Human GLUD1 and GLUD2 glutamate dehydrogenase localize to mitochondria and endoplasmic reticulum

2009 ◽  
Vol 87 (3) ◽  
pp. 505-516 ◽  
Author(s):  
Vasileios Mastorodemos ◽  
Dimitra Kotzamani ◽  
Ioannis Zaganas ◽  
Giovanna Arianoglou ◽  
Helen Latsoudis ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Glutamate Dehydrogenase ◽  
Fluorescent Protein ◽  
Signal Sequence ◽  
Expression Vectors ◽  
Subcellular Targeting ◽  
Western Blots ◽  
Green Fluorescent ◽  
Coarse Structures ◽  
Egfp Fluorescence

Mammalian glutamate dehydrogenase (GDH), an enzyme central to glutamate metabolism, is thought to localize to the mitochondrial matrix, although there are also suggestions for the extramitochondrial presence of this protein. Whereas GDH in mammals is encoded by the GLUD1 gene, humans and the great apes have, in addition, a GLUD2 gene showing a distinct expression pattern. The encoded hGDH1 and hGDH2 isoenzymes are highly homologous, but their leader sequences are more divergent. To explore their subcellular targeting, we constructed expression vectors in which hGDH1 or hGDH2 was fused with the enhanced green fluorescent protein (EGFP) and used these to transfect COS 7, HeLa, CHO, HEK293, or neuroblastoma SHSY-5Y cells. Confocal microscopy revealed GDH-EGFP fluorescence in the cytoplasm within coarse structures. Cotransfection experiments using organelle-specific markers revealed that hGDH1 or hGDH2 colocalized with the mitochondrial marker DsRed2-Mito and to a lesser extent with the endoplasmic reticulum marker DsRed2-ER. Western blots detected two GDH-EGFP specific bands: a ~90 kDa band and a ~95 kDa band associated with the mitochondria and the endoplasmic reticulum containing cytosol, respectively. Deletion of the signal sequence, while altering drastically the fluoresce distribution within the cell, prevented GDH from entering the mitochondria, with the ~90 kDa band being retained in the cytosol. In addition, the deletion eliminated the ~95 kDa band from cell lysates, thus confirming that it represents the full-length GDH. Hence, while most of the hGDHs translocate into the mitochondria (a process associated with cleavage of the signal sequence), part of the protein localizes to the endoplasmic reticulum, probably serving additional functions.

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.

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 New Recombinant Mycobacterium bovis BCG Expression Vectors: Improving Genetic Control over Mycobacterial Promoters

2016 ◽  
Vol 82 (8) ◽  
pp. 2240-2246 ◽  
Author(s):  
Alex I. Kanno ◽  
Cibelly Goulart ◽  
Henrique K. Rofatto ◽  
Sergio C. Oliveira ◽  
Luciana C. C. Leite ◽  
...  
Keyword(s):  
Mycobacterium Bovis ◽  
Fluorescent Protein ◽  
Vaccine Development ◽  
Expression Vectors ◽  
Content Type ◽  
Expression Levels ◽  
Wide Range ◽  
Mycobacteriophage L5 ◽  
Green Fluorescent ◽  
Selection Of

ABSTRACTThe expression of many antigens, stimulatory molecules, or even metabolic pathways in mycobacteria such asMycobacterium bovisBCG orM. smegmatiswas made possible through the development of shuttle vectors, and several recombinant vaccines have been constructed. However, gene expression in any of these systems relied mostly on the selection of natural promoters expected to provide the required level of expression by trial and error. To establish a systematic selection of promoters with a range of strengths, we generated a library of mutagenized promoters through error-prone PCR of the strong PL5promoter, originally from mycobacteriophage L5. These promoters were cloned upstream of the enhanced green fluorescent protein reporter gene, and recombinantM. smegmatisbacteria exhibiting a wide range of fluorescence levels were identified. A set of promoters was selected and identified as having high (pJK-F8), intermediate (pJK-B7, pJK-E6, pJK-D6), or low (pJK-C1) promoter strengths in bothM. smegmatisandM. bovisBCG. The sequencing of the promoter region demonstrated that it was extensively modified (6 to 11%) in all of the plasmids selected. To test the functionality of the system, two different expression vectors were demonstrated to allow corresponding expression levels of theSchistosoma mansoniantigen Sm29 in BCG. The approach used here can be used to adjust expression levels for synthetic and/or systems biology studies or for vaccine development to maximize the immune response.

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

scholarly journals Chaperone-mediated reflux of secretory proteins to the cytosol during endoplasmic reticulum stress

2019 ◽  
Vol 116 (23) ◽  
pp. 11291-11298 ◽  
Author(s):  
Aeid Igbaria ◽  
Philip I. Merksamer ◽  
Ala Trusina ◽  
Firehiwot Tilahun ◽  
Jeffrey R. Johnson ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Secretory Pathway ◽  
Protein Quality ◽  
Fluorescent Protein ◽  
Control Process ◽  
Secretory Proteins ◽  
Quality Control Process ◽  
Folded Proteins ◽  
Green Fluorescent

Diverse perturbations to endoplasmic reticulum (ER) functions compromise the proper folding and structural maturation of secretory proteins. To study secretory pathway physiology during such “ER stress,” we employed an ER-targeted, redox-responsive, green fluorescent protein—eroGFP—that reports on ambient changes in oxidizing potential. Here we find that diverse ER stress regimes cause properly folded, ER-resident eroGFP (and other ER luminal proteins) to “reflux” back to the reducing environment of the cytosol as intact, folded proteins. By utilizing eroGFP in a comprehensive genetic screen in Saccharomyces cerevisiae, we show that ER protein reflux during ER stress requires specific chaperones and cochaperones residing in both the ER and the cytosol. Chaperone-mediated ER protein reflux does not require E3 ligase activity, and proceeds even more vigorously when these ER-associated degradation (ERAD) factors are crippled, suggesting that reflux may work in parallel with ERAD. In summary, chaperone-mediated ER protein reflux may be a conserved protein quality control process that evolved to maintain secretory pathway homeostasis during ER protein-folding stress.

scholarly journals Genetic Transformation of the Obligate Parasite Plasmodiophora brassicae

2013 ◽  
Vol 103 (10) ◽  
pp. 1052-1057 ◽  
Author(s):  
J. Feng ◽  
Sheau-Fang Hwang ◽  
S. E. Strelkov
Keyword(s):  
Genetic Transformation ◽  
Fluorescent Protein ◽  
Plasmodiophora Brassicae ◽  
Expression Vectors ◽  
Obligate Parasite ◽  
Putative Transformants ◽  
Resting Spores ◽  
Selection Step ◽  
Protoplast Preparation ◽  
Green Fluorescent

A protocol for genetic transformation of the obligate parasite Plasmodiophora brassicae, causal agent of clubroot of crucifers, was developed. In this protocol, protoplast preparation was superseded with lithium acetate treatment and the selection step was omitted. In two independent experiments, germinating resting spores of P. brassicae were transformed by two fungal expression vectors containing either a green fluorescent protein (gfp) gene or a hygromycin resistance (hph) gene. Putative transformants were produced from both transformations, with ≈50% of the obtained galls containing resting spores from which transforming DNA could be detected by polymerase chain reaction (PCR). PCR, quantitative PCR (qPCR), and genome walking conducted on selected transformants indicated that the transforming DNA was intergraded into the P. brassicae genome. Transcript of hph but not gfp was detected by reverse-transcription qPCR from selected transformants. From all galls produced by transformants, no GFP activity could be identified. Verified transformants were inoculated on canola and new galls were generated. PCR and qPCR analyses based on these galls indicated that transforming DNA was still resident in P. brassicae. This is the first report on genetic transformation of P. brassicae. The information and data generated from this study will facilitate research in multiple areas of the clubroot pathosystem.

scholarly journals Double-Labeled Rabies Virus: Live Tracking of Enveloped Virus Transport

2007 ◽  
Vol 82 (1) ◽  
pp. 237-245 ◽  
Author(s):  
Yvonne Klingen ◽  
Karl-Klaus Conzelmann ◽  
Stefan Finke
Keyword(s):  
Cell Surface ◽  
Rabies Virus ◽  
Fluorescent Protein ◽  
Signal Sequence ◽  
Surface Membrane ◽  
Fusion Construct ◽  
Virus Particles ◽  
Enveloped Virus ◽  
Green Fluorescent

ABSTRACT Here we describe a strategy to fluorescently label the envelope of rabies virus (RV), of the Rhabdoviridae family, in order to track the transport of single enveloped viruses in living cells. Red fluorescent proteins (tm-RFP) were engineered to comprise the N-terminal signal sequence and C-terminal transmembrane spanning and cytoplasmic domain sequences of the RV glycoprotein (G). Two variants of tm-RFP were transported to and anchored in the cell surface membrane, independent of glycosylation. As shown by confocal microscopy, tm-RFP colocalized at the cell surface with the RV matrix and G protein and was incorporated into G gene-deficient virus particles. Recombinant RV expressing the membrane-anchored tm-RFP in addition to G yielded infectious viruses with mosaic envelopes containing both tm-RFP and G. Viable double-labeled virus particles comprising a red fluorescent envelope and a green fluorescent ribonucleoprotein were generated by expressing in addition an enhanced green fluorescent protein-phosphoprotein fusion construct (S. Finke, K. Brzozka, and K. K. Conzelmann, J. Virol. 78:12333-12343, 2004). Individual enveloped virus particles were observed under live cell conditions as extracellular particles and inside endosomal vesicles. Importantly, double-labeled RVs were transported in the retrograde direction over long distances in neurites of in vitro-differentiated NS20Y neuroblastoma cells. This indicates that the typical retrograde axonal transport of RV to the central nervous system involves neuronal transport vesicles in which complete enveloped RV particles are carried as a cargo.

scholarly journals Use of Green Fluorescent Protein To Tag Lactic Acid Bacterium Strains under Development as Live Vaccine Vectors

2000 ◽  
Vol 66 (1) ◽  
pp. 383-391 ◽  
Author(s):  
Marie-Claude Geoffroy ◽  
Cyril Guyard ◽  
Brigitte Quatannens ◽  
Sonia Pavan ◽  
Marc Lange ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Live Vaccine ◽  
Expression Vectors ◽  
Fermented Foods ◽  
Vaccine Vectors ◽  
Green Fluorescent

ABSTRACT The lactic acid bacteria (LAB) are safe microorganisms which are mainly used for the preparation of fermented foods and for probiotic applications. The potential of LAB as live vehicles for the production and delivery of therapeutic molecules such as antigens is also being actively investigated today. However, very little is known about the fate of live LAB when administered in vivo and about the interaction of these microorganisms with the nasal or gastrointestinal ecosystem. For future applications, it is essential to be able to discriminate the biotherapeutic strain from the endogenous microflora and to unravel the mechanisms underlying the postulated health-beneficial effect. We therefore started to investigate both aspects in a mouse model with two LAB species presently under development as live vaccine vectors, i.e.,Lactococcus lactis and Lactobacillus plantarum. We have constructed different expression vectors carrying thegfp (green fluorescent protein [GFP]) gene from the jellyfish Aequoria victoria, and we found that this visible marker was best expressed when placed under the control of the inducible strong nisA promoter from L. lactis. Notably, a threshold amount of GFP was necessary to obtain a bright fluorescent phenotype. We further demonstrated that fluorescentL. plantarum NCIMB8826 can be enumerated and sorted by flow cytometry. Moreover, tagging of this strain with GFP allowed us to visualize its phagocytosis by macrophages in vitro and ex vivo and to trace it in the gastrointestinal tract of mice upon oral administration.

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