scholarly journals Hantavirus Nucleocapsid Protein Is Expressed as a Membrane-Associated Protein in the Perinuclear Region

2001 ◽  
Vol 75 (4) ◽  
pp. 1808-1815 ◽  
Author(s):  
Eugene V. Ravkov ◽  
Richard W. Compans
Keyword(s):  
Nucleocapsid Protein ◽  
New World ◽  
Fluorescent Protein ◽  
Intracellular Localization ◽  
Cell Fractionation ◽  
Perinuclear Region ◽  
La Crosse Virus ◽  
Microsomal Membranes ◽  
The Difference ◽  
Green Fluorescent

ABSTRACT Black Creek Canal virus (BCCV) is a New World hantavirus which is associated with hantavirus pulmonary syndrome. We have examined the site of expression of the BCCV nucleocapsid protein (NBCCV) in the absence of BCCV glycoproteins and found that the majority of the protein is localized to the Golgi region. Immunofluorescence analysis of BHK21 cells expressing the NBCCV and La Crosse virus nucleocapsid protein (NLACV) showed different intracellular localization patterns of these proteins within the same cell: NLACV is cytoplasmic, whereas NBCCV is perinuclear. NBCCV was found to be colocalized with α-mannosidase II, a marker for the Golgi complex. Also, NBCCV was found to be associated with microsomal membranes following cell fractionation. Sedimentation analysis in density gradients revealed that the membrane association of NBCCV is sensitive to treatments with high-salt and high-pH solutions, which indicates that NBCCV is a peripheral membrane protein. Analysis of NBCCV truncation mutants revealed that the 141-amino-acid C-terminal portion of this protein was capable of targeting green fluorescent protein to the perinuclear region. The difference in the intracellular localization between the NBCCV and NLACV proteins suggests that the mechanisms involved in the morphogenesis of New World hantaviruses are distinct from that documented for other members of theBunyaviridae family.

scholarly journals Characterization of Signal That Directs C-Tail–anchored Proteins to Mammalian Mitochondrial Outer Membrane

2002 ◽  
Vol 13 (5) ◽  
pp. 1615-1625 ◽  
Author(s):  
Chika Horie ◽  
Hiroyuki Suzuki ◽  
Masao Sakaguchi ◽  
Katsuyoshi Mihara
Keyword(s):  
Outer Membrane ◽  
Fluorescent Protein ◽  
Structural Characteristics ◽  
Intracellular Localization ◽  
Basic Amino Acid ◽  
Mitochondrial Outer Membrane ◽  
Cell Fractionation ◽  
Mitochondrial Targeting ◽  
Intracellular Membranes ◽  
Green Fluorescent

We analyzed the signal that directs the outer membrane protein with the C-terminal transmembrane segment (TMS) to mammalian mitochondria by using yeast Tom5 as a model and green fluorescent protein as a reporter. Deletions or mutations were systematically introduced into the TMS or the flanking regions and their intracellular localization in COS-7 cells was examined using confocal microscopy and cell fractionation. 1) Three basic amino acid residues within the C-terminal five-residue segment (C-segment) contained the information required for mitochondrial-targeting. Reduction of the net positive charge in this segment decreased mitochondrial specificity, and the mutants were distributed throughout the intracellular membranes. 2) Elongation of the TMS interfered with the function of the C-segment and the mutants were delivered to the intracellular membranes. 3) Separation of the TMS and C-segment by linker insertion severely impaired mitochondrial targeting function, leading to mislocalization to the cytoplasm. 4) Mutations or small deletions in the region of the TMS flanking the C-segment also impaired the mitochondrial targeting. Therefore, the moderate length of the TMS, the positive charges in the C-segment, and the distance between or context of the TMS and C-segment are critical for the targeting signal. The structural characteristics of the signal thus defined were also confirmed with mammalian C-tail–anchored protein OMP25.

scholarly journals GluA1 trafficking and metabotropic NMDA: addressing results from other laboratories inconsistent with ours

2014 ◽  
Vol 369 (1633) ◽  
pp. 20130145 ◽  
Author(s):  
Sadegh Nabavi ◽  
Rocky Fox ◽  
Stephanie Alfonso ◽  
Jonathan Aow ◽  
Roberto Malinow
Keyword(s):  
Fluorescent Protein ◽  
Long Term Potentiation ◽  
Long Term Depression ◽  
Mk 801 ◽  
Over Expression ◽  
Term Potentiation ◽  
The Difference ◽  
Green Fluorescent ◽  
Gfp Tag

We have previously shown that when over-expressed in neurons, green fluorescent protein (GFP) tagged GluA1 (GluA1-GFP) delivery into synapses is dependent on plasticity. A recent study suggests that GluA1 over-expression leads to its incorporation into the synapse, in the absence of additional long-term potentiation-like manipulations. It is possible that a GFP tag was responsible for the difference. Using rectification index as a measure of synaptic delivery of GluA1, we found no difference in the synaptic delivery of GluA1-GFP versus untagged GluA1. We recently published a study showing that while D-APV blocks NMDAr-dependent long-term depression (LTD), MK-801 and 7-chloro kynurenate (7CK) fail to block LTD. We propose a metabotropic function for the NMDA receptor in LTD induction. In contrast to our observations, recent unpublished data suggest that the above antagonists are equally effective in blocking LTD. We noticed different methodology in their study. Here, we show that their methodology has complex effects on synaptic transmission. Therefore, it is not possible to conclude that 7CK is effective in blocking LTD from their type of experiment.

Intracellular localization and in vivo trafficking of p24A and p23

1999 ◽  
Vol 112 (4) ◽  
pp. 537-548 ◽  
Author(s):  
R. Blum ◽  
F. Pfeiffer ◽  
P. Feick ◽  
W. Nastainczyk ◽  
B. Kohler ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Intracellular Localization ◽  
Maximum Speed ◽  
Heterotrimeric G Proteins ◽  
P24 Protein ◽  
Intermediate Compartment ◽  
Copi Vesicle ◽  
Green Fluorescent ◽  
Golgi Network

Recently, p24A and p23 (also termed Tmp21), two members of the p24 protein family, have been proposed to function as integral receptors for the COPI-vesicle coat. This study describes the intracellular localization and trafficking of p24A in comparison to p23. For immunolocalization of p24A and p23, strong reduction and denaturation conditions were necessary to allow antibody interaction. Both p24A and p23 cycle continuously between intermediate compartment (IC) elements and the cis-Golgi network. In vivo trafficking of p24A and p23 tagged to green fluorescent protein (GFP) revealed that both proteins travel by large (up to 1 micrometer in length) microtubule-dependent pre-Golgi carriers with a maximum speed of up to 1.6 micrometer s-1 from the IC to the Golgi cisternae. Aluminum fluoride, a general activator of heterotrimeric G-proteins, blocked peripheral pre-Golgi movements of GFP-p24A/p23 and inhibited fluorescence recovery after photobleaching in the perinuclear Golgi area. p24A and p23 are predominantly colocalized. Overexpression of GFP-p24A, to an extent which did not destroy the Golgi complex, induced delocalization of part of the proteins into ER elements. This study therefore gives new insights into the localization and trafficking behavior of the two COPI-binding proteins p24A and p23.

Several protein regions contribute to determine the nuclear and cytoplasmic localization of the influenza A virus nucleoprotein

Microbiology ◽  
2000 ◽  
Vol 81 (1) ◽  
pp. 135-142 ◽  
Author(s):  
Rosario Bullido ◽  
Paulino Gómez-Puertas ◽  
Carmen Albo ◽  
Agustín Portela
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Fluorescent Protein ◽  
Intracellular Localization ◽  
Phosphorylation Site ◽  
Nuclear Accumulation ◽  
Systematic Analysis ◽  
Ribonucleoprotein Complexes ◽  
Cytoplasmic Transport ◽  
Green Fluorescent

A systematic analysis was carried out to identify the amino acid signals that regulate the nucleo-cytoplasmic transport of the influenza A virus nucleoprotein (NP). The analysis involved determining the intracellular localization of eight deleted recombinant NP proteins and 14 chimeric proteins containing the green fluorescent protein fused to different NP fragments. In addition, the subcellular distribution of NP derivatives that contained specific substitutions at serine-3, which is the major phosphorylation site of the A/Victoria/3/75 NP, were analysed. From the results obtained, it is concluded that the NP contains three signals involved in nuclear accumulation and two regions that cause cytoplasmic accumulation of the fusion proteins. One of the karyophilic signals was located at the N terminus of the protein, and the data obtained suggest that the functionality of this signal can be modified by phosphorylation at serine-3. These findings are discussed in the context of the transport of influenza virus ribonucleoprotein complexes into and out of the nucleus.

scholarly journals Release of Immunity Protein Requires Functional Endonuclease Colicin Import Machinery

2006 ◽  
Vol 188 (24) ◽  
pp. 8593-8600 ◽  
Author(s):  
Denis Duché ◽  
Aurélie Frenkian ◽  
Valérie Prima ◽  
Roland Lloubès
Keyword(s):  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Cell Envelope ◽  
Membrane Surface ◽  
Cell Fractionation ◽  
Extracellular Medium ◽  
Immunity Protein ◽  
Hybrid Complex ◽  
Colicin E2 ◽  
Green Fluorescent

ABSTRACT Bacteria producing endonuclease colicins are protected against the cytotoxic activity by a small immunity protein that binds with high affinity and specificity to inactivate the endonuclease. This complex is released into the extracellular medium, and the immunity protein is jettisoned upon binding of the complex to susceptible cells. However, it is not known how and at what stage during infection the immunity protein release occurs. Here, we constructed a hybrid immunity protein composed of the enhanced green fluorescent protein (EGFP) fused to the colicin E2 immunity protein (Im2) to enhance its detection. The EGFP-Im2 protein binds the free colicin E2 with a 1:1 stoichiometry and specifically inhibits its DNase activity. The addition of this hybrid complex to susceptible cells reveals that the release of the hybrid immunity protein is a time-dependent process. This process is achieved 20 min after the addition of the complex to the cells. We showed that complex dissociation requires a functional translocon formed by the BtuB protein and one porin (either OmpF or OmpC) and a functional import machinery formed by the Tol proteins. Cell fractionation and protease susceptibility experiments indicate that the immunity protein does not cross the cell envelope during colicin import. These observations suggest that dissociation of the immunity protein occurs at the outer membrane surface and requires full translocation of the colicin E2 N-terminal domain.

scholarly journals A Yeast STE11 Homologue CoMEKK1 Is Essential for Pathogenesis-Related Morphogenesis in Colletotrichum orbiculare

2010 ◽  
Vol 23 (12) ◽  
pp. 1563-1572 ◽  
Author(s):  
Ayumu Sakaguchi ◽  
Gento Tsuji ◽  
Yasuyuki Kubo
Keyword(s):  
Signal Transduction ◽  
Fluorescent Protein ◽  
Intracellular Localization ◽  
Active Form ◽  
Hyphal Growth ◽  
Mitogen Activated Protein Kinase ◽  
Colletotrichum Orbiculare ◽  
Pathogenesis Related ◽  
Mitogen Activated Protein ◽  
Green Fluorescent

Several signal transduction pathways, including mitogen-activated protein kinase (MAPK) pathways, are involved in appressorium development in Colletotrichum orbiculare, the causal agent of cucumber anthracnose disease. In this study, CoMEKK1, a yeast MAPK kinases (MAPKK) kinase STE11 homolog, was identified as a disrupted gene in an Agrobacterium tumefaciens-mediated transformation mutant. The phenotype of comekk1 disruptant was similar to that of cmk1, a Saccharomyces cerevisiae Fus3/Kss1 MAPK homolog mutant. Moreover, comekk1 and cmk1 mutants were sensitive to high osmotic and salinity stresses, indicating that Comekk1p/Cmk1p signal transduction is involved in stress tolerance. The transformants of the wild type and the comekk1 mutant expressing a constitutively active form of the CoMEKK1 showed slower hyphal growth and abnormal appressorium formation, whereas those of the cmk1 disruptant did not. A Cmk1p-green fluorescent protein (GFP) intracellular localization experiment indicated that nuclear localization of the Cmk1p-GFP fusion protein induced by salt stress was diminished in comekk1 mutants. These results indicate that Comekk1p functions upstream of Cmk1p.

scholarly journals Movement of Bax from the Cytosol to Mitochondria during Apoptosis

1997 ◽  
Vol 139 (5) ◽  
pp. 1281-1292 ◽  
Author(s):  
Keith G. Wolter ◽  
Yi-Te Hsu ◽  
Carolyn L. Smith ◽  
Amotz Nechushtan ◽  
Xu-Guang Xi ◽  
...  
Keyword(s):  
Soluble Protein ◽  
Fluorescent Protein ◽  
Intracellular Localization ◽  
Integral Membrane Protein ◽  
Mitochondrial Marker ◽  
Nuclear Condensation ◽  
Hydrophobic Domain ◽  
Induction Of Apoptosis ◽  
Green Fluorescent ◽  
Promote Cell Death

Bax, a member of the Bcl-2 protein family, accelerates apoptosis by an unknown mechanism. Bax has been recently reported to be an integral membrane protein associated with organelles or bound to organelles by Bcl-2 or a soluble protein found in the cytosol. To explore Bcl-2 family member localization in living cells, the green fluorescent protein (GFP) was fused to the NH2 termini of Bax, Bcl-2, and Bcl-XL. Confocal microscopy performed on living Cos-7 kidney epithelial cells and L929 fibroblasts revealed that GFP–Bcl-2 and GFP–Bcl-XL had a punctate distribution and colocalized with a mitochondrial marker, whereas GFP–Bax was found diffusely throughout the cytosol. Photobleaching analysis confirmed that GFP–Bax is a soluble protein, in contrast to organelle-bound GFP–Bcl-2. The diffuse localization of GFP–Bax did not change with coexpression of high levels of Bcl-2 or Bcl-XL. However, upon induction of apoptosis, GFP–Bax moved intracellularly to a punctate distribution that partially colocalized with mitochondria. Once initiated, this Bax movement was complete within 30 min, before cellular shrinkage or nuclear condensation. Removal of a COOH-terminal hydrophobic domain from GFP–Bax inhibited redistribution during apoptosis and inhibited the death-promoting activity of both Bax and GFP– Bax. These results demonstrate that in cells undergoing apoptosis, an early, dramatic change occurs in the intracellular localization of Bax, and this redistribution of soluble Bax to organelles appears important for Bax to promote cell death.

scholarly journals Intracellular Targeting of Gag Proteins of the Drosophila Telomeric Retrotransposons

2003 ◽  
Vol 77 (11) ◽  
pp. 6376-6384 ◽  
Author(s):  
S. Rashkova ◽  
A. Athanasiadis ◽  
M.-L. Pardue
Keyword(s):  
Protein Interactions ◽  
Fluorescent Protein ◽  
Cluster Formation ◽  
Intracellular Localization ◽  
Ltr Retrotransposons ◽  
Protein Protein Interactions ◽  
Gag Proteins ◽  
Intracellular Targeting ◽  
Identify Amino Acid ◽  
Green Fluorescent

ABSTRACT Drosophila has two non-long-terminal-repeat (non-LTR) retrotransposons that are unique because they have a defined role in chromosome maintenance. These elements, HeT-A and TART, extend chromosome ends by successive transpositions, producing long arrays of head-to-tail repeat sequences. These arrays appear to be analogous to the arrays produced by telomerase on chromosomes of other organisms. While other non-LTR retrotransposons transpose to many chromosomal sites, HeT-A and TART transpose only to chromosome ends. Although HeT-A and TART belong to different subfamilies of non-LTR retrotransposons, they encode very similar Gag proteins, which suggests that Gag proteins are involved in their unique transposition targeting. We have recently shown that both Gags localize efficiently to nuclei where HeT-A Gag forms structures associated with telomeres. TART Gag does not associate with telomeres unless HeT-A Gag is present, suggesting a symbiotic relationship in which HeT-A Gag provides telomeric targeting. We now report studies to identify amino acid regions responsible for different aspects of the intracellular targeting of these proteins. Green fluorescent protein-tagged deletion derivatives were expressed in cultured Drosophila cells. The intracellular localization of these proteins shows the following. (i) Several regions that direct subcellular localizations or cluster formation are found in both Gags and are located in equivalent regions of the two proteins. (ii) Regions important for telomere association are present only in HeT-A Gag. These are present at several places in the protein, are not redundant, and cannot be complemented in trans. (iii) Regions containing zinc knuckle and major homology region motifs, characteristic of retroviral Gags, are involved in protein-protein interactions of the telomeric Gags, as they are in retroviral Gags.

scholarly journals The Coronavirus Nucleocapsid Protein Is Dynamically Associated with the Replication-Transcription Complexes

2010 ◽  
Vol 84 (21) ◽  
pp. 11575-11579 ◽  
Author(s):  
Monique H. Verheije ◽  
Marne C. Hagemeijer ◽  
Mustafa Ulasli ◽  
Fulvio Reggiori ◽  
Peter J. M. Rottier ◽  
...  
Keyword(s):  
Viral Replication ◽  
Nucleocapsid Protein ◽  
Fluorescent Protein ◽  
Nonstructural Protein ◽  
Structural Protein ◽  
Independent Manner ◽  
N Protein ◽  
Nonstructural Protein 2 ◽  
Transcription Complexes ◽  
Green Fluorescent

ABSTRACT The coronavirus nucleocapsid (N) protein is a virion structural protein. It also functions, however, in an unknown way in viral replication and localizes to the viral replication-transcription complexes (RTCs). Here we investigated, using recombinant murine coronaviruses expressing green fluorescent protein (GFP)-tagged versions of the N protein, the dynamics of its interactions with the RTCs and the domain(s) involved. Using fluorescent recovery after photobleaching, we showed that the N protein, unlike the nonstructural protein 2, is dynamically associated with the RTCs. Recruitment of the N protein to the RTCs requires the C-terminal N2b domain, which interacts with other N proteins in an RNA-independent manner.

scholarly journals Regulation of ZAP-70 Intracellular Localization: Visualization with the Green Fluorescent Protein

1997 ◽  
Vol 186 (10) ◽  
pp. 1713-1724 ◽  
Author(s):  
Joanne Sloan-Lancaster ◽  
Weiguo Zhang ◽  
John Presley ◽  
Brandi L. Williams ◽  
Robert T. Abraham ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
T Cell Receptor ◽  
Kinase Activity ◽  
Fluorescent Protein ◽  
Intracellular Localization ◽  
Cell Receptor ◽  
Cellular Activation ◽  
Intracellular Location ◽  
Green Fluorescent ◽  
Biologic Function

To investigate the cellular dynamics of ZAP-70, we have studied the distribution and regulation of its intracellular location using a ZAP-70 green fluorescent protein chimera. Initial experiments in epithelial cells indicated that ZAP-70 is diffusely located throughout the quiescent cell, and accumulates at the plasma membrane upon cellular activation, a phenotype enhanced by the coexpression of Lck and the initiation of ZAP-70 kinase activity. Subsequent studies in T cells confirmed this phenotype. Intriguingly, a large amount of ZAP-70, both chimeric and endogenous, resides in the nucleus of quiescent and activated cells. Nuclear ZAP-70 becomes tyrosine phosphorylated upon stimulation via the T cell receptor, indicating that it may have an important biologic function.

Sign in / Sign up

Export Citation Format

Share Document