scholarly journals Roles for α2p24 and COPI in Endoplasmic Reticulum Cargo Exit Site Formation

1999 ◽  
Vol 146 (2) ◽  
pp. 285-299 ◽  
Author(s):  
C. Lavoie ◽  
J. Paiement ◽  
M. Dominguez ◽  
L. Roy ◽  
S. Dahan ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Family Member ◽  
De Novo ◽  
Brefeldin A ◽  
Site Formation ◽  
Low Density ◽  
Exit Site ◽  
Intermediate Compartment ◽  
Hydrolysis Of

A two-step reconstitution system for the generation of ER cargo exit sites from starting ER-derived low density microsomes (LDMs; 1.17 g/cc) is described. The first step is mediated by the hydrolysis of Mg2+ATP and Mg2+GTP, leading to the formation of a transitional ER (tER) with the soluble cargo albumin, transferrin, and the ER-to-Golgi recycling membrane proteins α2p24 and p58 (ERGIC-53, ER-Golgi intermediate compartment protein) enriched therein. Upon further incubation (step two) with cytosol and mixed nucleotides, interconnecting smooth ER tubules within tER transforms into vesicular tubular clusters (VTCs). The cytosolic domain of α2p24 and cytosolic COPI coatomer affect VTC formation. This is deduced from the effect of antibodies to the COOH-terminal tail of α2p24, but not of antibodies to the COOH-terminal tail of calnexin on this reconstitution, as well as the demonstrated recruitment of COPI coatomer to VTCs, its augmentation by GTPγS, inhibition by Brefeldin A (BFA), or depletion of β-COP from cytosol. Therefore, the p24 family member, α2p24, and its cytosolic coat ligand, COPI coatomer, play a role in the de novo formation of VTCs and the eneration of ER cargo exit sites.

scholarly journals Capacity of the Golgi Apparatus for Biogenesis from the Endoplasmic Reticulum

2003 ◽  
Vol 14 (12) ◽  
pp. 5011-5018 ◽  
Author(s):  
Sapna Puri ◽  
Adam D. Linstedt
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Self Assembly ◽  
De Novo ◽  
Brefeldin A ◽  
The Other ◽  
Matrix Proteins ◽  
Er Export ◽  
Golgi Matrix

It is unclear whether the mammalian Golgi apparatus can form de novo from the ER or whether it requires a preassembled Golgi matrix. As a test, we assayed Golgi reassembly after forced redistribution of Golgi matrix proteins into the ER. Two conditions were used. In one, ER redistribution was achieved using a combination of brefeldin A (BFA) to cause Golgi collapse and H89 to block ER export. Unlike brefeldin A alone, which leaves matrix proteins in relatively large remnant structures outside the ER, the addition of H89 to BFA-treated cells caused ER accumulation of all Golgi markers tested. In the other, clofibrate treatment induced ER redistribution of matrix and nonmatrix proteins. Significantly, Golgi reassembly after either treatment was robust, implying that the Golgi has the capacity to form de novo from the ER. Furthermore, matrix proteins reemerged from the ER with faster ER exit rates. This, together with the sensitivity of BFA remnants to ER export blockade, suggests that presence of matrix proteins in BFA remnants is due to cycling via the ER and preferential ER export rather than their stable assembly in a matrix outside the ER. In summary, the Golgi apparatus appears capable of efficient self-assembly.

scholarly journals The discrepancy between presenilin subcellular localization and γ-secretase processing of amyloid precursor protein

2001 ◽  
Vol 154 (4) ◽  
pp. 731-740 ◽  
Author(s):  
Philippe Cupers ◽  
Mustapha Bentahir ◽  
Katleen Craessaerts ◽  
Isabelle Orlans ◽  
Hugo Vanderstichele ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Amyloid Precursor Protein ◽  
Primary Cultures ◽  
Brefeldin A ◽  
Precursor Protein ◽  
Subcellular Compartment ◽  
Marker Proteins ◽  
Intermediate Compartment ◽  
The Relationship

We investigated the relationship between PS1 and γ-secretase processing of amyloid precursor protein (APP) in primary cultures of neurons. Increasing the amount of APP at the cell surface or towards endosomes did not significantly affect PS1-dependent γ-secretase cleavage, although little PS1 is present in those subcellular compartments. In contrast, almost no γ-secretase processing was observed when holo-APP or APP-C99, a direct substrate for γ-secretase, were specifically retained in the endoplasmic reticulum (ER) by a double lysine retention motif. Nevertheless, APP-C99-dilysine (KK) colocalized with PS1 in the ER. In contrast, APP-C99 did not colocalize with PS1, but was efficiently processed by PS1-dependent γ-secretase. APP-C99 resides in a compartment that is negative for ER, intermediate compartment, and Golgi marker proteins. We conclude that γ-secretase cleavage of APP-C99 occurs in a specialized subcellular compartment where little or no PS1 is detected. This suggests that at least one other factor than PS1, located downstream of the ER, is required for the γ-cleavage of APP-C99. In agreement, we found that intracellular γ-secretase processing of APP-C99-KK both at the γ40 and the γ42 site could be restored partially after brefeldin A treatment. Our data confirm the “spatial paradox” and raise several questions regarding the PS1 is γ-secretase hypothesis.

scholarly journals Evidence against an Essential Role of COPII-Mediated Cargo Transport to the Endoplasmic Reticulum-Golgi Intermediate Compartment in the Formation of the Primary Membrane of Vaccinia Virus

2003 ◽  
Vol 77 (21) ◽  
pp. 11754-11766 ◽  
Author(s):  
Matloob Husain ◽  
Bernard Moss
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Vaccinia Virus ◽  
Essential Role ◽  
Recombinant Vaccinia Virus ◽  
Dominant Negative ◽  
Cargo Transport ◽  
Dominant Negative Form ◽  
Intermediate Compartment

ABSTRACT Vaccinia virus assembles two distinct lipoprotein membranes. The primary membrane contains nonglycosylated proteins, appears as crescents in the cytoplasm, and delimits immature and mature intracellular virions. The secondary or wrapping membrane contains glycoproteins, is derived from virus-modified trans-Golgi or endosomal cisternae, forms a loose coat around some intracellular mature virions, and becomes the envelope of extracellular virions. Although the mode of formation of the wrapping membrane is partially understood, we know less about the primary membrane. Recent reports posit that the primary membrane originates from the endoplasmic reticulum-Golgi intermediate compartment (ERGIC). According to this model, viral primary membrane proteins are cotranslationally inserted into the ER and accumulate in the ERGIC. To test the ERGIC model, we employed Sar1H79G, a dominant negative form of the Sar1 protein, which is an essential component of coatomer protein II (COPII)-mediated cargo transport from the ER to the ERGIC and other post-ER compartments. Overexpression of Sar1H79G by transfection or by a novel recombinant vaccinia virus with an inducible Sar1H79G gene resulted in retention of ERGIC 53 in the ER but did not interfere with localization of viral primary membrane proteins in factory regions or with formation of viral crescent membranes and infectious intracellular mature virions. Wrapping of intracellular mature virions and formation of extracellular virions did not occur, however, because some proteins that are essential for the secondary membrane were retained in the ER as a consequence of Sar1H79G overexpression. Our data argue against an essential role of COPII-mediated cargo transport and the ERGIC in the formation of the viral primary membrane. Instead, viral membranes may be derived directly from the ER or by a novel mechanism.

scholarly journals cis-Golgi proteins accumulate near the ER exit sites and act as the scaffold for Golgi regeneration after brefeldin A treatment in tobacco BY-2 cells

2012 ◽  
Vol 23 (16) ◽  
pp. 3203-3214 ◽  
Author(s):  
Yoko Ito ◽  
Tomohiro Uemura ◽  
Keiko Shoda ◽  
Masaru Fujimoto ◽  
Takashi Ueda ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Fluorescent Proteins ◽  
Plant Cells ◽  
Brefeldin A ◽  
Eukaryotic Cells ◽  
Er Exit Sites ◽  
Intermediate Compartment ◽  
Golgi Protein ◽  
And Function

The Golgi apparatus forms stacks of cisternae in many eukaryotic cells. However, little is known about how such a stacked structure is formed and maintained. To address this question, plant cells provide a system suitable for live-imaging approaches because individual Golgi stacks are well separated in the cytoplasm. We established tobacco BY-2 cell lines expressing multiple Golgi markers tagged by different fluorescent proteins and observed their responses to brefeldin A (BFA) treatment and BFA removal. BFA treatment disrupted cis, medial, and trans cisternae but caused distinct relocalization patterns depending on the proteins examined. Medial- and trans-Golgi proteins, as well as one cis-Golgi protein, were absorbed into the endoplasmic reticulum (ER), but two other cis-Golgi proteins formed small punctate structures. After BFA removal, these puncta coalesced first, and then the Golgi stacks regenerated from them in the cis-to-trans order. We suggest that these structures have a property similar to the ER-Golgi intermediate compartment and function as the scaffold of Golgi regeneration.

The Role of Membrane Proteins and Phospholipids in the Interaction of Ribosomes with Endoplasmic Reticulum Membranes

1975 ◽  
Vol 53 (9) ◽  
pp. 1039-1045 ◽  
Author(s):  
Serge Jothy ◽  
Jean-Louis Bilodeau ◽  
Henry Simpkins
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Rat Liver ◽  
Rough Endoplasmic Reticulum ◽  
Binding Sites ◽  
Molecular Weights ◽  
Low Concentrations ◽  
Phospholipid Headgroups ◽  
Hydrolysis Of

Hydrolysis of the membrane proteins and phospholipid headgroups of rat liver rough endoplasmic reticulum membranes showed that the ribosomal binding sites involve membrane proteins susceptible to low concentrations of trypsin, chymotrypsin, and papain. Three membrane proteins having molecular weights of 120 000, 93 000 and 36 000 are found to be altered by trypsin and chymotrypsin treatment. Also the polar headgroup of phosphatidylinositol appears to play a role in the binding process.

scholarly journals Secretion and apparent activation of human hepatic lipase requires proper oligosaccharide processing in the endoplasmic reticulum

1998 ◽  
Vol 337 (1) ◽  
pp. 133-140 ◽  
Author(s):  
Adrie J. M. VERHOEVEN ◽  
Bernadette P. NEVE ◽  
Hans JANSEN
Keyword(s):  
Endoplasmic Reticulum ◽  
Intracellular Trafficking ◽  
De Novo ◽  
Hepatic Lipase ◽  
Brefeldin A ◽  
Apparent Increase ◽  
Triacylglycerol Hydrolase ◽  
Oligosaccharide Processing ◽  
Carbonyl Cyanide ◽  
Catalytically Active

Human hepatic lipase (HL) is a glycoprotein with four N-linked oligosaccharide side chains. The importance of glycosylation for the secretion of catalytically active HL was studied in HepG2 cells by using inhibitors of intracellular trafficking, N-glycosylation and oligosaccharide processing. Secretion of HL was inhibited by carbonyl cyanide m-chlorophenylhydrazone (CCCP), monensin, brefeldin A (BFA), tunicamycin, castanospermine and N-methyldeoxynojirimycin, but not by 1-deoxymannojirimycin. Secretion of α1-antitrypsin, an unrelated N-glycoprotein, was also inhibited by monensin, BFA and tunicamycin, but not by CCCP, castanospermine or N-methyldeoxynojirimycin. Intracellular HL activity decreased with CCCP, tunicamycin, castanospermine and N-methyldeoxynojirimycin, but increased with monensin and BFA. In the absence of protein synthesis de novo, HL activity secreted into the medium was 7.8±2.1-fold higher (mean±S.D., n = 7) than the simultaneous fall in intracellular HL activity. In cells pretreated with monensin or BFA, this factor decreased to 1.3±0.5, indicating that the apparent increase in HL activity had already occurred within these cells. After chromatography on Sepharose–heparin, the specific triacylglycerol hydrolase activity of secreted HL was only 1.7±0.3-fold higher than that of intracellular HL, indicating that the secretion-coupled increase in HL activity is only partly explained by true activation. We conclude that oligosaccharide processing by glucosidases in the endoplasmic reticulum is necessary for the transport of newly synthesized human HL, but not α1-antitrypsin, to the Golgi, where the catalytic activity of HL is unmasked.

Leptin biosynthetic pathway in white adipocytes

2006 ◽  
Vol 84 (2) ◽  
pp. 207-214 ◽  
Author(s):  
Philippe G Cammisotto ◽  
Ludwik J Bukowiecki ◽  
Yves Deshaies ◽  
Moise Bendayan
Keyword(s):  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
De Novo ◽  
Brefeldin A ◽  
Mrna Levels ◽  
De Novo Synthesis ◽  
Cellular Content ◽  
Constitutive Secretion

The aim of this study was to determine through morphological and biochemical means the biosynthetic and secretory pathway followed by leptin in adipocytes. Immunocytochemistry revealed the presence of leptin in the rough endoplasmic reticulum, the Golgi apparatus, and in numerous small vesicles along the plasma membrane of white adipo cytes. In vitro, isolated adipocytes under nonstimulated conditions (basal) continuously secreted leptin while their intra cellular content remained unchanged. When adipocytes were stimulated with insulin, leptin cellular content and secretion increased in parallel and were significantly different from basal secretion only after 45 min. L-leucine and L-glutamate also strongly stimulated leptin synthesis and secretion. These stimulating effects were abolished by cycloheximide and brefeldin A. The transcriptional inhibitor actinomycin D did not have any effects in either basal or stimulated conditions. Leptin mRNA levels were not affected by any stimulating or inhibiting agents. Finally, norepinephrine, isoproterenol, CL316243, and palmitate inhibited the effects of insulin, L-leucine, and L-glutamate on leptin synthesis. We thus conclude that (i) adipocytes continuously synthesize and secrete leptin along a rough endoplasmic reticulum–Golgi secretory vesicles pathway, (ii) an increase in leptin secretion requires increased de novo synthesis, and (iii) short-term leptin secretion does not involve changes in mRNA levels.Key words: leptin, vesicles, constitutive secretion, de novo synthesis, transcription.

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 The Cargo Receptors Surf4, Endoplasmic Reticulum-Golgi Intermediate Compartment (ERGIC)-53, and p25 Are Required to Maintain the Architecture of ERGIC and Golgi

2008 ◽  
Vol 19 (5) ◽  
pp. 1976-1990 ◽  
Author(s):  
Sandra Mitrovic ◽  
Houchaima Ben-Tekaya ◽  
Eva Koegler ◽  
Jean Gruenberg ◽  
Hans-Peter Hauri
Keyword(s):  
Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
Brefeldin A ◽  
Matrix Proteins ◽  
Anterograde Transport ◽  
Early Secretory Pathway ◽  
Partial Redistribution ◽  
Intermediate Compartment ◽  
Human Orthologue ◽  
Golgi Matrix

Rapidly cycling proteins of the early secretory pathway can operate as cargo receptors. Known cargo receptors are abundant proteins, but it remains mysterious why their inactivation leads to rather limited secretion phenotypes. Studies of Surf4, the human orthologue of the yeast cargo receptor Erv29p, now reveal a novel function of cargo receptors. Surf4 was found to interact with endoplasmic reticulum-Golgi intermediate compartment (ERGIC)-53 and p24 proteins. Silencing Surf4 together with ERGIC-53 or silencing the p24 family member p25 induced an identical phenotype characterized by a reduced number of ERGIC clusters and fragmentation of the Golgi apparatus without effect on anterograde transport. Live imaging showed decreased stability of ERGIC clusters after knockdown of p25. Silencing of Surf4/ERGIC-53 or p25 resulted in partial redistribution of coat protein (COP) I but not Golgi matrix proteins to the cytosol and partial resistance of the cis-Golgi to brefeldin A. These findings imply that cargo receptors are essential for maintaining the architecture of ERGIC and Golgi by controlling COP I recruitment.

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