scholarly journals Cloning and Characterization of an Ovine Intracellular Seven Transmembrane Receptor for Progesterone that Mediates Calcium Mobilization

Endocrinology ◽  
2006 ◽  
Vol 147 (9) ◽  
pp. 4151-4159 ◽  
Author(s):  
R. L. Ashley ◽  
C. M. Clay ◽  
T. A. Farmerie ◽  
G. D. Niswender ◽  
T. M. Nett
Keyword(s):  
Endoplasmic Reticulum ◽  
Cho Cells ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Specific Binding ◽  
Protein Fusion ◽  
Acid Protein ◽  
Modulation Of Gene Expression ◽  
Hydroxy Progesterone ◽  
Green Fluorescent

Classically, progesterone has been thought to act only through the well-known genomic pathway involving hormone binding to nuclear receptors (nPR) and subsequent modulation of gene expression. However, there is increasing evidence for rapid, nongenomic effects of progesterone in a variety of tissues in mammals, and it seems likely that a membrane PR (mPR) is causing these events. The objective of this study was to isolate and characterize an ovine mPR distinct from the nPR. A cDNA clone was isolated from ovine genomic DNA by PCR. The ovine mPR is a 350-amino acid protein that, based on computer hydrophobicity analysis, possesses seven transmembrane domains and is distinct from the nPR. Message for the ovine mPR was detected in hypothalamus, pituitary, uterus, ovary, and corpus luteum by RT-PCR. In CHO cells that overexpressed a mPR-green fluorescent protein fusion protein, the ovine mPR was localized to the endoplasmic reticulum and not the plasma membrane. Specific binding of 3H-progesterone to membrane fractions was demonstrated in CHO cells that expressed the ovine mPR but not in nontransfected cells. Furthermore, progesterone and 17α-hydroxy-progesterone stimulated intracellular Ca2+ mobilization in CHO cells that expressed ovine mPR in Ca2+-free medium (P < 0.05) but not in CHO cells transfected with empty vector. This rise in intracellular Ca2+ is believed to be from the endoplasmic reticulum as intracellular Ca2+ mobilization is absent when mPR transfected cells are first treated with thapsigargin to deplete Ca2+ stores from the endoplasmic reticulum. Isolation, identification, tissue distribution, cellular localization, steroid binding, and a functional response for a unique intracellular mPR in the sheep are presented.

scholarly journals Saccharomyces cerevisiae Nip7p is required for efficient 60S ribosome subunit biogenesis.

1997 ◽  
Vol 17 (9) ◽  
pp. 5001-5015 ◽  
Author(s):  
N I Zanchin ◽  
P Roberts ◽  
A DeSilva ◽  
F Sherman ◽  
D S Goldfarb
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fluorescent Protein ◽  
Open Reading Frames ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Protein Fusion ◽  
Acid Protein ◽  
Conserved Gene ◽  
Green Fluorescent

The Saccharomyces cerevisiae temperature-sensitive (ts) allele nip7-1 exhibits phenotypes associated with defects in the translation apparatus, including hypersensitivity to paromomycin and accumulation of halfmer polysomes. The cloned NIP7+ gene complemented the nip7-1 ts growth defect, the paromomycin hypersensitivity, and the halfmer defect. NIP7 encodes a 181-amino-acid protein (21 kDa) with homology to predicted products of open reading frames from humans, Caenorhabditis elegans, and Arabidopsis thaliana, indicating that Nip7p function is evolutionarily conserved. Gene disruption analysis demonstrated that NIP7 is essential for growth. A fraction of Nip7p cosedimented through sucrose gradients with free 60S ribosomal subunits but not with 80S monosomes or polysomal ribosomes, indicating that it is not a ribosomal protein. Nip7p was found evenly distributed throughout the cytoplasm and nucleus by indirect immunofluorescence; however, in vivo localization of a Nip7p-green fluorescent protein fusion protein revealed that a significant amount of Nip7p is present inside the nucleus, most probably in the nucleolus. Depletion of Nip7-1p resulted in a decrease in protein synthesis rates, accumulation of halfmers, reduced levels of 60S subunits, and, ultimately, cessation of growth. Nip7-1p-depleted cells showed defective pre-rRNA processing, including accumulation of the 35S rRNA precursor, presence of a 23S aberrant precursor, decreased 20S pre-rRNA levels, and accumulation of 27S pre-rRNA. Delayed processing of 27S pre-rRNA appeared to be the cause of reduced synthesis of 25S rRNA relative to 18S rRNA, which may be responsible for the deficit of 60S subunits in these cells.

scholarly journals Endoplasmic Reticulum Dynamics, Inheritance, and Cytoskeletal Interactions in Budding Yeast

2002 ◽  
Vol 13 (3) ◽  
pp. 854-865 ◽  
Author(s):  
K. L. Fehrenbacher ◽  
D. Davis ◽  
M. Wu ◽  
I. Boldogh ◽  
Liza A. Pon
Keyword(s):  
Endoplasmic Reticulum ◽  
Fluorescent Protein ◽  
Yeast Cells ◽  
Cell Cortex ◽  
Filamentous Actin ◽  
Protein Fusion ◽  
Directed Movement ◽  
Apical Bud ◽  
M Phase ◽  
Green Fluorescent

The endoplasmic reticulum (ER) in Saccharomyces cerevisiae consists of a reticulum underlying the plasma membrane (cortical ER) and ER associated with the nuclear envelope (nuclear ER). We used a Sec63p-green fluorescent protein fusion protein to study motility events associated with inheritance of cortical ER and nuclear ER in living yeast cells. During M phase before nuclear migration, we observed thick, apparently rigid tubular extensions emanating from the nuclear ER that elongate, undergo sweeping motions along the cell cortex, and shorten. Two findings support a role for microtubules in this process. First, extension of tubular structures from the nuclear ER is inhibited by destabilization of microtubules. Second, astral microtubules, structures that undergo similar patterns of extension, cortical surveillance and retraction, colocalize with nuclear ER extensions. During S and G2 phases of the cell cycle, we observed anchorage of the cortical ER at the site of bud emergence and apical bud growth. Thin tubules of the ER that extend from the anchored cortical ER display undulating, apparently random movement and move into the bud as it grows. Finally, we found that cortical ER morphology is sensitive to a filamentous actin–destabilizing drug, latrunculin-A, and to mutations in the actin-encoding ACT1 gene. Our observations support 1) different mechanisms and cytoskeletal mediators for the inheritance of nuclear and cortical ER elements and 2) a mechanism for cortical ER inheritance that is cytoskeleton dependent but relies on anchorage, not directed movement.

scholarly journals In VivoSelf-Assembly of Stable Green Fluorescent Protein Fusion Particles and Their Uses in Enzyme Immobilization

2014 ◽  
Vol 80 (10) ◽  
pp. 3062-3071 ◽  
Author(s):  
Mark Venning-Slater ◽  
David O. Hooks ◽  
Bernd H. A. Rehm
Keyword(s):  
Green Fluorescent Protein ◽  
Enzyme Immobilization ◽  
Self Assembly ◽  
Fluorescent Protein ◽  
Specific Binding ◽  
Biomass Conversion ◽  
Protein Fusion ◽  
Content Type ◽  
Green Fluorescent

ABSTRACTBacterial inclusion bodies are aggregations of mostly inactive and misfolded proteins. However, previously thein vivoself-assembly of green fluorescent protein (GFP) fusions into fluorescent particles which displayed specific binding sites suitable for applications in bioseparation and diagnostics was demonstrated. Here, the suitability of GFP particles for enzyme immobilization was assessed. The enzymes tested were a thermostable α-amylase fromBacillus licheniformis,N-acetyl-d-neuraminic acid aldolase (NanA) fromEscherichia coli, and organophosphohydrolase (OpdA) fromAgrobacterium radiobacter. Respective GFP particles were isolated and could be stably maintained outside the cell. These enzyme-bearing GFP particles exhibited considerable stability across a range of temperature, pH, and storage conditions and could be recycled. The α-amylase-bearing particles retained activity after treatments at 4 to 85°C and at pHs 4 to 10, were stable for 3 months at 4°C, and could be recycled up to three times. OpdA-bearing particles retained degradation activity after treatments at 4 to 45°C and at pHs 5 to 10 and were able to be recycled up to four times. In contrast, the performance of NanA-bearing particles rapidly declined (>50% loss) after each recycling step and 3 months storage at 4°C. However, they were still able to convertN-acetylmannosamine and pyruvate toN-acetylneuraminic acid after treatment at 4 to 85°C and at pHs 4 to 11. Fluorescent GFP fusion particles represent a novel method for the immobilization and display of enzymes. Potential applications include diagnostic assays, biomass conversion, pharmaceutical production, and bioremediation.

scholarly journals Evidence of a Continuous Endoplasmic Reticulum in the Protozoan Parasite Entamoeba histolytica

2008 ◽  
Vol 7 (7) ◽  
pp. 1222-1226 ◽  
Author(s):  
Jose E. Teixeira ◽  
Christopher D. Huston
Keyword(s):  
Endoplasmic Reticulum ◽  
Green Fluorescent Protein ◽  
Fusion Protein ◽  
Entamoeba Histolytica ◽  
Fluorescent Protein ◽  
Protozoan Parasite ◽  
Green Fluorescent Protein Fusion ◽  
Protein Fusion ◽  
Green Fluorescent

ABSTRACT Entamoeba histolytica, the cause of amebiasis, is believed to have no continuous endoplasmic reticulum (ER), with ER functions occurring in vesicles. Here, using an ER-targeted green fluorescent protein fusion protein and fluorescence loss in photobleaching, we have unambiguously demonstrated the presence of a continuous ER compartment in living E. histolytica trophozoites.

scholarly journals Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein

1999 ◽  
Vol 339 (2) ◽  
pp. 299-307 ◽  
Author(s):  
Arthur L. KRUCKEBERG ◽  
Ling YE ◽  
Jan A. BERDEN ◽  
Karel van DAM
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Glucose Transport ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Hexose Transporter ◽  
High Concentrations ◽  
Green Fluorescent

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4×105 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 °C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

scholarly journals Synaptotagmins at the endoplasmic reticulum–plasma membrane contact sites maintain diacylglycerol homeostasis during abiotic stress

2021 ◽  
Author(s):  
Noemi Ruiz-Lopez ◽  
Jessica Pérez-Sancho ◽  
Alicia Esteban del Valle ◽  
Richard P Haslam ◽  
Steffen Vanneste ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Abiotic Stress ◽  
Fluorescent Protein ◽  
Mechanical Damage ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Membrane Contact ◽  
Green Fluorescent

Abstract Endoplasmic reticulum-plasma membrane contact sites (ER-PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis thaliana mutants lacking the ER-PM protein tether synaptotagmin1 (SYT1) exhibit decreased plasma membrane (PM) integrity under multiple abiotic stresses such as freezing, high salt, osmotic stress and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER-PM tether that also functions in maintaining PM integrity. The ER-PM CS localization of SYT1 and SYT3 is dependent on PM phosphatidylinositol-4-phosphate and is regulated by abiotic stress. Lipidomic analysis revealed that cold stress increased the accumulation of diacylglycerol at the PM in a syt1/3 double mutant relative to wild type while the levels of most glycerolipid species remain unchanged. Additionally, the SYT1-green fluorescent protein (GFP) fusion preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work uncovers a SYT-dependent mechanism of stress adaptation counteracting the detrimental accumulation of diacylglycerol at the PM produced during episodes of abiotic stress.

scholarly journals Rtn1p Is Involved in Structuring the Cortical Endoplasmic Reticulum

2006 ◽  
Vol 17 (7) ◽  
pp. 3009-3020 ◽  
Author(s):  
Johan-Owen De Craene ◽  
Jeff Coleman ◽  
Paula Estrada de Martin ◽  
Marc Pypaert ◽  
Scott Anderson ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Fluorescent Protein ◽  
Cell Cortex ◽  
Proteomic Screen ◽  
Wide Range ◽  
Membrane Spanning ◽  
Green Fluorescent ◽  
Hydrophilic Loop ◽  
Yeast Mutants

The endoplasmic reticulum (ER) contains both cisternal and reticular elements in one contiguous structure. We identified rtn1Δ in a systematic screen for yeast mutants with altered ER morphology. The ER in rtn1Δ cells is predominantly cisternal rather than reticular, yet the net surface area of ER is not significantly changed. Rtn1-green fluorescent protein (GFP) associates with the reticular ER at the cell cortex and with the tubules that connect the cortical ER to the nuclear envelope, but not with the nuclear envelope itself. Rtn1p overexpression also results in an altered ER structure. Rtn proteins are found on the ER in a wide range of eukaryotes and are defined by two membrane-spanning domains flanking a conserved hydrophilic loop. Our results suggest that Rtn proteins may direct the formation of reticulated ER. We independently identified Rtn1p in a proteomic screen for proteins associated with the exocyst vesicle tethering complex. The conserved hydophilic loop of Rtn1p binds to the exocyst subunit Sec6p. Overexpression of this loop results in a modest accumulation of secretory vesicles, suggesting impaired exocyst function. The interaction of Rtn1p with the exocyst at the bud tip may trigger the formation of a cortical ER network in yeast buds.

scholarly journals Identification of a Novel Saturable Endoplasmic Reticulum Localization Mechanism Mediated by the C-Terminus of aDictyostelium Protein Disulfide Isomerase

2000 ◽  
Vol 11 (10) ◽  
pp. 3469-3484 ◽  
Author(s):  
Jean Monnat ◽  
Eva M. Neuhaus ◽  
Marius S. Pop ◽  
David M. Ferrari ◽  
Barbara Kramer ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Disulfide Isomerase ◽  
Fluorescent Protein ◽  
Null Mutation ◽  
Disulfide Isomerase ◽  
Isomerase Activity ◽  
C Terminus ◽  
Complementary Action ◽  
Green Fluorescent ◽  
Protein Disulfide

Localization of soluble endoplasmic reticulum (ER) resident proteins is likely achieved by the complementary action of retrieval and retention mechanisms. Whereas the machinery involving the H/KDEL and related retrieval signals in targeting escapees back to the ER is well characterized, other mechanisms including retention are still poorly understood. We have identified a protein disulfide isomerase (Dd-PDI) lacking the HDEL retrieval signal normally found at the C terminus of ER residents in Dictyostelium discoideum. Here we demonstrate that its 57 residue C-terminal domain is necessary for intracellular retention of Dd-PDI and sufficient to localize a green fluorescent protein (GFP) chimera to the ER, especially to the nuclear envelope. Dd-PDI and GFP-PDI57 are recovered in similar cation-dependent complexes. The overexpression of GFP-PDI57 leads to disruption of endogenous PDI complexes and induces the secretion of PDI, whereas overexpression of a GFP-HDEL chimera induces the secretion of endogenous calreticulin, revealing the presence of two independent and saturable mechanisms. Finally, low-level expression of Dd-PDI but not of PDI truncated of its 57 C-terminal residues complements the otherwise lethal yeast TRG1/PDI1 null mutation, demonstrating functional disulfide isomerase activity and ER localization. Altogether, these results indicate that the PDI57 peptide contains ER localization determinants recognized by a conserved machinery present in D. discoideum and Saccharomyces cerevisiae.

Increased Redox-Sensitive Green Fluorescent Protein Reduction Potential in the Endoplasmic Reticulum following Glutathione-Mediated Dimerization

Biochemistry ◽  
2013 ◽  
Vol 52 (19) ◽  
pp. 3332-3345 ◽  
Author(s):  
Deboleena Dipak Sarkar ◽  
Sarah K. Edwards ◽  
Justin A. Mauser ◽  
Allen M. Suarez ◽  
Maxwell A. Serowoky ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Green Fluorescent Protein ◽  
Reduction Potential ◽  
Fluorescent Protein ◽  
Green Fluorescent ◽  
Protein Reduction

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.

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