Nucleocytoplasmic Shuttling of p53 Is Essential for MDM2-Mediated Cytoplasmic Degradation but Not Ubiquitination

ABSTRACT As a shuttling protein, p53 is constantly transported through the nuclear pore complex. p53 nucleocytoplasmic transport is carried out by a bipartite nuclear localization signal (NLS) located at its C-terminal domain and two nuclear export signals (NES) located in its N- and C-terminal regions, respectively. The role of nucleocytoplasmic shuttling in p53 ubiquitination and degradation has been a subject of debate. Here we show that the two basic amino acid groups in the p53 bipartite NLS function collaboratively to import p53. Mutations disrupting individual amino acids in the NLS, although causing accumulation of p53 in the cytoplasm to various degrees, reduce but do not eliminate the NLS activity, and these mutants remain sensitive to MDM2 degradation. However, disrupting both parts of the bipartite NLS completely blocks p53 from entering the nucleus and causes p53 to become resistant to MDM2-mediated degradation. Similarly, mutations disrupting four conserved hydrophobic amino acids in the p53 C-terminal NES block p53 export and prohibit it from MDM2 degradation. We also show that colocalization of a nonshuttling p53 with MDM2 either in the nucleus or in the cytoplasm is sufficient for MDM2-induced p53 polyubiquitination but not degradation. Our data provide new insight into the mechanism and regulation of p53 nucleocytoplasmic shuttling and degradation.

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

The Journal of Cell Biology ◽

10.1083/jcb.140.3.499 ◽

1998 ◽

Vol 140 (3) ◽

pp. 499-509 ◽

Cited By ~ 316

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.

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RNA-Regulated Interaction of Transportin-1 and Exportin-5 with the Double-Stranded RNA-Binding Domain Regulates Nucleocytoplasmic Shuttling of ADAR1

Molecular and Cellular Biology ◽

10.1128/mcb.01519-08 ◽

2009 ◽

Vol 29 (6) ◽

pp. 1487-1497 ◽

Cited By ~ 72

Author(s):

Jutta Fritz ◽

Alexander Strehblow ◽

Andreas Taschner ◽

Sandy Schopoff ◽

Pawel Pasierbek ◽

...

Keyword(s):

Nuclear Export ◽

Rna Binding ◽

Nuclear Export Signal ◽

Nucleocytoplasmic Transport ◽

Nucleocytoplasmic Shuttling ◽

Double Stranded Rna ◽

Binding Domains ◽

Dsrna Binding ◽

Localization Signal ◽

Editing Enzyme

ABSTRACT Double-stranded RNA (dsRNA)-binding proteins interact with substrate RNAs via dsRNA-binding domains (dsRBDs). Several proteins harboring these domains exhibit nucleocytoplasmic shuttling and possibly remain associated with their substrate RNAs bound in the nucleus during nuclear export. In the human RNA-editing enzyme ADAR1-c, the nuclear localization signal overlaps the third dsRBD, while the corresponding import factor is unknown. The protein also lacks a clear nuclear export signal but shuttles between the nucleus and the cytoplasm. Here we identify transportin-1 as the import receptor for ADAR1. Interestingly, dsRNA binding interferes with transportin-1 binding. At the same time, each of the dsRBDs in ADAR1 interacts with the export factor exportin-5. RNA binding stimulates this interaction but is not a prerequisite. Thus, our data demonstrate a role for some dsRBDs as RNA-sensitive nucleocytoplasmic transport signals. dsRBD3 in ADAR1 can mediate nuclear import, while interaction of all dsRBDs might control nuclear export. This finding may have implications for other proteins containing dsRBDs and suggests a selective nuclear export mechanism for substrates interacting with these proteins.

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

Molecular and Cellular Biology ◽

10.1128/mcb.18.11.6805 ◽

1998 ◽

Vol 18 (11) ◽

pp. 6805-6815 ◽

Cited By ~ 88

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.

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

10.1101/2021.07.11.451941 ◽

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.

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Nucleocytoplasmic Shuttling of Porcine Parvovirus NS1 Protein Mediated by the CRM1 Nuclear Export Pathway and the Importin α/β Nuclear Import Pathway

Journal of Virology ◽

10.1128/jvi.01481-21 ◽

2021 ◽

Author(s):

Liyan Cao ◽

Fang Fu ◽

Jianfei Chen ◽

Hongyan Shi ◽

Xin Zhang ◽

...

Keyword(s):

Amino Acids ◽

Nuclear Import ◽

Nuclear Export ◽

Nonstructural Protein ◽

Nuclear Pore ◽

Porcine Parvovirus ◽

Ns1 Protein ◽

Nucleocytoplasmic Shuttling ◽

Export Pathway ◽

Importin Α

Porcine parvovirus (PPV) NS1, the major nonstructural protein of this virus, plays an important role in PPV replication. We show, for the first time, that NS1 dynamically shuttles between the nucleus and cytoplasm, although its subcellular localization is predominantly nuclear. NS1 contains two nuclear export signals (NESs) at amino acids 283–291 (designated NES2) and 602–608 (designated NES1). NES1 and NES2 are both functional and transferable NESs, and their nuclear export activity is blocked by leptomycin B (LMB), suggesting that the export of NS1 from the nucleus is dependent upon the chromosome region maintenance 1 (CRM1) pathway. Deletion and site-directed mutational analyses showed that NS1 contains a bipartite nuclear localization signal (NLS) at amino acids 256–274. Coimmunoprecipitation assays showed that NS1 interacts with importins α5 and α7 through its NLS. The overexpression of CRM1, importins α5 and α7 significantly promoted PPV replication, whereas the inhibition of CRM1 and importin α/β-mediated transport by specific inhibitors (LMB, importazole and ivermectin) clearly blocked PPV replication. The mutant viruses of delete NESs or NLS motif of the NS1 by using reverse genetics could not be rescued, suggesting that NESs and NLS are essential for PPV replication. Collectively, these findings suggest that NS1 shuttles between the nucleus and cytoplasm, mediated by its functional NESs and NLS, via the CRM1-dependent nuclear export pathway and the importin α/β-mediated nuclear import pathway, and PPV proliferation was inhibited if blocking NS1 nuclear import or export. Importance PPV replicates in the nucleus, and the nuclear envelope is a barrier to its entry into and egress from the nucleus. PPV NS1 is a nucleus-targeting protein that is important for viral DNA replication. Because the NS1 molecule is large (> 50 kDa), it cannot pass through the nuclear pore complex by diffusion alone, and requires specific transport receptors to permit its nucleocytoplasmic shuttling. In this study, the two functional NESs in the NS1 protein were identified, and its dependence on the CRM1 pathway for nuclear export demonstrated. The nuclear import of NS1 utilizes importins α5 and α7 in the importin α/β nuclear import pathway.

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The Yeast Nucleoporin Nup53p Specifically Interacts with Nic96p and Is Directly Involved in Nuclear Protein Import

Molecular Biology of the Cell ◽

10.1091/mbc.11.11.3885 ◽

2000 ◽

Vol 11 (11) ◽

pp. 3885-3896 ◽

Cited By ~ 22

Author(s):

Birthe Fahrenkrog ◽

Wolfgang Hübner ◽

Anna Mandinova ◽

Nelly Panté ◽

Walter Keller ◽

...

Keyword(s):

Nuclear Export ◽

Protein Import ◽

Nuclear Protein ◽

Genomic Library ◽

Nuclear Periphery ◽

Nucleocytoplasmic Transport ◽

Immunogold Electron Microscopy ◽

Nuclear Pore ◽

Nuclear Protein Import

The bidirectional nucleocytoplasmic transport of macromolecules is mediated by the nuclear pore complex (NPC) which, in yeast, is composed of ∼30 different proteins (nucleoporins). Pre-embedding immunogold-electron microscopy revealed that Nic96p, an essential yeast nucleoporin, is located about the cytoplasmic and the nuclear periphery of the central channel, and near or at the distal ring of the yeast NPC. Genetic approaches further implicated Nic96p in nuclear protein import. To more specifically explore the potential role of Nic96p in nuclear protein import, we performed a two-hybrid screen withNIC96 as the bait against a yeast genomic library to identify transport factors and/or nucleoporins involved in nuclear protein import interacting with Nic96p. By doing so, we identified the yeast nucleoporin Nup53p, which also exhibits multiple locations within the yeast NPC and colocalizes with Nic96p in all its locations. Whereas Nup53p is directly involved in NLS-mediated protein import by its interaction with the yeast nuclear import receptor Kap95p, it appears not to participate in NES-dependent nuclear export.

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Role of the Ndc1 interaction network in yeast nuclear pore complex assembly and maintenance

The Journal of Cell Biology ◽

10.1083/jcb.200810030 ◽

2009 ◽

Vol 185 (3) ◽

pp. 475-491 ◽

Cited By ~ 97

Author(s):

Evgeny Onischenko ◽

Leslie H. Stanton ◽

Alexis S. Madrid ◽

Thomas Kieselbach ◽

Karsten Weis

Keyword(s):

Nuclear Pore Complex ◽

Structural Integrity ◽

Fluorescent Protein ◽

Interaction Network ◽

Nucleocytoplasmic Transport ◽

Nuclear Pore ◽

Binding Partners ◽

Interaction Partners ◽

Redundant Functions

The nuclear pore complex (NPC) mediates all nucleocytoplasmic transport, yet its structure and biogenesis remain poorly understood. In this study, we have functionally characterized interaction partners of the yeast transmembrane nucleoporin Ndc1. Ndc1 forms a distinct complex with the transmembrane proteins Pom152 and Pom34 and two alternative complexes with the soluble nucleoporins Nup53 and Nup59, which in turn bind to Nup170 and Nup157. The transmembrane and soluble Ndc1-binding partners have redundant functions at the NPC, and disruption of both groups of interactions causes defects in Ndc1 targeting and in NPC structure accompanied by significant pore dilation. Using photoconvertible fluorescent protein fusions, we further show that the depletion of Pom34 in cells that lack NUP53 and NUP59 blocks new NPC assembly and leads to the reversible accumulation of newly made nucleoporins in cytoplasmic foci. Therefore, Ndc1 together with its interaction partners are collectively essential for the biosynthesis and structural integrity of yeast NPCs.

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

Molecular Biology of the Cell ◽

10.1091/mbc.e04-07-0634 ◽

2006 ◽

Vol 17 (2) ◽

pp. 931-943 ◽

Cited By ~ 20

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.

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