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.

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 Molecular cloning, characterization, subcellular localization and dynamics of p23, the mammalian KDEL receptor.

1993 ◽  
Vol 120 (2) ◽  
pp. 325-338 ◽  
Author(s):  
B L Tang ◽  
S H Wong ◽  
X L Qi ◽  
S H Low ◽  
W Hong
Keyword(s):  
Golgi Apparatus ◽  
Brefeldin A ◽  
Cell Types ◽  
In Vitro Translation ◽  
Tubular Structures ◽  
Human Sequence ◽  
Intermediate Compartment ◽  
Membrane Spanning ◽  
Membrane Spanning Domains

We have isolated a cDNA clone (mERD2) for the mammalian (bovine) homologue of the yeast ERD2 gene, which codes for the yeast HDEL receptor. The deduced amino acid sequence bears extensive homology to its yeast counterpart and is almost identical to a previously described human sequence. The sequence predicts a very hydrophobic protein with multiple membrane spanning domains, as confirmed by analysis of the in vitro translation product. The protein encoded by mERD2 (p23) has widespread occurrence, being present in all the cell types examined. p23 was localized to the cis-side of the Golgi apparatus and to a spotty intermediate compartment which mediates ER to Golgi transport. A majority of the intracellular staining could be accumulated in the intermediate compartment by a low temperature (15 degrees C) or brefeldin A. During recovery from these treatments, the spotty intermediate compartment staining of p23 was shifted to the perinuclear staining of the Golgi apparatus and tubular structures marked by p23 were observed. These tubular structures may serve to mediate transport between the intermediate compartment and the Golgi apparatus.

scholarly journals The rubella virus E1 glycoprotein is arrested in a novel post-ER, pre-Golgi compartment.

1992 ◽  
Vol 118 (4) ◽  
pp. 795-811 ◽  
Author(s):  
T C Hobman ◽  
L Woodward ◽  
M G Farquhar
Keyword(s):  
Rubella Virus ◽  
Secretory Pathway ◽  
Brefeldin A ◽  
Exit Site ◽  
Golgi Stack ◽  
E2 Glycoprotein ◽  
Golgi Compartment ◽  
Intermediate Compartment ◽  
Rough Er ◽  
Glycoprotein Transport

Evidence is accumulating that a distinct compartment(s) exists in the secretory pathway interposed between the rough ER (RER) and the Golgi stack. In this study we have defined a novel post-RER, pre-Golgi compartment where unassembled subunits of rubella virus (RV) E1 glycoprotein accumulate. When RV E1 is expressed in CHO cells in the absence of E2 glycoprotein, transport of E1 to the Golgi complex is arrested. The compartment in which E1 accumulates consists of a tubular network of smooth membranes which is in continuity with the RER but has distinctive properties from either the RER, Golgi, or previously characterized intermediate compartments. It lacks RER and Golgi membrane proteins and is not disrupted by agents which disrupt either the RER (thapsigargin, ionomycin) or Golgi (nocodazole and brefeldin A). However, luminal ER proteins bearing the KDEL signal have access to this compartment. Kinetically the site of E1 arrest lies distal to or at the site where palmitylation occurs and proximal to the low temperature 15 degrees C block. Taken together the findings suggest that the site of E1 arrest corresponds to, or is located close to the exit site from the ER. This compartment could be identified morphologically because it is highly amplified in cells overexpressing unassembled E1 subunits, but it may have its counterpart among the transitional elements of non-transfected cells. We conclude that the site of E1 arrest may represent a new compartment or a differentiated proximal moiety of the intermediate compartment.

scholarly journals A membrane glycoprotein, Sec12p, required for protein transport from the endoplasmic reticulum to the Golgi apparatus in yeast.

1988 ◽  
Vol 107 (3) ◽  
pp. 851-863 ◽  
Author(s):  
A Nakano ◽  
D Brada ◽  
R Schekman
Keyword(s):  
Amino Acids ◽  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Membrane Fraction ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Genomic Library ◽  
Membrane Glycoprotein ◽  
Mobility Shift ◽  
Cloned Gene

SEC12, a gene that is required for secretory, membrane, and vacuolar proteins to be transported from the endoplasmic reticulum to the Golgi apparatus, has been cloned from a genomic library by complementation of a sec12 ts mutation. Genetic analysis has shown that the cloned gene integrates at the SEC12 locus and that a null mutation at the locus is lethal. The DNA sequence predicts a protein of 471 amino acids containing a hydrophobic stretch of 19 amino acids near the COOH terminus. To characterize the gene product (Sec12p) in detail, a lacZ-SEC12 gene fusion has been constructed and a polyclonal antibody raised against the hybrid protein. The antibody recognizes Sec12p as a approximately 70-kD protein that sediments in a mixed membrane fraction that includes endoplasmic reticulum. Sec12p is not removed from the membrane fraction by treatment at high pH and high salt and is not degraded by exogenous protease unless detergent is present. Glycosylation of Sec12p during biogenesis is indicated by an electrophoretic mobility shift of the protein that is influenced by tunicamycin and by imposition of an independent secretory pathway block. We suggest that Sec12p is an integral membrane glycoprotein with a prominent domain that faces the cytoplasm where it functions to promote protein transport to the Golgi apparatus. In the process of transport, Sec12p itself may migrate to the Golgi apparatus and function in subsequent transport events.

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.

scholarly journals Retrograde Transport from the Pre-Golgi Intermediate Compartment and the Golgi Complex Is Affected by the Vacuolar H+-ATPase Inhibitor Bafilomycin A1

1998 ◽  
Vol 9 (12) ◽  
pp. 3561-3578 ◽  
Author(s):  
Harri Palokangas ◽  
Ming Ying ◽  
Kalervo Väänänen ◽  
Jaakko Saraste
Keyword(s):  
Brefeldin A ◽  
Retrograde Transport ◽  
Small Gtpase ◽  
Bafilomycin A1 ◽  
Atpase Inhibitor ◽  
Marker Proteins ◽  
Golgi Region ◽  
Intermediate Compartment ◽  
Acidic Compartments

The effect of the vacuolar H+-ATPase inhibitor bafilomycin A1 (Baf A1) on the localization of pre-Golgi intermediate compartment (IC) and Golgi marker proteins was used to study the role of acidification in the function of early secretory compartments. Baf A1 inhibited both brefeldin A- and nocodazole-induced retrograde transport of Golgi proteins to the endoplasmic reticulum (ER), whereas anterograde ER-to-Golgi transport remained largely unaffected. Furthermore, p58/ERGIC-53, which normally cycles between the ER, IC, and cis-Golgi, was arrested in pre-Golgi tubules and vacuoles, and the number of p58-positive ∼80-nm Golgi (coatomer protein I) vesicles was reduced, suggesting that the drug inhibits the retrieval of the protein from post-ER compartments. In parallel, redistribution of β-coatomer protein from the Golgi to peripheral pre-Golgi structures took place. The small GTPase rab1p was detected in short pre-Golgi tubules in control cells and was efficiently recruited to the tubules accumulating in the presence of Baf A1. In contrast, these tubules showed no enrichment of newly synthesized, anterogradely transported proteins, indicating that they participate in retrograde transport. These results suggest that the pre-Golgi structures contain an active H+-ATPase that regulates retrograde transport at the ER–Golgi boundary. Interestingly, although Baf A1 had distinct effects on peripheral pre-Golgi structures, only more central, p58-containing elements accumulated detectable amounts of 3-(2,4-dinitroanilino)-3′-amino-N-methyldipropylamine (DAMP), a marker for acidic compartments, raising the possibility that the lumenal pH of the pre-Golgi structures gradually changes in parallel with their translocation to the Golgi region.

Abstract 273: P2Y2 Receptor-Mediated Lymphotoxin-α Secretion Regulates ICAM-1 Expression in Smooth Muscle Cells

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Cheikh Seye ◽  
Wilbert Derbigny ◽  
Shaomin Qian
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Protein Transport ◽  
Brefeldin A ◽  
Muscle Cells ◽  
Secretory Vesicle ◽  
Actin Binding ◽  
Filamin A ◽  
Artery Disease ◽  
Lymphotoxin Α

Rationale: Single nucleotide polymorphism (SNP) in the LGASL2 galectin-2 (Gal-2) gene leads to altered secretion of lymphotoxin-α (LT-α) and is associated with coronary artery disease. Objective:Our aim was to determine whether factors other than genetic variations in LGASL2 regulate LT-α release and to define the role of this pro-inflammatory in vascular smooth muscle cells (SMC). Methods and results: The proinflammatory cytokine lymphotoxin-alpha (LTA) is thought to contribute to the pathogenesis of atherosclerosis. However, the mechanisms that regulate its expression in VSMC are poorly understood. The ability of exogenous nucleotides to stimulate LTA production was evaluated in VSMC by ELISA. The P2Y 2 nucleotide receptor (P2Y 2 R) agonist UTP stimulates a strong and sustained release of LTA from wild-type but not P2Y 2 R -/- SMC. Assessment of LTA gene transcription by LTA promoter-luciferase construct indicated that LTA levels are controlled at the level of transcription. We show using RNAi techniques that knockdown of the actin-binding protein filamin-A (FLNa) severely impaired nucleotide-induced Rho activation and consequent Rho-mediated LTA secretion. Re-introduction of FLNa in FLNa RNAi SMC rescued UTP-induced LTA expression. In addition, we found UTP-stimulated LTA secretion is not sensitive to brefeldin A (BFA), which blocks the formation of vesicles involved in protein transport from the ER to the Golgi apparatus, suggesting that P2Y 2 R/filamin-mediated secretion of LTA is independent of the ER/Golgi secretory vesicle route. Furthermore, UTP selectively induces ICAM-1 expression in WT but not SMC expressing a truncated P2Y 2 R deficient in LTA secretion. Conclusion: These data suggest that P2Y 2 R recruits FLNa to provide a cytoskeletal scaffold necessary for Rho signaling pathway upstream of LTA release and subsequent stimulation of ICAM-1 expression on VSMC.

Complementation of a polyamine-deficient Escherichia coli mutant by expression of mouse ornithine decarboxylase

1987 ◽  
Vol 7 (1) ◽  
pp. 564-567
Author(s):  
M Macrae ◽  
P Coffino
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Enzymatic Activity ◽  
Ornithine Decarboxylase ◽  
Polyamine Biosynthesis ◽  
Coli Mutant ◽  
Terminal Amino ◽  
High Level ◽  
Carboxy Terminal ◽  
Exogenous Source

Mouse ornithine decarboxylase (ODCase) cDNA was expressed at a high level in an Escherichia coli mutant deficient in polyamine biosynthesis. The expression of mouse ornithine decarboxylase relieved the dependence of the mutant on an exogenous source of polyamines, presumably by providing putrescine, the product of the enzyme. The effect on the enzymatic activity of deletions that removed carboxy-terminal amino acids of the protein was determined.

scholarly journals Characterization of the budding compartment of mouse hepatitis virus: evidence that transport from the RER to the Golgi complex requires only one vesicular transport step

1994 ◽  
Vol 124 (1) ◽  
pp. 55-70 ◽  
Author(s):  
J Krijnse-Locker ◽  
M Ericsson ◽  
PJ Rottier ◽  
G Griffiths
Keyword(s):  
Golgi Complex ◽  
Hepatitis Virus ◽  
Vesicular Transport ◽  
Helix Pomatia ◽  
M Protein ◽  
Membrane Structures ◽  
Golgi Stack ◽  
Permeabilized Cells ◽  
Intermediate Compartment ◽  
Rough Er

Mouse hepatitis coronavirus (MHV) buds into pleomorphic membrane structures with features expected of the intermediate compartment between the ER and the Golgi complex. Here, we characterize the MHV budding compartment in more detail in mouse L cells using streptolysin O (SLO) permeabilization which allowed us to better visualize the membrane structures at the ER-Golgi boundary. The MHV budding compartment shares membrane continuities with the rough ER as well as with cisternal elements on one side of the Golgi stack. It also labeled with p58 and rab2, two markers of the intermediate compartment, and with PDI, usually considered to be a marker of the rough ER. The membranes of the budding compartment, as well as the budding virions themselves, but not the rough ER, labeled with the N-acetyl-galactosamine (GalNAc)-specific lectin Helix pomatia. When the SLO-permeabilized cells were treated with guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), the budding compartment accumulated a large number of beta-cop-containing buds and vesicular profiles. Complementary biochemical experiments were carried out to determine whether vesicular transport was required for the newly synthesized M protein, that contains only O-linked oligosaccharides, to acquire first, GalNAc and second, the Golgi modifications galactose and sialic acid. The results from both in vivo studies and from the use of SLO-permeabilized cells showed that, while GalNAc addition occurred under conditions which block vesicular transport, both cytosol and ATP were prerequisites for the M protein oligosaccharides to acquire Golgi modifications. Collectively, our data argue that transport from the rough ER to the Golgi complex requires only one vesicular transport step and that the intermediate compartment is a specialized domain of the endoplasmatic reticulum that extends to the first cisterna on the cis side of the Golgi stack.

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