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.

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.

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 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.

Analytical Subcellular Fractionation Studies on Different Cell Types Isolated from Normal Rat Liver

1978 ◽  
Vol 55 (5) ◽  
pp. 423-427
Author(s):  
Clare Selden ◽  
A. M. Wootton ◽  
D. W. Moss ◽  
T. J. Peters
Keyword(s):  
Biliary Tract ◽  
Rat Liver ◽  
Gradient Centrifugation ◽  
Cell Types ◽  
Sucrose Density Gradient ◽  
Subcellular Fractionation ◽  
Marker Enzymes ◽  
Sucrose Density Gradient Centrifugation ◽  
Normal Rat ◽  
Parenchymal Cells

1. Parenchymal, Kupffer and biliary tract cells were isolated from normal rat liver by perfusion with collagenase solution. 2. The specific activities (munits of enzyme activity/mg of protein) of marker enzymes for the principal subcellular organelles were determined in the isolated cell homogenates and compared with whole liver homogenates. 3. The cells were disrupted and the extracts subjected to analytical subcellular fractionation by sucrose-density-gradient centrifugation. Lysosomal integrity was determined by assaying latent β-N-acetylglucosaminidase in the extracts. 4. Similar subcellular distributions were found for lysosomal, endoplasmic reticulum and plasma membrane marker enzymes in the whole liver and in parenchymal and biliary tract cells. In Kupffer cells, the proportion of these enzymes in the cytosol was significantly increased compared with the other fractions. In addition the equilibrium densities of the various organelles in these cells were lower than those from parenchymal cells.

scholarly journals A reversibly palmitoylated resident protein (p63) of an ER-Golgi intermediate compartment is related to a circulatory shock resuscitation protein

1993 ◽  
Vol 104 (3) ◽  
pp. 685-694 ◽  
Author(s):  
A. Schweizer ◽  
J. Rohrer ◽  
P. Jeno ◽  
A. DeMaio ◽  
T.G. Buchman ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Transport ◽  
Brefeldin A ◽  
Circulatory Shock ◽  
Remarkable Feature ◽  
Intermediate Compartment ◽  
Rough Er ◽  
Membrane Spanning ◽  
High Level ◽  
Cloned Gene

The recently identified 63 kDa membrane protein, p63, is a resident protein of a membrane network interposed in between rough ER and Golgi apparatus. To characterize p63 at the molecular level a 2.91 kb cDNA encoding p63 has been isolated from a human placenta lambda gt10 cDNA library. Sequence analysis of tryptic peptides prepared from isolated p63 confirmed the identify of the cloned gene. The translated amino acid sequence consists of 601 amino acids (65.8 kDa) with a single putative membrane-spanning region and a N-terminal cytoplasmic domain of 106 amino acids. The human p63 cDNA exhibits a high level of sequence identify to the pig hepatic cDNA 3AL (accession number M27092) whose expression is enhanced after resuscitation from circulatory shock. An additional remarkable feature of p63 is that it becomes reversibly palmitoylated when intracellular protein transport is blocked by the drug brefeldin A. Overexpression of p63 in COS cells led to the development of a striking tubular membrane network in the cytoplasm. This suggests that the protein may be determinant for the structure of the p63 compartment.

scholarly journals Reassessment of the subcellular localization of p63

1995 ◽  
Vol 108 (6) ◽  
pp. 2477-2485 ◽  
Author(s):  
A. Schweizer ◽  
J. Rohrer ◽  
J.W. Slot ◽  
H.J. Geuze ◽  
S. Kornfeld
Keyword(s):  
Immunoelectron Microscopy ◽  
Integral Membrane Protein ◽  
Cell Fractionation ◽  
Marker Protein ◽  
Double Immunofluorescence ◽  
Cos Cells ◽  
Confocal Immunofluorescence ◽  
Intermediate Compartment ◽  
Rough Er ◽  
Protein Disulfide

p63 is a type II integral membrane protein that has previously been suggested to be a resident protein of a membrane network interposed between the ER and the Golgi apparatus. In the present study, we have produced a polyclonal antibody against the purified human p63 protein to reassess the subcellular distribution of p63 by confocal immunofluorescence, immunoelectron microscopy, and cell fractionation. Double immunofluorescence of COS cells showed significant colocalization of p63 and a KDEL-containing lumenal ER marker protein, except for differences in the staining of the outer nuclear membrane. Immunoelectron microscopy of native HepG2 cells and of COS cells transfected with p63 revealed that both endogenous and overexpressed p63 are predominantly localized in the rough ER. While p63 was colocalized with protein disulfide isomerase, an ER marker protein, very little overlap of p63 was found with ERGIC-53, an established marker for the ER-Golgi intermediate compartment. When rough and smooth membranes were prepared from rat liver, p63 was found to copurify with ribophorin II, a rough ER protein. Both p63 and ribophorin II were predominantly recovered in rough microsomes and were largely separated from the intermediate compartment marker protein p58. From these results it is concluded that p63 is localized in the rough ER.

scholarly journals Cytoskeleton-Associated Protein 4: Functions Beyond the Endoplasmic Reticulum in Physiology and Disease

2012 ◽  
Vol 2012 ◽  
pp. 1-11 ◽  
Author(s):  
Kevin M. Tuffy ◽  
Sonia Lobo Planey
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Types ◽  
Lipid Homeostasis ◽  
Drug Induced ◽  
Microtubule Network ◽  
Major Function ◽  
Lung Lipid ◽  
Intermediate Compartment ◽  
Rough Er ◽  
Cellular Compartments

Cytoskeleton-associated protein 4 (CKAP4; also known as p63, CLIMP-63, or ERGIC-63) is a 63 kDa, reversibly palmitoylated and phosphorylated, type II transmembrane (TM) protein, originally identified as a resident of the endoplasmic reticulum (ER)/Golgi intermediate compartment (ERGIC). When localized to the ER, a major function of CKAP4 is to anchor rough ER to microtubules, organizing the overall structure of ER with respect to the microtubule network. There is also steadily accumulating evidence for diverse roles for CKAP4 localized outside the ER, including data demonstrating functionality of cell surface forms of CKAP4 in various cell types and of CKAP4 in the nucleus. We will review the recent studies that provide evidence for the existence of CKAP4 in multiple cellular compartments (i.e., ER, plasma membrane, and the nucleus) and discuss CKAP4’s role in the regulation of various physiological and pathological processes, such as interstitial cystitis, drug-induced cytotoxicity, pericullar proteolytic activity, and lung lipid homeostasis.

Differential susceptibilities of isolated hamster lung cell types to amiodarone toxicity

1998 ◽  
Vol 76 (7-8) ◽  
pp. 721-727 ◽  
Author(s):  
M W Bolt ◽  
W J Racz ◽  
J F Brien ◽  
T M Bray ◽  
T E Massey
Keyword(s):  
Alveolar Macrophages ◽  
Gradient Centrifugation ◽  
Cell Types ◽  
Clara Cell ◽  
Blue Dye ◽  
Alveolar Type ◽  
Specific Cell ◽  
Type Ii ◽  
Clara Cells

Treatment of cardiac dysrhythmias with the iodinated benzofuran derivative amiodarone (AM) is limited by pulmonary toxicity. The susceptibilities of different lung cell types of male Golden Syrian hamsters to AM-induced cytotoxicity were investigated in vitro. Bronchoalveolar lavage and protease digestion to release cells, followed by centrifugal elutriation and density gradient centrifugation, resulted in preparations enriched with alveolar macrophages (98%), alveolar type II cells (75-85%), and nonciliated bronchiolar epithelial (Clara) cells (35-50%). Alveolar type II cell and Clara cell preparations demonstrated decreased viability (by 0.5% trypan blue dye exclusion) when incubated with 50 µM AM for 36 h, and all AM-treated cell preparations demonstrated decreased viability when incubated with 100 or 200 µM AM. Based on a viability index ((viability of AM-treated cells ÷ viability of controls) × 100%), the Clara cell fraction was significantly (p < 0.05) more susceptible than all of the other cell types to 50 µM AM. However, AM cytotoxicity was greatest (p < 0.05) in alveolar macrophages following incubation with 100 or 200 µM AM. There was no difference between any of the enriched cell preparations in the amount of drug accumulated following 24 h of incubation with 50 µM AM, whereas alveolar macrophages accumulated the most drug during incubation with 100 µM AM. Thus, the most susceptible cell type was dependent on AM concentration. AM-induced cytotoxicity in specific cell types may initiate processes leading to inflammation and pulmonary fibrosis.Key words: amiodarone, susceptibility, alveolar macrophage, accumulation.

scholarly journals Butyrate formation from glucose by the rumen protozoon Dasytricha ruminantium

1985 ◽  
Vol 228 (1) ◽  
pp. 187-192 ◽  
Author(s):  
N Yarlett ◽  
D Lloyd ◽  
A G Williams
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Subcellular Fractionation ◽  
Small Granule ◽  
Metabolic Reactions ◽  
Pyruvate:Ferredoxin Oxidoreductase ◽  
Butyrate Kinase ◽  
Coa Reductase ◽  
Butyrate Production

Production of butyrate by the holotrich protozoon Dasytricha ruminantium involves the enzymes of glycolysis, pyruvate:ferredoxin oxidoreductase, acetyl-CoA:acetyl-CoA C-acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, 3-hydroxyacyl-CoA hydro-lyase, 3-hydroxyacyl-CoA reductase, phosphate butyryltransferase and butyrate kinase. Subcellular fractionation by differential and density-gradient centrifugation on sucrose gradients indicated that all those enzymes except pyruvate:ferredoxin oxidoreductase were non-sedimentable at 6 × 10(6) g-min. Butyrate kinase and phosphate butyryltransferase were associated with the large- and small-granule fractions. Thus, although metabolic reactions necessary for butyrate production proceed predominantly in the cytosol, hydrogenosomes play a key role in the conversion of pyruvate into acetyl-CoA.

Intracellular traffic of the ecto-nucleotide pyrophosphatase/phosphodiesterase NPP3 to the apical plasma membrane of MDCK and Caco-2 cells: apical targeting occurs in the absence of N-glycosylation

2000 ◽  
Vol 113 (23) ◽  
pp. 4193-4202 ◽  
Author(s):  
N.R. Meerson ◽  
V. Bello ◽  
J.L. Delaunay ◽  
T.A. Slimane ◽  
D. Delautier ◽  
...  
Keyword(s):  
Cell Surface ◽  
Mdck Cells ◽  
Transmembrane Proteins ◽  
Cell Types ◽  
Apical Plasma Membrane ◽  
Apical Surface ◽  
Intracellular Traffic ◽  
Transmembrane Glycoprotein ◽  
Direct Demonstration ◽  
Targeting Signal

Glycosylation was considered the major signal candidate for apical targeting of transmembrane proteins in polarized epithelial cells. However, direct demonstration of the role of glycosylation has proved difficult because non-glycosylated apical transmembrane proteins usually do not reach the cell surface. Here we were able to follow the targeting of the apical transmembrane glycoprotein NPP3 both when glycosylated and non-glycosylated. Transfected in polarized MDCK and Caco-2 cells, NPP3 was exclusively expressed at the apical membrane. The transport kinetics of the protein to the cell surface were studied after metabolic (35)S-labeling and surface immunoprecipitation. The newly synthesized protein was mainly targeted directly to the apical surface in MDCK cells, whereas 50% transited through the basolateral surface in Caco-2 cells. In both cell types, the basolaterally targeted pool was effectively transcytosed to the apical surface. In the presence of tunicamycin, NPP3 was not N-glycosylated. The non-glycosylated protein was partially retained intracellularly but the fraction that reached the cell surface was nevertheless predominantly targeted apically. However, transcytosis of the non-glycosylated protein was partially impaired in MDCK cells. These results provide direct evidence that glycosylation cannot be considered an apical targeting signal for NPP3, although glycosylation is necessary for correct trafficking of the protein to the cell surface.

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