scholarly journals Promotion of importin α–mediated nuclear import by the phosphorylation-dependent binding of cargo protein to 14-3-3

2005 ◽  
Vol 169 (3) ◽  
pp. 415-424 ◽  
Author(s):  
Christian Faul ◽  
Stefan Hüttelmaier ◽  
Jun Oh ◽  
Virginie Hachet ◽  
Robert H. Singer ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Nuclear Export ◽  
Protein Targeting ◽  
Underlying Mechanism ◽  
Intracellular Protein ◽  
Regulatory Processes ◽  
Cargo Protein ◽  
Cargo Proteins ◽  
Importin Α ◽  
Localization Signal

14-3-3 proteins are phosphoserine/threonine-binding proteins that play important roles in many regulatory processes, including intracellular protein targeting. 14-3-3 proteins can anchor target proteins in the cytoplasm and in the nucleus or can mediate their nuclear export. So far, no role for 14-3-3 in mediating nuclear import has been described. There is also mounting evidence that nuclear import is regulated by the phosphorylation of cargo proteins, but the underlying mechanism remains elusive. Myopodin is a dual-compartment, actin-bundling protein that functions as a tumor suppressor in human bladder cancer. In muscle cells, myopodin redistributes between the nucleus and the cytoplasm in a differentiation-dependent and stress-induced fashion. We show that importin α binding and the subsequent nuclear import of myopodin are regulated by the serine/threonine phosphorylation-dependent binding of myopodin to 14-3-3. These results establish a novel paradigm for the promotion of nuclear import by 14-3-3 binding. They provide a molecular explanation for the phosphorylation-dependent nuclear import of nuclear localization signal-containing cargo proteins.

scholarly journals Functional requirement of the Arabidopsis importin-α nuclear transport receptor family in autoimmunity mediated by the NLR protein SNC1

2020 ◽  
Author(s):  
Daniel Lüdke ◽  
Charlotte Roth ◽  
Sieglinde A. Kamrad ◽  
Jana Messerschmidt ◽  
Denise Hartken ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Nuclear Import ◽  
Nuclear Transport ◽  
Basal Resistance ◽  
Protein Protein Interaction ◽  
Cargo Proteins ◽  
Importin Α ◽  
Localization Signal ◽  
Receptor Mutant ◽  
Transport Receptor

SUMMARYIMPORTIN-α3/MOS6 (MODIFIER OF SNC1, 6) is one of nine importin-α isoforms in Arabidopsis that recruit nuclear localization signal (NLS)-containing cargo proteins to the nuclear import machinery. IMP-α3/MOS6 is required genetically for full autoimmunity of the nucleotide-binding leucine-rich repeat (NLR) immune receptor mutant snc1 (suppressor of npr1-1, constitutive 1) and MOS6 also contributes to basal disease resistance. Here, we investigated the contribution of the other importin-α genes to both types of immune responses, and we analyzed potential interactions of all importin-α isoforms with SNC1. By using reverse-genetic analyses in Arabidopsis and protein-protein interaction assays in N. benthamiana we provide evidence that among the nine α-importins in Arabidopsis, IMP-α3/MOS6 is the main nuclear transport receptor of SNC1, and that IMP-α3/MOS6 is required selectively for autoimmunity of snc1 and basal resistance to mildly virulent Pseudomonas syringae in Arabidopsis.SIGNIFICANCE STATEMENTSpecific requirement for the Arabidopsis α-importin MOS6 in snc1-mediated autoimmunity is explained by selective formation of MOS6-SNC1 nuclear import complexes.

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.

Use of a general purpose mammalian expression vector for studying intracellular protein targeting: identification of critical residues in the nuclear lamin A/C nuclear localization signal

1993 ◽  
Vol 105 (2) ◽  
pp. 481-488 ◽  
Author(s):  
J.V. Frangioni ◽  
B.G. Neel
Keyword(s):  
Amino Acid ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Protein Targeting ◽  
General Purpose ◽  
Expression Vectors ◽  
Intracellular Protein ◽  
Mammalian Expression ◽  
Nuclear Lamin ◽  
Localization Signal

We have constructed a general purpose mammalian expression vector for the study of intracellular protein targeting. The vector, p3PK, facilitates construction of N- and/or C-terminal fusions of an amino acid sequence of interest to the normally cytosolic protein chicken muscle pyruvate kinase (CMPK). The vector has been engineered such that any fusion construct can be subcloned into the versatile pJx omega family of mammalian expression vectors and into pGEX bacterial expression vectors, for the generation of affinity reagents. In this paper, we demonstrate the general utility of p3PK by redirecting CMPK to mitochondria (using the twelve amino acid pre-sequence of yeast cytochrome c oxidase subunit IV) and to the nucleus (using a putative eight amino acid nuclear localization signal from human nuclear lamins A and C). We also report that, contrary to the predictions of previously published work, substitution of a critical residue in the nuclear lamin A/C nuclear localization signal (the equivalent of lysine 128 in the SV40 large T nuclear localization signal) retains nuclear localization, and discuss how amino acid context might affect targeting to the nucleus.

scholarly journals The Karyopherin Kap142p/Msn5p Mediates Nuclear Import and Nuclear Export of Different Cargo Proteins

2001 ◽  
Vol 152 (4) ◽  
pp. 729-740 ◽  
Author(s):  
Kimihisa Yoshida ◽  
Günter Blobel
Keyword(s):  
Nuclear Import ◽  
Nuclear Export ◽  
Protein Import ◽  
Protein A ◽  
Double Strand Breaks ◽  
Wild Type ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Highly Sensitive ◽  
Cargo Proteins

We have identified a novel pathway for protein import into the nucleus. Although the product of Saccharomyces cerevisiae gene MSN5 was previously shown to function as a karyopherin (Kap) for nuclear export of various proteins, we discovered a nuclear import pathway mediated by Msn5p (also referred to as Kap142p). We have purified from yeast cytosol a complex containing Kap142p and the trimeric replication protein A (RPA), which is required for multiple aspects of DNA metabolism, including DNA replication, DNA repair, and recombination. In wild-type cells, RPA was localized primarily to the nucleus but, in a KAP142 deletion strain, RPA was mislocalized to the cytoplasm and the strain was highly sensitive to bleomycin (BLM). BLM causes DNA double-strand breaks and, in S. cerevisiae, the DNA damage is repaired predominantly by RPA-dependent homologous recombination. Therefore, our results indicate that in wild-type cells a critical portion of RPA was imported into the nucleus by Kap142p. Like several other import-related Kap–substrate complexes, the endogenous RPA–Kap142p complex was dissociated by RanGTP, but not by RanGDP. All three RPA genes are essential for viability, whereas KAP142 is not. Perhaps explaining this disparity, we observed an interaction between RPA and Kap95p in a strain lacking Kap142p. This interaction could provide a mechanism for import of RPA into the nucleus and cell viability in the absence of Kap142p. Together with published results (Kaffman, A., N.M. Rank, E.M. O'Neill, L.S. Huang, and E.K. O'Shea. 1998. Nature. 396:482–486; Blondel, M., P.M. Alepuz, L.S. Huang, S. Shaham, G. Ammerer, and M. Peter. 1999. Genes Dev. 13:2284–2300; DeVit, M.J., and M. Johnston. 1999. Curr. Biol. 9:1231–1241; Mahanty, S.K., Y. Wang, F.W. Farley, and E.A. Elion. 1999. Cell. 98:501–512) our data indicate that the karyopherin Kap142p is able to mediate nuclear import of one set of proteins and nuclear export of a different set of proteins.

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 CRM1-mediated Recycling of Snurportin 1 to the Cytoplasm

1999 ◽  
Vol 145 (2) ◽  
pp. 255-264 ◽  
Author(s):  
Efrosyni Paraskeva ◽  
Elisa Izaurralde ◽  
F. Ralf Bischoff ◽  
Jochen Huber ◽  
Ulrike Kutay ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Nuclear Export ◽  
Activation Domain ◽  
Large Domain ◽  
High Affinity ◽  
Importin Α ◽  
Localization Signal ◽  
Major Mediator ◽  
Rev Protein

Importin β is a major mediator of import into the cell nucleus. Importin β binds cargo molecules either directly or via two types of adapter molecules, importin α, for import of proteins with a classical nuclear localization signal (NLS), or snurportin 1, for import of m3G-capped U snRNPs. Both adapters have an NH2-terminal importin β–binding domain for binding to, and import by, importin β, and both need to be returned to the cytoplasm after having delivered their cargoes to the nucleus. We have shown previously that CAS mediates export of importin α. Here we show that snurportin 1 is exported by CRM1, the receptor for leucine-rich nuclear export signals (NESs). However, the interaction of CRM1 with snurportin 1 differs from that with previously characterized NESs. First, CRM1 binds snurportin 1 50-fold stronger than the Rev protein and 5,000-fold stronger than the minimum Rev activation domain. Second, snurportin 1 interacts with CRM1 not through a short peptide but rather via a large domain that allows regulation of affinity. Strikingly, snurportin 1 has a low affinity for CRM1 when bound to its m3G-capped import substrate, and a high affinity when substrate-free. This mechanism appears crucial for productive import cycles as it can ensure that CRM1 only exports snurportin 1 that has already released its import substrate in the nucleus.

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 Discrimination between NL1- and NL2-Mediated Nuclear Localization of the Glucocorticoid Receptor

1999 ◽  
Vol 19 (2) ◽  
pp. 1025-1037 ◽  
Author(s):  
Joanne G. A. Savory ◽  
Brian Hsu ◽  
Ian R. Laquian ◽  
Ward Giffin ◽  
Terry Reich ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Binding Domain ◽  
Leptomycin B ◽  
Importin Α ◽  
Agonist And Antagonist ◽  
Import And Export ◽  
Pathway Inhibition

ABSTRACT Glucocorticoid receptor (GR) cycles between a free liganded form that is localized to the nucleus and a heat shock protein (hsp)-immunophilin-complexed, unliganded form that is usually localized to the cytoplasm but that can also be nuclear. In addition, rapid nucleocytoplasmic exchange or shuttling of the receptor underlies its localization. Nuclear import of liganded GR is mediated through a well-characterized sequence, NL1, adjacent to the receptor DNA binding domain and a second, uncharacterized motif, NL2, that overlaps with the ligand binding domain. In this study we report that rapid nuclear import (half-life [t 1/2] of 4 to 6 min) of agonist- and antagonist-treated GR and the localization of unliganded, hsp-associated GRs to the nucleus in G0 are mediated through NL1 and correlate with the binding of GR to pendulin/importin α. By contrast, NL2-mediated nuclear transfer of GR occurred more slowly (t 1/2 = 45 min to 1 h), was agonist specific, and appeared to be independent of binding to importin α. Together, these results suggest that NL2 mediates the nuclear import of GR through an alternative nuclear import pathway. Nuclear export of GR was inhibited by leptomycin B, suggesting that the transfer of GR to the cytoplasm is mediated through the CRM1-dependent pathway. Inhibition of GR nuclear export by leptomycin B enhanced the nuclear localization of both unliganded, wild-type GR and hormone-treated NL1− GR. These results highlight that the subcellular localization of both liganded and unliganded GRs is determined, at least in part, by a flexible equilibrium between the rates of nuclear import and export.

scholarly journals Unconventional tonicity-regulated nuclear trafficking of NFAT5 mediated by KPNB1, XPOT and RUVBL2

2021 ◽  
Author(s):  
Chris Y. Cheung ◽  
Ting-Ting Huang ◽  
Ning Chow ◽  
Shuqi Zhang ◽  
Yanxiang Zhao ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Nuclear Export ◽  
Nuclear Pore ◽  
Nucleocytoplasmic Shuttling ◽  
Proteomics Analysis ◽  
Nuclear Trafficking ◽  
Localization Signal ◽  
Underlying Mechanisms ◽  
Sirna Screening

NFAT5 is the only known mammalian tonicity-responsive transcription factor functionally implicated in diverse physiological and pathological processes. NFAT5 activity is tightly regulated by extracellular tonicity but the underlying mechanisms remain elusive. We demonstrated that NFAT5 enters the nucleus via the nuclear pore complex. We also found that NFAT5 utilizes a non-canonical nuclear localization signal (NFAT5-NLS) for nuclear imports. siRNA screening revealed that karyopherin beta-1 (KPNB1) drives nuclear import of NFAT5 via directly interacting with NFAT5-NLS. Proteomics analysis and siRNA screening further revealed that nuclear export of NFAT5 under hypotonicity is mediated by Exportin-T, and that it requires RuvB-Like AAA type ATPase 2 (RUVBL2) as an indispensable chaperone. Our findings have identified KPNB1 and RUVBL2 as key molecules responsible for the unconventional tonicity-regulated nucleocytoplasmic shuttling of NFAT5. These findings offer an opportunity for developing novel NFAT5 targeting strategies that are potentially useful for the treatment of diseases associated with NFAT5 dysregulation.

scholarly journals Nuclear Shuttling of She2p Couples ASH1 mRNA Localization to its Translational Repression by Recruiting Loc1p and Puf6p

2009 ◽  
Vol 20 (8) ◽  
pp. 2265-2275 ◽  
Author(s):  
Zhifa Shen ◽  
Nicolas Paquin ◽  
Amélie Forget ◽  
Pascal Chartrand
Keyword(s):  
Nuclear Import ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Mrna Localization ◽  
Translation Regulation ◽  
Importin Α ◽  
Localization Signal ◽  
Specific Proteins ◽  
Nuclear Shuttling ◽  
Ash1 Mrna

The transport and localization of mRNAs results in the asymmetric synthesis of specific proteins. In yeast, the nucleocytoplasmic shuttling protein She2 binds the ASH1 mRNA and targets it for localization at the bud tip by recruiting the She3p–Myo4p complex. Although the cytoplasmic role of She2p in mRNA localization is well characterized, its nuclear function is still unclear. Here, we show that She2p contains a nonclassical nuclear localization signal (NLS) that is essential for its nuclear import via the importin α Srp1p. Exclusion of She2p from the nucleus by mutagenesis of its NLS leads to defective ASH1 mRNA localization and Ash1p sorting. Interestingly, these phenotypes mimic knockouts of LOC1 and PUF6, which encode for nuclear RNA-binding proteins that bind the ASH1 mRNA and control its translation. We find that She2p interacts with both Loc1p and Puf6p and that excluding She2p from the nucleus decreases this interaction. Absence of nuclear She2p disrupts the binding of Loc1p and Puf6p to the ASH1 mRNA, suggesting that nuclear import of She2p is necessary to recruit both factors to the ASH1 transcript. This study reveals that a direct coupling between localization and translation regulation factors in the nucleus is required for proper cytoplasmic localization of mRNAs.

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