Characterization of a novel 63 kDa membrane protein. Implications for the organization of the ER-to-Golgi pathway

1993 ◽  
Vol 104 (3) ◽  
pp. 671-683 ◽  
Author(s):  
A. Schweizer ◽  
M. Ericsson ◽  
T. Bachi ◽  
G. Griffiths ◽  
H.P. Hauri
Keyword(s):  
Membrane Protein ◽  
Laser Scanning ◽  
Protein Transport ◽  
Brefeldin A ◽  
Vero Cells ◽  
Subcellular Fractionation ◽  
Confocal Laser ◽  
Similar Degree ◽  
Fractionation Procedure ◽  
Intermediate Compartment

Owing to the lack of appropriate markers the structural organization of the ER-to-Golgi pathway and the dynamics of its membrane elements have been elusive. To elucidate this organization we have taken a monoclonal antibody (mAb) approach. A mAb against a novel 63 kDa membrane protein (p63) was produced that identifies a large tubular network of smooth membranes in the cytoplasm of primate cells. The distribution of p63 overlaps with the ER-Golgi intermediate compartment, defined by a previously described 53 kDa marker protein (here termed ERGIC-53), as visualized by confocal laser scanning immunofluorescence microscopy and immunoelectron microscopy. The p63 compartment mediates protein transport from the ER to Golgi apparatus, as indicated by partial colocalization of p63 and vesicular stomatitis virus G protein in Vero cells cultured at 15 degrees C. Low temperatures and brefeldin A had little effect on the cellular distribution of p63, suggesting that this novel marker is a stably anchored resident protein of these pre-Golgi membranes. p63 and ERGIC-53 were enriched to a similar degree by the same subcellular fractionation procedure. These findings demonstrate an unanticipated complexity of the ER-Golgi interface and suggest that the ER-Golgi intermediate compartment defined by ERGIC-53 may be part of a greater network of smooth membranes.

scholarly journals The isolated ER-Golgi intermediate compartment exhibits properties that are different from ER and cis-Golgi.

1991 ◽  
Vol 113 (1) ◽  
pp. 45-54 ◽  
Author(s):  
A Schweizer ◽  
K Matter ◽  
C M Ketcham ◽  
H P Hauri
Keyword(s):  
Protein Transport ◽  
Gradient Centrifugation ◽  
Cell Types ◽  
Vero Cells ◽  
Subcellular Fractionation ◽  
Fractionation Procedure ◽  
Targeting Signal ◽  
Intermediate Compartment ◽  
Rough Er ◽  
Protein Disulfide

A procedure has been established in Vero cells for the isolation of an intermediate compartment involved in protein transport from the ER to the Golgi apparatus. The two-step subcellular fractionation procedure consists of Percoll followed by Metrizamide gradient centrifugation. Using the previously characterized p53 as a marker protein, the average enrichment factor of the intermediate compartment was 41. The purified fraction displayed a unique polypeptide pattern. It was largely separated from the rough ER proteins ribophorin I, ribophorin II, BIP, and protein disulfide isomerase, as well as from the putative cis-Golgi marker N-acetylglucosamine-1-phosphodiester-alpha-N-acetylglucosaminidase, the second of the two enzymes generating the lysosomal targeting signal mannose-6-phosphate. The first enzyme, N-acetylglucosaminylphosphotransferase, for which previous biochemical evidence had suggested both a pre- and a cis-Golgi localization in other cell types, cofractionated with the cis-Golgi rather than the intermediate compartment in Vero cells. The results suggest that the intermediate compartment defined by p53 has unique properties and does not exhibit typical features of rough ER and cis-Golgi.

CaBP1, a calcium binding protein of the thioredoxin family, is a resident KDEL protein of the ER and not of the intermediate compartment

1994 ◽  
Vol 107 (10) ◽  
pp. 2719-2727 ◽  
Author(s):  
J. Fullekrug ◽  
B. Sonnichsen ◽  
U. Wunsch ◽  
K. Arseven ◽  
P. Nguyen Van ◽  
...  
Keyword(s):  
Calcium Binding ◽  
Laser Scanning ◽  
Vero Cells ◽  
Subcellular Fractionation ◽  
Laser Scanning Microscopy ◽  
Cos Cells ◽  
Marker Proteins ◽  
Intermediate Compartment ◽  
High Level ◽  
High Degree

A cDNA encoding rat CaBP1 has been isolated and sequenced. The deduced polypeptide chain consists of 440 amino acids including two internal thioredoxin-like domains and a C-terminal KDEL retention/retrieval signal. Regarding the high degree of identity to the hamster protein P5, CaBP1 is considered to be the homologous rat protein. Previous work has suggested that CaBP1 is a resident luminal protein of the intermediate compartment (Schweizer, A., Peter, F., Nguyen Van, P., Soling, H.D. and Hauri, H.P. (1993) Eur. J. Cell Biol. 60, 366–370). Our conclusion that CaBP1 is a resident protein of the endoplasmic reticulum and not of the intermediate compartment is based on three different approaches: subcellular fractionation, indirect immunofluorescence and overexpression of CaBP1. Subcellular fractionation of Vero cells in a velocity controlled step gradient led to copurification of CaBP1-containing vesicles and several marker proteins for the ER including calreticulin and alpha-SSRP. The intermediate compartment, as defined by a monoclonal antibody against the marker protein p53 (ERGIC-53), could be separated from these ER markers. Double immunofluorescence analysed by laser scanning microscopy showed no significant colocalization between CaBP1 and p53, but between CaBP1 and calreticulin. In addition experiments, Vero cells were infected with VSV tsO45. At 15 degrees C the VSV-G protein accumulated in punctuate structures representing the intermediate compartment, while CaBP1 maintained its original reticular localization. Even after high-level overexpression in COS cells, CaBP1 was not detected in the intermediate compartment, but was efficiently retained in the ER as judged by light microscopy.

scholarly journals Identification, characterization and subcellular localization of TcPDE1, a novel cAMP-specific phosphodiesterase from Trypanosoma cruzi

2004 ◽  
Vol 378 (1) ◽  
pp. 63-72 ◽  
Author(s):  
Maximiliano A. D'ANGELO ◽  
Santiago SANGUINETI ◽  
Jeffrey M. REECE ◽  
Lutz BIRNBAUMER ◽  
Héctor N. TORRES ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Subcellular Localization ◽  
Laser Scanning ◽  
Phosphodiesterase Inhibitor ◽  
Integral Membrane Protein ◽  
Subcellular Fractionation ◽  
Sodium Cholate ◽  
Confocal Laser ◽  
Acid Protein ◽  
Amino Acid Protein

Compartmentalization of cAMP phosphodiesterases plays a key role in the regulation of cAMP signalling in mammals. In the present paper, we report the characterization and subcellular localization of TcPDE1, the first cAMP-specific phosphodiesterase to be identified from Trypanosoma cruzi. TcPDE1 is part of a small gene family and encodes a 929-amino-acid protein that can complement a heat-shock-sensitive yeast mutant deficient in phospho-diesterase genes. Recombinant TcPDE1 strongly associates with membranes and cannot be released with NaCl or sodium cholate, suggesting that it is an integral membrane protein. This enzyme is specific for cAMP and its activity is not affected by cGMP, Ca2+, calmodulin or fenotiazinic inhibitors. TcPDE1 is sensitive to the phosphodiesterase inhibitor dipyridamole but is resistant to 3-isobutyl-1-methylxanthine, theophylline, rolipram and zaprinast. Papaverine, erythro-9-(2-hydroxy-3-nonyl)-adenine hydrochloride, and vinpocetine are poor inhibitors of this enzyme. Confocal laser scanning of T. cruzi epimastigotes showed that TcPDE1 is associated with the plasma membrane and concentrated in the flagellum of the parasite. The association of TcPDE1 with this organelle was confirmed by subcellular fractionation and cell-disruption treatments. The localization of this enzyme is a unique feature that distinguishes it from all the trypanosomatid phosphodiesterases described so far and indicates that compartmentalization of cAMP phosphodiesterases could also be important in these parasites.

scholarly journals Assessment of the In Vitro Antischistosomal Activities of the Extracts and Compounds from Solidago Microglossa DC (Asteraceae) and Aristolochia Cymbifera Mart. & Zucc. (Aristolochiaceae)

2020 ◽  
Vol 2020 ◽  
pp. 1-11
Author(s):  
Poliana da Silva Costa ◽  
Ohana Oliveira Zuza da Silva ◽  
Danilo de Souza Costa ◽  
Lara Aparecida de Oliveira Silva ◽  
Priscila de Faria Pinto ◽  
...  
Keyword(s):  
Methyl Ester ◽  
Motor Activity ◽  
Laser Scanning ◽  
Acid Methyl Ester ◽  
Vero Cells ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Acid Methyl ◽  
Antischistosomal Activity

Schistosomiasis, caused by helminth flatworms of the genus Schistosoma, is a neglected tropical disease that afflicts over 230 million people worldwide. Currently, treatment is achieved with only one drug, praziquantel (PZQ). In this regard, the roots of Solidago microglossa (Asteraceae) and Aristolochia cymbifera (Aristolochiaceae) are popularly used as anthelmintic. Despite their medicinal use against helminthiasis, such as schistosomiasis, A. cymbifera, and S. microglossa have not been evaluated against S. mansoni. Then, in this work, the in vitro antischistosomal activity of the crude extracts of A. cymbifera (Ac) and S. microglossa (Sm) and their isolated compounds were investigated against S. mansoni adult worms. Sm (200 μg/mL) and Ac (100–200 μg/mL) were lethal to all male and female worms at the 24 h incubation. In addition, Sm (10–50 μg/mL) and Ac (10 μg/mL) caused significant reduction in the parasite’s movements, showing no significant cytotoxicity to Vero cells at the same range of schistosomicidal concentrations. Confocal laser scanning microscopy revealed that Sm and Ac caused tegumental damages and reduced the numbers of tubercles of male schistosomes. Chromatographic fractionation of Sm leads to isolation of bauerenol, α-amirin, and spinasterol, while populifolic acid, cubebin, 2-oxopopulifolic acid methyl ester, and 2-oxopopulifolic acid were isolated from Ac. At concentrations of 25–100 μM, bauerenol, α-amirin, spinasterol, populifolic acid, and cubebin showed significant impact on motor activity of S. mansoni. 2-oxopopulifolic acid methyl ester and 2-oxopopulifolic acid caused 100% mortality and decreased the motor activity of adult schistosomes at 100 μM. This study has reported, for the first time, the in vitro antischistosomal effects of S. microglossa and A. cymbifera extracts, also showing promising compounds against adult schistosomes.

Rapid Mobilization of Intracellularly Stored RANTES in Response to Interferon-γ in Human Eosinophils

Blood ◽  
1999 ◽  
Vol 94 (1) ◽  
pp. 23-32 ◽  
Author(s):  
Paige Lacy ◽  
Salahaddin Mahmudi-Azer ◽  
Ben Bablitz ◽  
Stacey C. Hagen ◽  
Juan R. Velazquez ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Human Peripheral Blood ◽  
Subcellular Fractionation ◽  
Secretory Vesicle ◽  
Interferon Γ ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Enzyme Linked Immunosorbent Assay ◽  
Gm Csf ◽  
Ifn Γ

The CC chemokine RANTES is synthesized, stored, and upregulated in response to interferon-γ (IFN-γ) in human peripheral blood eosinophils. In this report, we propose that RANTES is rapidly mobilized from eosinophil crystalloid granules during agonist-induced degranulation. We stimulated purified eosinophils (>99%) from atopic asthmatics with 500 U/mL IFN-γ to analyze the kinetics of mobilization and release of RANTES (0 to 240 minutes). We used subcellular fractionation, immunogold analysis, two-color confocal laser scanning microscopy (CLSM), and enzyme-linked immunosorbent assay (ELISA) to trace the movement of eosinophil-derived RANTES from intracellular stores to release. RANTES was rapidly mobilized (10 minutes) and released after 120 minutes of stimulation (80 ± 15 pg/mL per 2 × 106 cells). RANTES appeared to be stored in at least two intracellular compartments: the matrix of crystalloid granules, detected by major basic protein and eosinophil peroxidase activities, and a specialized small secretory vesicle present in light membrane fractions. The extragranular RANTES was mobilized more rapidly than that of crystalloid granules during IFN-γ stimulation. This effect was not observed in eosinophils treated with IFN-, interleukin-3 (IL-3), IL-5, granulocyte-macrophage colony-stimulating factor (GM-CSF), or genistein followed by IFN-γ. Our findings suggest that RANTES may be mobilized and released by piecemeal degranulation upon stimulation, involving transport through a putative pool of small secretory vesicles.

scholarly journals Transport of an external Lys-Asp-Glu-Leu (KDEL) protein from the plasma membrane to the endoplasmic reticulum: studies with cholera toxin in Vero cells.

1996 ◽  
Vol 133 (4) ◽  
pp. 777-789 ◽  
Author(s):  
I V Majoul ◽  
P I Bastiaens ◽  
H D Söling
Keyword(s):  
Endoplasmic Reticulum ◽  
Cholera Toxin ◽  
Retrograde Transport ◽  
Vero Cells ◽  
Subcellular Fractionation ◽  
Pulse Treatment ◽  
Resistant Form ◽  
Immunofluorescence Studies ◽  
Intermediate Compartment ◽  
Kdel Sequence

The A2 chain of cholera toxin (CTX) contains a COOH-terminal Lys-Asp-Glu-Leu (KDEL) sequence. We have, therefore, analyzed by immunofluorescence and by subcellular fractionation in Vero cells whether CTX can used to demonstrate a retrograde transport of KDEL proteins from the Golgi to the ER. Immunofluorescence studies reveal that after a pulse treatment with CTX, the CTX-A and B subunits (CTX-A and CTX-B) reach Golgi-like structures after 15-20 min (maximum after 30 min). Between 30 and 90 min, CTX-A (but not CTX-B) appear in the intermediate compartment and in the ER, whereas the CTX-B are translocated to the lysosomes. Subcellular fractionation studies confirm these results: after CTX uptake for 15 min, CTX-A is associated only with endosomal and Golgi compartments. After 30 min, a small amount of CTX-A appears in the ER in a trypsin-resistant form, and after 60 min, a significant amount appears. CTX-A seems to be transported mainly in its oxidized form (CTX-A1-S-S-CTX-A2) from the Golgi to the ER, where it becomes slowly reduced to form free CTX A1 and CTX-A2, as indicated by experiments in which cells were homogenized 30 and 90 min after the onset of CTX uptake in the presence of N-ethylmaleimide. Nocodazol applied after accumulation of CTX in Golgi inhibits the appearance of CTX-A in the ER and delays the increase of 3',5'cAMP, indicating the participation of microtubules in the retrograde Golgi-ER transport.

Confocal laser scanning microscopical investigation of epithelial membrane polarity in 3-dimensional MDCK multicellular cysts

1992 ◽  
Vol 50 (1) ◽  
pp. 698-699
Author(s):  
Allan Z. Wang ◽  
Jane C. Wang ◽  
Herbert S. Diamond
Keyword(s):  
Membrane Protein ◽  
Laser Scanning ◽  
De Novo ◽  
Basal Membrane ◽  
Immunofluorescent Staining ◽  
Confocal Laser ◽  
Growth Media ◽  
Apical Surface ◽  
3 Dimensional ◽  
Domain Specific

We have previously described the process ol membrane polarity formation and reversal in MDCK epithelial cysts (Fig.1) using immunofluorescent staining on frozen sections. Apical surface of polarized epithelial cells faced the growth media and basl surface faced the cyst lumen by locating membrane domain-specific proteins (Fig.2). Polarity reversal was demonstrated by showing that polarized distribution of membrane proteins were reversed after the suspension-cultured cysts were transferred into collagen I gel embedding (Fig.3). There was loss of endogeneous basal membrane protein and appearance of apical membrane protein at the lumenal surface, indicating a novel mechanism for de novo formation of apical domain in the multicellular cysts.

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