scholarly journals Nuclear targeting of the β isoform of Type II phosphatidylinositol phosphate kinase (phosphatidylinositol 5-phosphate 4-kinase) by its α-helix 7

2000 ◽  
Vol 346 (3) ◽  
pp. 587-591 ◽  
Author(s):  
Antonio CIRUELA ◽  
Katherine A. HINCHLIFFE ◽  
Nullin DIVECHA ◽  
Robin F. IRVINE
Keyword(s):  
Nuclear Localization ◽  
Fluorescent Protein ◽  
Physiological Role ◽  
Nuclear Localization Sequence ◽  
Type Ii ◽  
Nuclear Targeting ◽  
Phosphatidylinositol Phosphate ◽  
Localization Sequence ◽  
Α Helix ◽  
Green Fluorescent

Type II phosphatidylinositol phosphate kinases (PIPkins) have recently been found to be primarily phosphatidylinositol 5-phosphate 4-kinases, and their physiological role remains unclear. We have previously shown that a Type II PIPkin [isoform(s) unknown], is localized partly in the nucleus [Divecha, Rhee, Letcher and Irvine (1993) Biochem. J. 289, 617-620], and here we show, by transfection of HeLa cells with green-fluorescent-protein-tagged Type II PIPkins, that this is likely to be the Type IIβ isoform. Type IIβ PIPkin has no obvious nuclear localization sequence, and a detailed analysis of the localization of chimaeras and mutants of the α (cytosolic) and β PIPkins shows that the nuclear localization requires the presence of a 17-amino-acid length of α-helix (α-helix 7) that is specific to the β isoform, and that this helix must be present in its entirety, with a precise orientation. This resembles the nuclear targeting of the HIV protein Vpr, and Type IIβ PIPkin is apparently therefore the first example of a eukaryotic protein that uses the same mechanism.

scholarly journals Degradation of SAMHD1 by Vpx Is Independent of Uncoating

2015 ◽  
Vol 89 (10) ◽  
pp. 5701-5713 ◽  
Author(s):  
Paula Jáuregui ◽  
Eric C. Logue ◽  
Megan L. Schultz ◽  
Stephanie Fung ◽  
Nathaniel R. Landau
Keyword(s):  
T Cells ◽  
Green Fluorescent Protein ◽  
Fusion Protein ◽  
Hela Cell ◽  
Nuclear Localization ◽  
Fluorescent Protein ◽  
Nuclear Localization Sequence ◽  
Hela Cell Lines ◽  
Localization Sequence ◽  
Green Fluorescent

ABSTRACTSterile alpha motif domain and HD domain-containing protein 1 (SAMHD1) restricts human immunodeficiency virus type 1 (HIV-1) replication in myeloid and resting T cells. Lentiviruses such as HIV-2 and some simian immunodeficiency viruses (SIVs) counteract the restriction by encoding Vpx or Vpr, accessory proteins that are packaged in virions and which, upon entry of the virus into the cytoplasm, induce the proteasomal degradation of SAMHD1. As a tool to study these mechanisms, we generated HeLa cell lines that express a fusion protein termed NLS.GFP.SAM595 in which the Vpx binding domain of SAMHD1 is fused to the carboxy terminus of green fluorescent protein (GFP) and a nuclear localization signal is fused to the amino terminus of GFP. Upon incubation of Vpx-containing virions with the cells, the NLS.GFP.SAM595 fusion protein was degraded over several hours and the levels remained low over 5 days as the result of continued targeting of the CRL4 E3 ubiquitin ligase. Degradation of the fusion protein required that it contain a nuclear localization sequence. Fusion to the cytoplasmic protein muNS rendered the protein resistant to Vpx-mediated degradation, confirming that SAMHD1 is targeted in the nucleus. Virions treated with protease inhibitors failed to release Vpx, indicating that Gag processing was required for Vpx release from the virion. Mutations in the capsid protein that altered the kinetics of virus uncoating and the Gag binding drug PF74 had no effect on the Vpx-mediated degradation. These results suggest that Vpx is released from virions without a need for uncoating of the capsid, allowing Vpx to transit to the nucleus rapidly upon entry into the cytoplasm.IMPORTANCESAMHD1 restricts lentiviral replication in myeloid cells and resting T cells. Its importance is highlighted by the fact that viruses such as HIV-2 encode an accessory protein that is packaged in the virion and is dedicated to inducing SAMHD1 degradation. Vpx needs to act rapidly upon infection to allow reverse transcription to proceed. The limited number of Vpx molecules in a virion also needs to clear the cell of SAMHD1 over a prolonged period of time. Using an engineered HeLa cell line that expresses a green fluorescent protein (GFP)-SAMHD1 fusion protein, we showed that the Vpx-dependent degradation occurs without a need for viral capsid uncoating. In addition, the fusion protein was degraded only when it was localized to the nucleus, confirming that SAMHD1 is targeted in the nucleus and thus explaining why Vpx also localizes to the nucleus.

scholarly journals Heterologous Expression of CLIBASIA_03915/CLIBASIA_04250 by Tobacco Mosaic Virus Resulted in Phloem Necrosis in the Senescent Leaves of Nicotiana benthamiana

2020 ◽  
Vol 21 (4) ◽  
pp. 1414 ◽  
Author(s):  
Hui Li ◽  
Xiaobao Ying ◽  
Lina Shang ◽  
Bryce Redfern ◽  
Nicholas Kypraios ◽  
...  
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Nicotiana Benthamiana ◽  
Fluorescent Protein ◽  
Nuclear Localization Sequence ◽  
Localization Sequence ◽  
Candidatus Liberibacter ◽  
Phloem Necrosis ◽  
Liberibacter Asiaticus ◽  
Green Fluorescent

Huanglongbing (HLB), also known as citrus greening, is the most notorious citrus disease worldwide. Candidatus Liberibacter asiaticus (CaLas) is a phloem-restricted bacterium associated with HLB. Because there is no mutant library available, the pathogenesis of CaLas is obscure. In this study, we employed tobacco mosaic virus (TMV) to express two mature secretion proteins CLIBASIA_03915 (m03915) and CLIBASIA_04250 (m04250) in Nicotiana benthamiana (N. benthamiana). Phloem necrosis was observed in the senescent leaves of N. benthamiana that expressed the two low molecular weight proteins, while no phloem necrosis was observed in the plants that expressed the control, green fluorescent protein (GFP). Additionally, no phloem necrosis was observed in the senescent leaves of N. benthamiana that expressed the null mutation of m03915 and frameshifting m04250. The subcellular localizations of m03915 and m04250 were determined by fusion with GFP using confocal microscopy. The subcellular localization of m03915 was found to be as free GFP without a nuclear localization sequence (NLS). However, m04250 did have an NLS. Yeast two-hybrid (Y2H) was carried out to probe the citrus proteins interacting with m03915 and m04250. Six citrus proteins were found to interact with m03915. The identified proteins were involved in the metabolism of compounds, transcription, response to abiotic stress, ubiquitin-mediated protein degradation, etc. The prey of m04250 was involved in the processing of specific pre-mRNAs. Identification of new virulence factors of CaLas will give insight into the pathogenesis of CaLas, and therefore, it will eventually help develop the HLB-resistant citrus.

scholarly journals Identification of the Nuclear Localization Signal inXenopus Cyclin E and Analysis of Its Role in Replication and Mitosis

2002 ◽  
Vol 13 (12) ◽  
pp. 4388-4400 ◽  
Author(s):  
Jonathan D. Moore ◽  
Sally Kornbluth ◽  
Tim Hunt
Keyword(s):  
Dna Replication ◽  
Nuclear Localization ◽  
Cyclin E ◽  
T Antigen ◽  
Cyclin B ◽  
Nuclear Accumulation ◽  
Nuclear Localization Sequence ◽  
Nuclear Targeting ◽  
Sv40 Large T Antigen ◽  
Localization Sequence

Cyclin-dependent kinase (Cdk)2/cyclin E is imported into nuclei assembled in Xenopus egg extracts by a pathway that requires importin-α and -β. Here, we identify a basic nuclear localization sequence (NLS) in the N-terminus ofXenopus cyclin E. Mutation of the NLS eliminated nuclear accumulation of both cyclin E and Cdk2, and such versions of cyclin E were unable to trigger DNA replication. Addition of a heterologous NLS from SV40 large T antigen restored both nuclear targeting of Cdk2/cyclin E and DNA replication. We present evidence indicating that Cdk2/cyclin E complexes must become highly concentrated within nuclei to support replication and find that cyclin A can trigger replication at much lower intranuclear concentrations. We confirmed that depletion of endogenous cyclin E increases the concentration of cyclin B necessary to promote entry into mitosis. In contrast to its inability to promote DNA replication, cyclin E lacking its NLS was able to cooperate with cyclin B in promoting mitotic entry.

scholarly journals Rhinovirus 16 2A Protease Affects Nuclear Localization of 3CD during Infection

2016 ◽  
Vol 90 (24) ◽  
pp. 11032-11042 ◽  
Author(s):  
Erin Walker ◽  
Lora Jensen ◽  
Sarah Croft ◽  
Kejun Wei ◽  
Alex J. Fulcher ◽  
...  
Keyword(s):  
Virus Replication ◽  
Nuclear Localization ◽  
Fluorescent Protein ◽  
Human Rhinovirus ◽  
Subcellular Targeting ◽  
Nuclear Targeting ◽  
3C Protease ◽  
2A Protease ◽  
Targeting Ability ◽  
Green Fluorescent

ABSTRACTThe human rhinovirus (HRV) 3C and 2A proteases (3Cproand 2Apro, respectively) are critical in HRV infection, as they are required for viral polyprotein processing as well as proteolysing key host factors to facilitate virus replication. Early in infection, 3Cprois present as its precursor 3CD, which, although the mechanism of subcellular targeting is unknown, is found in the nucleus as well as the cytoplasm. In this study, we use transfected and infected cell systems to show that 2Aproactivity is required for 3CD nuclear localization. Using green fluorescent protein (GFP)-tagged forms of 3Cpro, 3D, and mutant derivatives thereof, we show that 3Cprois located in the cytoplasm and the nucleus, whereas 3CD and 3D are localized predominantly in the cytoplasm, implying that 3D lacks nuclear targeting ability and that 3Cproactivity within 3CD is not sufficient to allow the larger protein into the nucleus. Importantly, by coexpressing mCherry-2Aprofusion proteins, we demonstrate formally that 2Aproactivity is required to allow HRV 3CD access to the nucleus. In contrast, mCherry-3Cprois insufficient to allow 3CD access to the nucleus. Finally, we confirm the relevance of these results to HRV infection by demonstrating that nuclear localization of 3CD correlates with 2Aproactivity and not 3Cproactivity, which is observed only later in infection. The results thus define the temporal activities of 2Aproand 3CD/3Cproactivities in HRV serotype16 infection.IMPORTANCEThe human rhinovirus genome encodes two proteases, 2A and 3C, as well as a precursor protease, 3CD. These proteases are essential for efficient virus replication. The 3CD protein is found in the nucleus early during infection, though the mechanism of subcellular localization is unknown. Here we show that 2A protease is required for this localization, the 3C protease activity of 3CD is not sufficient to allow 3CD entry into the nucleus, and 3D lacks nuclear targeting ability. This study demonstrates that both 2A and 3C proteases are required for the correct localization of proteins during infection and defines the temporal regulation of 2A and 3CD/3C protease activities during HRV16 infection.

scholarly journals Rat1p and Xrn1p are functionally interchangeable exoribonucleases that are restricted to and required in the nucleus and cytoplasm, respectively.

1997 ◽  
Vol 17 (10) ◽  
pp. 6122-6130 ◽  
Author(s):  
A W Johnson
Keyword(s):  
Fluorescent Protein ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Nuclear Localization Sequence ◽  
Wild Type ◽  
Gene Encoding ◽  
Cytoplasmic Rna ◽  
Localization Sequence ◽  
Green Fluorescent

XRN1 encodes an abundant cytoplasmic exoribonuclease, Xrn1p, responsible for mRNA turnover in yeast. A screen for bypass suppressors of the inviability of xrn1 ski2 double mutants identified dominant alleles of RAT1, encoding an exoribonuclease homologous with Xrn1p. These RAT1 alleles restored XRN1-like functions, including cytoplasmic RNA turnover, wild-type sensitivity to the microtubule-destabilizing drug benomyl, and sporulation. The mutations were localized to a region of the RAT1 gene encoding a putative bipartite nuclear localization sequence (NLS). Fusions to green fluorescent protein were used to demonstrate that wild-type Rat1p is localized to the nucleus and that the mutant alleles result in mislocalization of Rat1p to the cytoplasm. Conversely, targeting Xrn1p to the nucleus by the addition of the simian virus 40 large-T-antigen NLS resulted in complementation of the temperature sensitivity of a rat1-1 strain. These results indicate that Xrn1p and Rat1p are functionally interchangeable exoribonucleases that function in and are restricted to the cytoplasm and nucleus, respectively. It is likely that the higher eukaryotic homologs of these proteins will function similarly in the cytoplasm and nucleus.

scholarly journals Nuclear Import of Histone H2a and H2b Is Mediated by a Network of Karyopherins

2001 ◽  
Vol 153 (2) ◽  
pp. 251-262 ◽  
Author(s):  
Nima Mosammaparast ◽  
Kelley R. Jackson ◽  
Yurong Guo ◽  
Cynthia J. Brame ◽  
Jeffrey Shabanowitz ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Fluorescent Protein ◽  
Nuclear Localization Sequence ◽  
Temperature Sensitive ◽  
Histone H2a ◽  
Amino Terminal ◽  
Core Histones ◽  
Localization Sequence ◽  
Green Fluorescent

The first step in the assembly of new chromatin is the cell cycle–regulated synthesis and nuclear import of core histones. The core histones include H2A and H2B, which are assembled into nucleosomes as heterodimers. We show here that the import of histone H2A and H2B is mediated by several members of the karyopherin (Kap; importin) family. An abundant complex of H2A, H2B, and Kap114p was detected in cytosol. In addition, two other Kaps, Kap121p and Kap123p, and the histone chaperone Nap1p were isolated with H2A and H2B. Nap1p is not necessary for the formation of the Kap114p-H2A/H2B complex or for import of H2A and H2B. We demonstrate that both histones contain a nuclear localization sequence (NLS) in the amino-terminal tail. Fusions of the NLSs to green fluorescent protein were specifically mislocalized to the cytoplasm in kap mutant strains. In addition, we detected a specific mislocalization in a kap95 temperature-sensitive strain, suggesting that this Kap is also involved in the import of H2A and H2B in vivo. Importantly, we show that Kap114p, Kap121p, and Kap95 interact directly with both histone NLSs and that RanGTP inhibits this association. These data suggest that the import of H2A and H2B is mediated by a network of Kaps, in which Kap114p may play the major role.

scholarly journals A monopartite nuclear localization sequence regulates nuclear targeting of the actin binding protein myopodin

FEBS Letters ◽  
2005 ◽  
Vol 579 (29) ◽  
pp. 6673-6680 ◽  
Author(s):  
Ariane De Ganck ◽  
Thomas Hubert ◽  
Katrien Van Impe ◽  
Danny Geelen ◽  
Joël Vandekerckhove ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Binding Protein ◽  
Actin Binding ◽  
Nuclear Localization Sequence ◽  
Nuclear Targeting ◽  
Actin Binding Protein ◽  
Localization Sequence

scholarly journals Evidence for Interaction of Schizophyllum commune Y Mating-Type Proteins in Vivo

Genetics ◽  
2002 ◽  
Vol 160 (4) ◽  
pp. 1461-1467
Author(s):  
C Ian Robertson ◽  
Alexander McMahon Kende ◽  
Kurt Toenjes ◽  
Charles P Novotny ◽  
Robert C Ullrich
Keyword(s):  
Mating Type ◽  
Protein Interactions ◽  
Sexual Development ◽  
Fluorescent Protein ◽  
Schizophyllum Commune ◽  
Nuclear Localization Sequence ◽  
Type Locus ◽  
Localization Sequence ◽  
Green Fluorescent

Abstract The Aα mating-type locus of Schizophyllum commune regulates sexual development and contains the code for two proteins, Y and Z, which are thought to form a complex and function as a transcription factor. Import of these proteins into the nucleus may be an essential step in Aα-regulated sexual development. The Y proteins contain a bipartite basic sequence, which is an excellent candidate for a nuclear localization sequence (NLS), while Z proteins contain no such sequence. Here we describe experiments in which deletions were made in the putative NLS sequence of Y4. We show that this putative NLS is essential to the function of the Y protein and capable of mislocalizing green fluorescent protein (GFP) to the nucleus in Saccharomyces cerevisiae. Further, we describe genetic experiments that demonstrate the first Y-Y protein interactions in vivo. These results are consistent with our previously postulated hypothesis that the Y-Z complex is likely to be of a higher order than dimer.

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