scholarly journals Nucleocytoplasmic Shuttling Factors Including Ran and CRM1 Mediate Nuclear Export of NFAT In Vitro

1998 ◽  
Vol 141 (4) ◽  
pp. 863-874 ◽  
Author(s):  
Ralph H. Kehlenbach ◽  
Achim Dickmanns ◽  
Larry Gerace
Keyword(s):  
Nuclear Export ◽  
Fluorescent Protein ◽  
Dependent Manner ◽  
Nucleocytoplasmic Shuttling ◽  
Permeabilized Cell ◽  
Permeabilized Cells ◽  
Digitonin Permeabilization ◽  
Green Fluorescent ◽  
Rate Limiting

We have developed a permeabilized cell assay to study the nuclear export of the shuttling transcription factor NFAT, which contains a leucine-rich export signal. The assay uses HeLa cells that are stably transfected with NFAT fused to the green fluorescent protein (GFP). Nuclear export of GFP–NFAT in digitonin-permeabilized cells occurs in a temperature- and ATP-dependent manner and can be quantified by flow cytometry. In vitro NFAT export requires the GTPase Ran, which is released from cells during the digitonin permeabilization. At least one additional rate-limiting export factor is depleted from permeabilized cells by a preincubation at 30°C in the absence of cytosol. This activity can be provided by cytosolic or nucleoplasmic extracts in a subsequent export step. Using this assay, we have purified a second major export activity from cytosol. We found that it corresponds to CRM1, a protein recently reported to be a receptor for certain leucine-rich export sequences. CRM1 appears to be imported into the nucleus by a Ran-dependent mechanism that is distinct from conventional signaling pathways. Considered together, our studies directly demonstrate by fractionation and reconstitution that nuclear export of NFAT is mediated by multiple nucleocytoplasmic shuttling factors, including Ran and CRM1.

scholarly journals Modulation of Virulence by Two Acidified Nitrite-Responsive Loci of Salmonella enterica Serovar Typhimurium

2003 ◽  
Vol 71 (6) ◽  
pp. 3196-3205 ◽  
Author(s):  
Charles C. Kim ◽  
Denise Monack ◽  
Stanley Falkow
Keyword(s):  
Salmonella Enterica ◽  
Fluorescent Protein ◽  
Dependent Manner ◽  
Wild Type ◽  
Independent Manner ◽  
Inducible Promoters ◽  
Serovar Typhimurium ◽  
Bacterial Genes ◽  
Green Fluorescent

ABSTRACT Two acidified nitrite-inducible genes of Salmonella enterica serovar Typhimurium were identified with a green fluorescent protein-based promoter-trap screen. The nitrite-inducible promoters were located upstream of loci that we designated nipAB and nipC, which correspond to hcp-hcr (hybrid cluster protein) of Escherichia coli and norA of Alcaligenes eutrophus, respectively. Maximal induction of the promoters by nitrite was dependent on pH. The nipAB promoter was regulated by oxygen in an Fnr-dependent manner. The nipC promoter was also regulated by oxygen but in an Fnr-independent manner. The promoters were upregulated in activated RAW264.7 macrophage-like cells, which produce NO via the inducible nitric oxide synthase (iNOS), and the induction was inhibited by aminoguanidine, an inhibitor of iNOS. Although the nipAB and nipC mutants displayed no defects under a variety of in vitro conditions or in tissue culture infections, they exhibited lower oral 50% lethal doses (LD50s) than did the wild type in C57BL/6J mouse infections. The lower LD50s reflected an unexpected increased ability of small inoculating doses of the mutant bacteria to cause lethal infection 2 to 3 weeks after challenge, compared to a similar challenge dose of wild-type bacteria. We conclude that these genes are regulated by physiological nitrogen oxides and that the absence of these bacterial genes in some way diminishes the ability of mice to clear a low dose infection.

scholarly journals Characterization of the Streptococcal C5a Peptidase Using a C5a-Green Fluorescent Protein Fusion Protein Substrate

2000 ◽  
Vol 182 (11) ◽  
pp. 3254-3258 ◽  
Author(s):  
D. K. Stafslien ◽  
P. P. Cleary
Keyword(s):  
Green Fluorescent Protein ◽  
Fusion Protein ◽  
Fluorescent Protein ◽  
High Sensitivity ◽  
Dependent Manner ◽  
Protein Fusion ◽  
Group B Streptococci ◽  
Mutant Proteins ◽  
Green Fluorescent

ABSTRACT A glutathione-S-transferase (GST)–C5a–green fluorescent protein (GFP) fusion protein was designed for use as a substrate for the streptococcal C5a peptidase (SCPA). The substrate was immobilized on a glutathione-Sepharose affinity matrix and used to measure wild-type SCPA activity in the range of 0.8 to 800 nM. The results of the assay demonstrated that SCPA is highly heat stable and has optimal activity on the synthetic substrate at or above pH 8.0. SCPA activity was unaffected by 0.1 to 10 mM Ca2+, Mg2+, and Mn2+ but was inhibited by the same concentrations of Zn2+. The assay shows high sensitivity to ionic strength; NaCl inhibits SCPA cleavage of GST-C5a-GFP in a dose-dependent manner. Based on previously published computer homology modeling, four substitutions were introduced into the putative active site of SCPA: Asp130-Ala, His193-Ala, Asn295-Ala, and Ser512-Ala. All four mutant proteins had over 1,000-fold less proteolytic activity on C5a in vitro, as determined both by the GFP assay described here and by a polymorphonuclear cell adherence assay. In addition, recombinant SCPA1 and SCPA49, from two distinct lineages of Streptococcus pyogenes (group A streptococci), and recombinant SCPB, fromStreptococcus agalactiae (group B streptococci), were compared in the GFP assay. The three enzymes had similar activities, all cleaving approximately 6 mol of C5a mmol of SCP−1liter−1 min−1.

Activity of Cardiorespiratory Networks Revealed by Transsynaptic Virus Expressing GFP

2001 ◽  
Vol 85 (1) ◽  
pp. 435-438 ◽  
Author(s):  
Mustapha Irnaten ◽  
Robert A. Neff ◽  
Jijiang Wang ◽  
Arthur D. Loewy ◽  
Thomas C. Mettenleiter ◽  
...  
Keyword(s):  
Pseudorabies Virus ◽  
Fluorescent Protein ◽  
Nucleus Ambiguus ◽  
Dependent Manner ◽  
Neural Basis ◽  
Electrophysiological Properties ◽  
Functional Studies ◽  
Neuronal Systems ◽  
Green Fluorescent

A fluorescent transneuronal marker capable of labeling individual neurons in a central network while maintaining their normal physiology would permit functional studies of neurons within entire networks responsible for complex behaviors such as cardiorespiratory reflexes. The Bartha strain of pseudorabies virus (PRV), an attenuated swine alphaherpesvirus, can be used as a transsynaptic marker of neural circuits. Bartha PRV invades neuronal networks in the CNS through peripherally projecting axons, replicates in these parent neurons, and then travels transsynaptically to continue labeling the second- and higher-order neurons in a time-dependent manner. A Bartha PRV mutant that expresses green fluorescent protein (GFP) was used to visualize and record from neurons that determine the vagal motor outflow to the heart. Here we show that Bartha PRV-GFP-labeled neurons retain their normal electrophysiological properties and that the labeled baroreflex pathways that control heart rate are unaltered by the virus. This novel transynaptic virus permits in vitro studies of identified neurons within functionally defined neuronal systems including networks that mediate cardiovascular and respiratory function and interactions. We also demonstrate superior laryngeal motorneurons fire spontaneously and synapse on cardiac vagal neurons in the nucleus ambiguus. This cardiorespiratory pathway provides a neural basis of respiratory sinus arrhythmias.

scholarly journals A novel histone exchange factor, protein phosphatase 2Cγ, mediates the exchange and dephosphorylation of H2A–H2B

2006 ◽  
Vol 175 (3) ◽  
pp. 389-400 ◽  
Author(s):  
Hiroshi Kimura ◽  
Nanako Takizawa ◽  
Eric Allemand ◽  
Tetsuya Hori ◽  
Francisco J. Iborra ◽  
...  
Keyword(s):  
Dna Replication ◽  
Protein Phosphatase ◽  
Fluorescent Protein ◽  
Histone Variants ◽  
Permeabilized Cells ◽  
Cellular Factors ◽  
Core Histones ◽  
Green Fluorescent ◽  
Histone Exchange

In eukaryotic nuclei, DNA is wrapped around a protein octamer composed of the core histones H2A, H2B, H3, and H4, forming nucleosomes as the fundamental units of chromatin. The modification and deposition of specific histone variants play key roles in chromatin function. In this study, we established an in vitro system based on permeabilized cells that allows the assembly and exchange of histones in situ. H2A and H2B, each tagged with green fluorescent protein (GFP), are incorporated into euchromatin by exchange independently of DNA replication, and H3.1-GFP is assembled into replicated chromatin, as found in living cells. By purifying the cellular factors that assist in the incorporation of H2A–H2B, we identified protein phosphatase (PP) 2C γ subtype (PP2Cγ/PPM1G) as a histone chaperone that binds to and dephosphorylates H2A–H2B. The disruption of PP2Cγ in chicken DT40 cells increased the sensitivity to caffeine, a reagent that disturbs DNA replication and damage checkpoints, suggesting the involvement of PP2Cγ-mediated histone dephosphorylation and exchange in damage response or checkpoint recovery in higher eukaryotes.

scholarly journals Calreticulin Is a Receptor for Nuclear Export

2001 ◽  
Vol 152 (1) ◽  
pp. 127-140 ◽  
Author(s):  
James M. Holaska ◽  
Ben E. Black ◽  
Dona C. Love ◽  
John A. Hanover ◽  
John Leszyk ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Hormone Receptors ◽  
Fluorescent Protein ◽  
Kinase Inhibitor ◽  
Steroid Hormone ◽  
Nuclear Export Signal ◽  
Steroid Hormone Receptors ◽  
Permeabilized Cell ◽  
Green Fluorescent

In previous work, we used a permeabilized cell assay that reconstitutes nuclear export of protein kinase inhibitor (PKI) to show that cytosol contains an export activity that is distinct from Crm1 (Holaska, J.M., and B.M. Paschal. 1995. Proc. Natl. Acad. Sci. USA. 95: 14739–14744). Here, we describe the purification and characterization of the activity as calreticulin (CRT), a protein previously ascribed to functions in the lumen of the ER. We show that cells contain both ER and cytosolic pools of CRT. The mechanism of CRT-dependent export of PKI requires a functional nuclear export signal (NES) in PKI and involves formation of an export complex that contains RanGTP. Previous studies linking CRT to downregulation of steroid hormone receptor function led us to examine its potential role in nuclear export of the glucocorticoid receptor (GR). We found that CRT mediates nuclear export of GR in permeabilized cell, microinjection, and transfection assays. GR export is insensitive to the Crm1 inhibitor leptomycin B in vivo, and it does not rely on a leucine-rich NES. Rather, GR export is facilitated by its DNA-binding domain, which is shown to function as an NES when transplanted to a green fluorescent protein reporter. CRT defines a new export pathway that may regulate the transcriptional activity of steroid hormone receptors.

scholarly journals In vitro FRAP reveals the ATP-dependent nuclear mobilization of the exon junction complex protein SRm160

2004 ◽  
Vol 164 (6) ◽  
pp. 843-850 ◽  
Author(s):  
Stefan Wagner ◽  
Simion Chiosea ◽  
Maria Ivshina ◽  
Jeffrey A. Nickerson
Keyword(s):  
Rna Splicing ◽  
Fluorescence Recovery After Photobleaching ◽  
Fluorescent Protein ◽  
Fluorescence Recovery ◽  
Exon Junction Complex ◽  
Permeabilized Cells ◽  
Exon Junction ◽  
Rna Export ◽  
Green Fluorescent

We present a new in vitro system for characterizing the binding and mobility of enhanced green fluorescent protein (EGFP)–labeled nuclear proteins by fluorescence recovery after photobleaching in digitonin-permeabilized cells. This assay reveals that SRm160, a splicing coactivator and component of the exon junction complex (EJC) involved in RNA export, has an adenosine triphosphate (ATP)–dependent mobility. Endogenous SRm160, lacking the EGFP moiety, could also be released from sites at splicing speckled domains by an ATP-dependent mechanism. A second EJC protein, RNPS1, also has an ATP-dependent mobility, but SRm300, a protein that binds to SRm160 and participates with it in RNA splicing, remains immobile after ATP supplementation. This finding suggests that SRm160-containing RNA export, but not splicing, complexes have an ATP-dependent mobility. We propose that RNA export complexes have an ATP-regulated mechanism for release from binding sites at splicing speckled domains. In vitro fluorescence recovery after photobleaching is a powerful tool for identifying cofactors required for nuclear binding and mobility.

scholarly journals A Membrane-Destabilizing Peptide in Capsid Protein L2 Is Required for Egress of Papillomavirus Genomes from Endosomes

2006 ◽  
Vol 80 (2) ◽  
pp. 759-768 ◽  
Author(s):  
Nadine Kämper ◽  
Patricia M. Day ◽  
Thorsten Nowak ◽  
Hans-Christoph Selinka ◽  
Luise Florin ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Fluorescent Protein ◽  
Dependent Manner ◽  
Permeabilized Cells ◽  
Viral Genomes ◽  
Amino Acid Peptide ◽  
C Terminus ◽  
Endosomal Membranes ◽  
Exogenous Addition ◽  
Green Fluorescent

ABSTRACT Papillomaviruses are internalized via clathrin-dependent endocytosis. However, the mechanism by which viral genomes pass endosomal membranes has not been elucidated. In this report we show that the minor capsid protein L2 is required for egress of viral genomes from endosomes but not for initial uptake and uncoating and that a 23-amino-acid peptide at the C terminus of L2 is necessary for this function. Pseudogenomes encapsidated by L1 and L2 lacking this peptide accumulated in vesicular compartments similar to that observed with L1-only viral particles, and these mutant pseudoviruses were noninfectious. This L2 peptide displayed strong membrane-disrupting activity, induced cytolysis of bacteria and eukaryotic cells in a pH-dependent manner, and permeabilized cells after exogenous addition. Fusions between green fluorescent protein and the L2 peptide integrated into cellular membranes like the wild type but not like C-terminal mutants of L2. Our data indicate that the L2 C terminus facilitates escape of viral genomes from the endocytic compartment and that this feature is conserved among papillomaviruses. Furthermore, the characteristic of this peptide differs from the classical virus-encoded membrane-penetrating peptides.

scholarly journals CRM1 Mediates the Export of ADAR1 through a Nuclear Export Signal within the Z-DNA Binding Domain

2001 ◽  
Vol 21 (22) ◽  
pp. 7862-7871 ◽  
Author(s):  
Hanne Poulsen ◽  
Jakob Nilsson ◽  
Christian K. Damgaard ◽  
Jan Egebjerg ◽  
Jørgen Kjems
Keyword(s):  
Nuclear Export ◽  
Fluorescent Protein ◽  
Protein A ◽  
Nuclear Export Signal ◽  
Nuclear Accumulation ◽  
Adenosine Deaminases ◽  
Editing Activity ◽  
Green Fluorescent ◽  
Export Signal

ABSTRACT RNA editing of specific residues by adenosine deamination is a nuclear process catalyzed by adenosine deaminases acting on RNA (ADAR). Different promoters in the ADAR1 gene give rise to two forms of the protein: a constitutive promoter expresses a transcript encoding (c)ADAR1, and an interferon-induced promoter expresses a transcript encoding an N-terminally extended form, (i)ADAR1. Here we show that (c)ADAR1 is primarily nuclear whereas (i)ADAR1 encompasses a functional nuclear export signal in the N-terminal part and is a nucleocytoplasmic shuttle protein. Mutation of the nuclear export signal or treatment with the CRM1-specific drug leptomycin B induces nuclear accumulation of (i)ADAR1 fused to the green fluorescent protein and increases the nuclear editing activity. In concurrence, CRM1 and RanGTP interact specifically with the (i)ADAR1 nuclear export signal to form a tripartite export complex in vitro. Furthermore, our data imply that nuclear import of (i)ADAR1 is mediated by at least two nuclear localization sequences. These results suggest that the nuclear editing activity of (i)ADAR1 is modulated by nuclear export.

The non-catalytic domain of the Xenopus laevis auroraA kinase localises the protein to the centrosome

2001 ◽  
Vol 114 (11) ◽  
pp. 2095-2104
Author(s):  
Régis Giet ◽  
Claude Prigent
Keyword(s):  
Xenopus Laevis ◽  
Fluorescent Protein ◽  
Catalytic Domain ◽  
Dependent Manner ◽  
Bacterial Cells ◽  
Terminal Domain ◽  
Egg Extracts ◽  
Green Fluorescent ◽  
Xenopus Egg Extracts

Aurora kinases are involved in mitotic events that control chromosome segregation. All members of this kinase subfamily possess two distinct domains, a highly conserved catalytic domain and an N-terminal non-catalytic extension that varies in size and sequence. To investigate the role of this variable non-catalytic region we overexpressed and purified Xenopus laevis auroraA (pEg2) histidine-tagged N-terminal peptide from bacterial cells. The peptide has no effect on the in vitro auroraA kinase activity, but it inhibits both bipolar spindle assembly and stability in Xenopus egg extracts. Unlike the full-length protein, the N-terminal domain shows only low affinity for paclitaxel-stabilised microtubules in vitro, but localises to the centrosomes in a microtubule-dependent manner. When expressed in Xenopus XL2 cells, it is able to target the green fluorescent protein to centrosomes. Surprisingly, this is also true of the pEg2 catalytic domain, although to a lesser extent. The centrosome localisation of the N-terminal peptide was disrupted by nocodazole whereas localisation of the catalytic domain was not, suggesting that in order to efficiently localise to the centrosome, pEg2 kinase required the non-catalytic N-terminal domain and the presence of microtubules.

scholarly journals Salicylic Acid Activates Sigma Factor B by rsbU-Dependent and -Independent Mechanisms

2006 ◽  
Vol 188 (16) ◽  
pp. 5896-5903 ◽  
Author(s):  
Marco Palma ◽  
Arnold Bayer ◽  
Leon I. Kupferwasser ◽  
Tammy Joska ◽  
Michael R. Yeaman ◽  
...  
Keyword(s):  
Salicylic Acid ◽  
Parental Strain ◽  
Fluorescent Protein ◽  
Toxin Production ◽  
Dependent Manner ◽  
Alpha Toxin ◽  
Reporter System ◽  
Fibronectin Binding ◽  
Green Fluorescent

ABSTRACT Salicylic acid (SAL) may impact Staphylococcus aureus virulence by activating the sigB operon (rsbU-V-W-sigB), thus leading to reductions in alpha-toxin production and decreased fibronectin binding (L. I. Kupferwasser et al., J. Clin. Investig. 112:222-233, 2003). As these prior studies were performed in strain RN6390 (an rsbU mutant) and its rsbU-repaired variant, SH1000, the current investigation was designed to determine if the SAL effect occurs via rsbU- and/or rsbV-dependent pathways in an rsbU-intact S. aureus strain (FDA486). We thus quantified the transcription from two sigB-dependent promoters (asp23 and sarA P3) in FDA486 in response to SAL exposure in vitro, using isogenic single-knockout constructs of rsbU, rsbV, or rsbW and a green fluorescent protein reporter system. SAL induced sarA P3 and asp23 promoter activities in a dose-dependent manner in the parental strain. In contrast, sigB activation by SAL was progressively more mitigated in the rsbU and rsbV mutants. As predicted, SAL caused significant reductions in both alpha-toxin production and fibrinogen and fibronectin binding in the parental strain. The extent of these reductions, compared with the parent, was reduced in the rsb mutants (rsbV > rsbU), especially at low SAL concentrations. Since generation of the free SigB protein usually requires a sequential rsbU-V-W-sigB activation cascade, the present phenotypic and genotypic data suggest key roles for both rsbU and rsbV in SAL-mediated activation of sigB in strains with a fully intact sigB operon.

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