-Novatt ◽  
Diana S. Agate ◽  
Rosemary Williams ◽  
Wenzhu Zhang ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Nuclear Pore Complex ◽  
Nuclear Import ◽  
Yeast Cells ◽  
Limiting Factor ◽  
Nuclear Pore ◽  
Nuclear Localization Signals ◽  
The Poor ◽  
Cytoplasmic Proteins

Many cargoes destined for nuclear import carry nuclear localization signals that are recognized by karyopherins (Kaps). We present methods to quantitate import rates and measure Kap and cargo concentrations in single yeast cells in vivo, providing new insights into import kinetics. By systematically manipulating the amounts, types, and affinities of Kaps and cargos, we show that import rates in vivo are simply governed by the concentrations of Kaps and their cargo and the affinity between them. These rates fit to a straightforward pump–leak model for the import process. Unexpectedly, we deduced that the main limiting factor for import is the poor ability of Kaps and cargos to find each other in the cytoplasm in a background of overwhelming nonspecific competition, rather than other more obvious candidates such as the nuclear pore complex and Ran. It is likely that most of every import round is taken up by Kaps and nuclear localization signals sampling other cytoplasmic proteins as they locate each other in the cytoplasm.

scholarly journals GTP-dependent Binding and Nuclear Transport of RNA Polymerase II by Npa3 Protein

2011 ◽  
Vol 286 (41) ◽  
pp. 35553-35561 ◽  
Author(s):  
Lidija Staresincic ◽  
Jane Walker ◽  
A. Barbara Dirac-Svejstrup ◽  
Richard Mitter ◽  
Jesper Q. Svejstrup
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Nuclear Import ◽  
Chromatin Immunoprecipitation ◽  
Nuclear Transport ◽  
Proteomic Screen ◽  
Genome Wide ◽  
Importin Α

We identified XAB1 in a proteomic screen for factors that interact with human RNA polymerase II (RNAPII). Because XAB1 has a conserved Saccharomyces cerevisiae homologue called Npa3, yeast genetics and biochemical analysis were used to dissect the significance of the interaction. Degron-dependent Npa3 depletion resulted in genome-wide transcription decreases, correlating with a loss of RNAPII from genes as measured by chromatin immunoprecipitation. Surprisingly, however, transcription in vitro was unaffected by Npa3, suggesting that it affects a process that is not required for transcription in yeast extracts. Indeed, Npa3 depletion in vivo affects nuclear localization of RNAPII; the polymerase accumulates in the cytoplasm. Npa3 is a member of the GPN-LOOP family of GTPases. Npa3 mutants that either cannot bind GTP or that bind but cannot hydrolyze it are inviable and unable to support nuclear transport of RNAPII. Surprisingly, we were unable to detect interactions between Npa3 and proteins in the classical importin α/β pathway for nuclear import. Interestingly, Npa3-RNAPII binding is significantly increased by the addition of GTP or its slowly hydrolyzable analogue guanosine 5′-3-O-(thio)triphosphate (GTPγS). Moreover, the Npa3 mutant that binds GTP, but cannot hydrolyze it, binds RNAPII even in the absence of added GTP, whereas the mutant that cannot bind GTP is unable to bind the polymerase. Together, our data suggest that Npa3 defines an unconventional pathway for nuclear import of RNAPII, which involves GTP-dependent binding of Npa3 to the polymerase.

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.

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