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 Requirement of the N-Terminal Region of Orthobunyavirus Nonstructural Protein NSm for Virus Assembly and Morphogenesis

2006 ◽  
Vol 80 (16) ◽  
pp. 8089-8099 ◽  
Author(s):  
Xiaohong Shi ◽  
Alain Kohl ◽  
Vincent H. J. Léonard ◽  
Ping Li ◽  
Angela McLees ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Virus Assembly ◽  
Signal Sequence ◽  
Nonstructural Protein ◽  
Intracellular Localization ◽  
Integral Membrane Protein ◽  
Terminal Region ◽  
Coding Region ◽  
Hydrophobic Domain

ABSTRACT The nonstructural protein NSm of Bunyamwera virus (BUNV), the prototype of the Bunyaviridae family, is encoded by the M segment in a polyprotein precursor, along with the virion glycoproteins, in the order Gn-NSm-Gc. As little is known of its function, we examined the intracellular localization, membrane integrality, and topology of NSm and its role in virus replication. We confirmed that NSm is an integral membrane protein and that it localizes in the Golgi complex, together with Gn and Gc. Coimmunoprecipitation assays and yeast two-hybrid analysis demonstrated that NSm was able to interact with other viral proteins. NSm is predicted to contain three hydrophobic (I, III, and V) and two nonhydrophobic (II and IV) domains. The N-terminal nonhydrophobic domain II was found in the lumen of an intracellular compartment. A novel BUNV assembly assay was developed to monitor the formation of infectious virus-like-particles (VLPs). Using this assay, we showed that deletions of either the complete NSm coding region or domains I, II, and V individually seriously compromised VLP production. Consistently, we were unable to rescue viable viruses by reverse genetics from cDNA constructs that contained the same deletions. However, we could generate mutant BUNV with deletions in NSm domains III and IV and also a recombinant virus with the green fluorescent protein open reading frame inserted into NSm domain IV. The mutant viruses displayed differences in their growth properties. Overall, our data showed that the N-terminal region of NSm, which includes domain I and part of domain II, is required for virus assembly and that the C-terminal hydrophobic domain V may function as an internal signal sequence for the Gc glycoprotein.

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.

scholarly journals Autophagy and the cvt Pathway Both Depend onAUT9

2000 ◽  
Vol 182 (8) ◽  
pp. 2125-2133 ◽  
Author(s):  
Thomas Lang ◽  
Steffen Reiche ◽  
Michael Straub ◽  
Monika Bredschneider ◽  
Michael Thumm
Keyword(s):  
Protein Turnover ◽  
Protein Transport ◽  
Fluorescent Protein ◽  
Genomic Library ◽  
Integral Membrane Protein ◽  
Biologically Active ◽  
Transport Pathways ◽  
Rich Media ◽  
Green Fluorescent ◽  
Mutant Cells

ABSTRACT In growing cells of the yeast Saccharomyces cerevisiae, proaminopeptidase I reaches the vacuole via the selective cytoplasm-to-vacuole targeting (cvt) pathway. During nutrient limitation, autophagy is also responsible for the transport of proaminopeptidase I. These two nonclassical protein transport pathways to the vacuole are distinct in their characteristics but in large part use identical components. We expanded our initial screen foraut − mutants and isolated aut9-1cells, which show a defect in both pathways, the vacuolar targeting of proaminopeptidase I and autophagy. By complementation of the sporulation defect of homocygous diploid aut9-1 mutant cells with a genomic library, in this study we identified and characterized the AUT9 gene, which is allelic withCVT7. aut9-deficient cells have no obvious defects in growth on rich media, vacuolar biogenesis, and acidification, but like other mutant cells with a defect in autophagy, they exhibit a reduced survival rate and reduced total protein turnover during starvation. Aut9p is the first putative integral membrane protein essential for autophagy. A biologically active green fluorescent protein-Aut9 fusion protein was visualized at punctate structures in the cytosol of growing cells.

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 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 Nucleus–Vacuole Junctions in Saccharomyces cerevisiae Are Formed Through the Direct Interaction of Vac8p with Nvj1p

2000 ◽  
Vol 11 (7) ◽  
pp. 2445-2457 ◽  
Author(s):  
Xiaozhou Pan ◽  
Paul Roberts ◽  
Yan Chen ◽  
Erik Kvam ◽  
Natalyia Shulga ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Fluorescent Protein ◽  
Close Contact ◽  
Direct Interaction ◽  
Integral Membrane Protein ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Importin Α ◽  
Green Fluorescent ◽  
Pore Complexes

Vac8p is a vacuolar membrane protein that is required for efficient vacuole inheritance and fusion, cytosol-to-vacuole targeting, and sporulation. By analogy to other armadillo domain proteins, including β-catenin and importin α, we hypothesize that Vac8p docks various factors at the vacuole membrane. Two-hybrid and copurfication assays demonstrated that Vac8p does form complexes with multiple binding partners, including Apg13p, Vab2p, and Nvj1p. Here we describe the surprising role of Vac8p-Nvj1p complexes in the formation of nucleus–vacuole (NV) junctions. Nvj1p is an integral membrane protein of the nuclear envelope and interacts with Vac8p in the cytosol through its C-terminal 40–60 amino acids (aa). Nvj1p green fluorescent protein (GFP) concentrated in small patches or rafts at sites of close contact between the nucleus and one or more vacuoles. Previously, we showed that Vac8p-GFP concentrated in intervacuole rafts, where is it likely to facilitate vacuole-vacuole fusion, and in “orphan” rafts at the edges of vacuole clusters. Orphan rafts of Vac8p red-sifted GFP (YFP) colocalize at sites of NV junctions with Nvj1p blue-sifted GFP (CFP). GFP-tagged nuclear pore complexes (NPCs) were excluded from NV junctions. In vac8-Δ cells, Nvj1p-GFP generally failed to concentrate into rafts and, instead, encircled the nucleus. NV junctions were absent in both nvj1-Δ andvac8-Δ cells. Overexpression of Nvj1p caused the profound proliferation of NV junctions. We conclude that Vac8p and Nvj1p are necessary components of a novel interorganelle junction apparatus.

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 The sorting sequence of the peroxisomal integral membrane protein PMP47 is contained within a short hydrophilic loop.

1996 ◽  
Vol 133 (2) ◽  
pp. 269-280 ◽  
Author(s):  
J M Dyer ◽  
J A McNew ◽  
J M Goodman
Keyword(s):  
Amino Acids ◽  
Fluorescent Protein ◽  
Membrane Surface ◽  
Integral Membrane Protein ◽  
The Matrix ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Green Fluorescent ◽  
Necessary And Sufficient ◽  
Hydrophilic Loop

No targeting sequence for peroxisomal integral membrane proteins has yet been identified. We have previously shown that a region of 67 amino acids is necessary to target Pmp47, a protein that spans the membrane six times, to peroxisomes. This region comprises two membrane spans and the intervening loop. We now demonstrate that the 20 amino acid loop, which is predicted to face the matrix, is both necessary and sufficient for peroxisomal targeting. Sufficiency was demonstrated with both chloramphenicol acetyltransferase and green fluorescent protein as carriers. There is a cluster of basic amino acids in the middle of the loop that we predict protrudes from the membrane surface into the matrix by a flanking stem structure. We show that the targeting signal is composed of this basic cluster and a block of amino acids immediately down-stream from it.

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 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.

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