scholarly journals Intracellular targeting and homotetramer formation of a truncated inositol 1,4,5-trisphosphate receptor–green fluorescent protein chimera in Xenopus laevis oocytes: evidence for the involvement of the transmembrane spanning domain in endoplasmic reticulum targeting and homotetramer complex formation

1997 ◽  
Vol 323 (1) ◽  
pp. 273-280 ◽  
Author(s):  
Lee G. SAYERS ◽  
Atsushi MIYAWAKI ◽  
Akira MUTO ◽  
Hiroshi TAKESHITA ◽  
Akitsugu YAMAMOTO ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Intracellular Localization ◽  
Animal Pole ◽  
Inositol Trisphosphate Receptor ◽  
Intracellular Targeting ◽  
Fluorescence Confocal Microscopy ◽  
Inositol 1,4,5 Trisphosphate ◽  
Microscopy Data ◽  
Green Fluorescent ◽  
Trisphosphate Receptor

In an attempt to define structural regions of the type I inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptor [Ins(1,4,5)P3R] involved in its intracellular targeting to the endoplasmic reticulum (ER), we have employed the use of green fluorescent protein (GFP) to monitor the localization of a truncated Ins(1,4,5)P3R mutant containing just the putative transmembrane spanning domain and the C-terminal cytoplasmic domain [amino acids 2216-2749; termed inositol trisphosphate receptor(ES)]. We expressed a chimeric GFP-Ins(1,4,5)P3R(ES) fusion protein in Xenopus laevisoocytes, and used fluorescence confocal microscopy to monitor its intracellular localization. Fluorescence confocal microscopy data showed an intense fluorescence in the perinuclear region and in a reticular-network under the animal pole of the oocyte, consistent with the targeting of expressed GFP-Ins(1,4,5)P3R(ES) to perinuclear ER and ER under the animal pole. These findings are consistent with the intracellular localization of the endogenous Xenopus Ins(1,4,5)P3R shown previously. Furthermore, electron microscopy data indicate that expressed GFP-Ins(1,4,5)P3R(ES) is in fact targeted to the ER. Sodium carbonate extraction of microsomal membranes and cross-linking experiments indicate that the expressed chimeric protein is in fact membrane anchored and able to form a homotetrameric complex. Our data provides evidence that Ins(1,4,5)P3R(ES) constitutes the membrane spanning domain of the Ins(1,4,5)P3R and is able to mediate homotetramer formation, without the need for the large N-terminal cytoplasmic domain. Furthermore, the localization of GFP-Ins(1,4,5)P3R(ES) on the ER indicates that an ER retention/targeting signal is contained within the transmembrane spanning domain of the inositol trisphosphate receptor.

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 three isoenzymes of human inositol-1,4,5-trisphosphate 3-kinase show specific intracellular localization but comparable Ca2+ responses on transfection in COS-7 cells

2003 ◽  
Vol 374 (1) ◽  
pp. 41-49 ◽  
Author(s):  
Valérie DEWASTE ◽  
Colette MOREAU ◽  
Florence De SMEDT ◽  
Françoise BEX ◽  
Humbert De SMEDT ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Intracellular Localization ◽  
Metabolizing Enzymes ◽  
Transfected Cells ◽  
Extracellular Signals ◽  
Inositol 1,4,5 Trisphosphate ◽  
Low Levels ◽  
Green Fluorescent ◽  
Mutant Cells

Inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] 3-kinase catalyses the phosphorylation of InsP3 to inositol 1,3,4,5-tetrakisphosphate. cDNAs encoding three human isoenzymes of InsP3 3-kinase (A, B and C) have been reported previously [Choi, Kim, Lee, Moon, Sim, Kim, Chung and Rhee (1990) Science 248, 64–66; Dewaste, Pouillon, Moreau, Shears, Takazawa and Erneux (2000) Biochem. J. 352, 343–351; Dewaste, Roymans, Moreau and Erneux (2002) Biochem. Biophys. Res. Commun. 291, 400–405; Takazawa, Perret, Dumont and Erneux (1991) Biochem. Biophys. Res. Commun. 174, 529–535]. The localization of InsP3 3-kinase isoenzymes fused at their N-terminus to the green fluorescent protein has been studied by confocal microscopy. The A isoform appeared to associate with the cytoskeleton, whereas the C isoform was totally cytoplasmic. The B isoform had a more complex localization: it appeared in the plasma membrane, cytoskeleton and in the endoplasmic reticulum. The three human isoenzymes of InsP3 3-kinase can thus be distinguished by their N-terminal sequence, sensitivity to Ca2+/calmodulin and localization on transfection in COS-7 cells. We have compared the cytosolic Ca2+ responses induced by ATP in COS-7 cells transfected with the three isoenzymes. Cells expressing high levels of any of the three isoforms no longer respond to ATP, whereas cells expressing low levels of each enzyme showed a reduced response consisting of one to three Ca2+ spikes in response to 100 μM ATP. These effects were seen only in wild-type InsP3 3-kinase-transfected cells. 3-Kinase-dead mutant cells behaved as vector-transfected cells. The results highlight the potential role of the three isoforms of InsP3 3-kinase as direct InsP3 metabolizing enzymes and direct regulators of Ca2+ responses to extracellular signals.

scholarly journals Functional analysis of the green fluorescent protein-tagged inositol 1,4,5-trisphosphate receptor type 3 in Ca2+ release and entry in DT40 B lymphocytes

2004 ◽  
Vol 382 (3) ◽  
pp. 793-801 ◽  
Author(s):  
Takao MORITA ◽  
Akihiko TANIMURA ◽  
Akihiro NEZU ◽  
Tomohiro KUROSAKI ◽  
Yosuke TOJYO
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Cell Receptor ◽  
Receptor Type ◽  
Intact Cells ◽  
Entry Pathway ◽  
Inositol 1,4,5 Trisphosphate ◽  
Green Fluorescent ◽  
Trisphosphate Receptor ◽  
Type 3

We examined the function of GFP-IP3R3 (green fluorescent protein-tagged inositol 1,4,5-trisphosphate receptor type 3) in Ca2+ release and entry using a mutant DT40 cell line (IP3R-KO) in which all three IP3R genes had been disrupted. GFP-IP3R3 fluorescence largely overlapped with the distribution of endoplasmic reticulum, whereas a portion of GFP-IP3R3 apparently co-localized with the plasma membrane. The application of IP3 to permeabilized WT (wild-type) DT40 cells induced Ca2+ release from internal stores. Although this did not occur in IP3R-KO cells it was restored by expression of GFP-IP3R3. In intact cells, application of anti-IgM, an activator of the BCR (B-cell receptor), or trypsin, a protease-activated receptor 2 agonist, did not cause any Ca2+ response in IP3R-KO cells, whereas these treatments induced oscillatory or transient Ca2+ responses in GFP-IP3R3-expressing IP3R-KO cells, as well as in WT cells. In addition, BCR activation elicited Ca2+ entry in WT and GFP-IP3R3-expressing IP3R-KO cells but not in IP3R-KO cells. This BCR-mediated Ca2+ entry was observed in the presence of La3+, which blocks capacitative Ca2+ entry. Thapsigargin depleted Ca2+ stores and led to Ca2+ entry in IP3R-KO cells irrespective of GFP-IP3R3 expression. In contrast with BCR stimulation, thapsigargin-induced Ca2+ entry was completely blocked by La3+, suggesting that the BCR-mediated Ca2+ entry pathway is distinct from the capacitative Ca2+ entry pathway. The present study demonstrates that GFP-IP3R3 could compensate for native IP3R in both IP3-induced Ca2+ release and BCR-mediated Ca2+ entry.

scholarly journals Targeting of inositol 1,4,5-trisphosphate receptor to the endoplasmic reticulum by its first transmembrane domain

2009 ◽  
Vol 425 (1) ◽  
pp. 61-74 ◽  
Author(s):  
Evangelia Pantazaka ◽  
Colin W. Taylor
Keyword(s):  
Endoplasmic Reticulum ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Signal Sequence ◽  
Subcellular Fractionation ◽  
Hydrophobic Residues ◽  
Inositol 1,4,5 Trisphosphate ◽  
Stable Association ◽  
Green Fluorescent ◽  
Trisphosphate Receptor

Targeting of IP3R (inositol 1,4,5-trisphosphate receptors) to membranes of the ER (endoplasmic reticulum) and their retention within ER or trafficking to other membranes underlies their ability to generate spatially organized Ca2+ signals. N-terminal fragments of IP3R1 (type 1 IP3R) were tagged with enhanced green fluorescent protein, expressed in COS-7 cells and their distribution was determined by confocal microscopy and subcellular fractionation. Localization of IP3R1 in the ER requires translation of between 26 and 34 residues beyond the end of the first transmembrane domain (TMD1), a region that includes TMD2 (second transmembrane domain). Replacement of these post-TMD1 residues with unrelated sequences of similar length (24–36 residues) partially mimicked the native residues. We conclude that for IP3R approx. 30 residues after TMD1 must be translated to allow a signal sequence within TMD1 to be extruded from the ribosome and mediate co-translational targeting to the ER. Hydrophobic residues within TMD1 and TMD2 then ensure stable association with the ER membrane.

Complex regulation and nuclear localization of JRK protein

2004 ◽  
Vol 32 (6) ◽  
pp. 920-923 ◽  
Author(s):  
R. Waldron ◽  
T. Moore
Keyword(s):  
Cellular Localization ◽  
Fluorescent Protein ◽  
Binding Protein ◽  
Distinct Pattern ◽  
Nuclear Bodies ◽  
Null Mouse ◽  
Fluorescence Confocal Microscopy ◽  
Pml Nuclear Bodies ◽  
Generalized Epilepsy ◽  
Green Fluorescent

The mouse jerky gene and its human orthologue, JRK/JH8, encode a putative DNA-binding protein with homology to the CENP-B (centromere-binding protein B). Disruption of the mouse jerky gene by transgene insertion causes generalized recurrent seizures reminiscent of human idiopathic generalized epilepsy. In addition (and similar to a cenp-b null mouse) jerky null mice exhibit postnatal weight loss and reduced fertility. Using fluorescence confocal microscopy, the cellular localization of a JRK–GFP fusion (where GFP stands for green fluorescent protein) was investigated in HeLa cells. JRK–GFP has a dynamic expression pattern in the interphase nucleus, localizing in a small number of punctate nuclear foci and in the nucleolus. The JRK–GFP foci number changes during the cell cycle, but a distinct pattern of three JRK–GFP foci is observed at G2. The endogenous protein behaves in a similar manner to the GFP-fusion protein. JRK–GFP was found to co-localize with CREST antigens (which recognize the centromere-binding proteins, CENP-A, -B and -C) through S and G2 phases of interphase and co-localized completely with a subset of PML nuclear bodies at G2. We speculate that JRK protein associates with a specific chromosomal centromeric locus in G2, where it associates fully with PML bodies. Research is underway to identify this locus.

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 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.

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