scholarly journals Synthesis and trafficking of prion proteins in cultured cells.

1992 ◽  
Vol 3 (8) ◽  
pp. 851-863 ◽  
Author(s):  
A Taraboulos ◽  
A J Raeber ◽  
D R Borchelt ◽  
D Serban ◽  
S B Prusiner
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Cultured Cells ◽  
Prion Proteins ◽  
Brefeldin A ◽  
Endocytic Pathway ◽  
Chromosomal Gene ◽  
Golgi Stack ◽  
Mouse Neuroblastoma ◽  
N Terminus

Scrapie prions are composed largely, if not entirely, of the scrapie prion protein (PrPSc) that is encoded by a chromosomal gene. Scrapie-infected mouse neuroblastoma (ScN2a) and hamster brain (ScHaB) cells synthesize PrPSc from the normal PrP isoform (PrPC) or a precursor through a posttranslational process. In pulse-chase radiolabeling experiments, we found that presence of brefeldin A (BFA) during both the pulse and the chase periods prevented the synthesis of PrPSc. Removal of BFA after the chase permitted synthesis of PrPSc to resume. BFA also blocked the export of nascent PrPC to the cell surface but did not alter the distribution of intracellular deposits of PrPSc. Under the same conditions, BFA caused the redistribution of the Golgi marker MG160 into the endoplasmic reticulum (ER). Using monensin as an inhibitor of mid-Golgi glycosylation, we determined that PrP traverses the mid-Golgi stack before acquiring protease resistance. About 1 h after the formation of PrPSc, its N-terminus was removed by a proteolytic process that was inhibited by ammonium chloride, chloroquine, and monensin, arguing that this is a lysosomal event. These results suggest that the ER is not competent for the synthesis of PrPSc and that the synthesis of PrPSc occurs during the transit of PrP between the mid-Golgi stack and lysosomes. Presumably, the endocytic pathway features in the synthesis of PrPSc.

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 Overexpression of a Mutant Form of EhRabA, a Unique Rab GTPase of Entamoeba histolytica, Alters Endoplasmic Reticulum Morphology and Localization of the Gal/GalNAc Adherence Lectin

2009 ◽  
Vol 8 (7) ◽  
pp. 1014-1026 ◽  
Author(s):  
B. H. Welter ◽  
L. A. Temesvari
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Entamoeba Histolytica ◽  
Vesicle Trafficking ◽  
Brefeldin A ◽  
Surface Proteins ◽  
Secretory Proteins ◽  
Rab Gtpase ◽  
Mutant Form ◽  
Rab Gtpases

ABSTRACT Entamoeba histolytica is a protozoan parasite that causes amoebic dysentery and liver abscess. Vesicle trafficking events, such as phagocytosis and delivery of plasma membrane proteins, have been implicated in pathogenicity. Rab GTPases are proteins whose primary function is to regulate vesicle trafficking; therefore, understanding the function of Rabs in this organism may provide insight into virulence. E. histolytica possesses a number of unique Rabs that exhibit limited homology to host Rabs. In this study we examined the function of one such Rab, EhRabA, by characterizing a mutant overexpressing a constitutively GTP-bound version of the protein. Overexpression of mutant EhRabA resulted in decreased adhesion to and phagocytosis of human red blood cells and in the appearance of large tubular organelles that could be stained with endoplasmic reticulum (ER)-specific but not Golgi complex-specific antibodies. Consistent with the adhesion defect, two subunits of a cell surface adhesin, the galactose/N-acetylgalactosamine lectin, were mislocalized to the novel organelle. A cysteine protease, EhCP2, was also localized to the ER-like compartment in the mutant; however, the localization of two additional cell surface proteins, Igl and SREHP, remained unchanged in the mutant. The phenotype of the mutant could be recapitulated by treatment with brefeldin A, a cellular toxin that disrupts ER-to-Golgi apparatus vesicle traffic. This suggests that EhRabA influences vesicle trafficking pathways that are also sensitive to brefeldin A. Together, the data indicate that EhRabA directly or indirectly influences the morphology of secretory organelles and regulates trafficking of a subset of secretory proteins in E. histolytica.

scholarly journals Cyclic AMP Increases Cell Surface Expression of Functional Na,K-ATPase Units in Mammalian Cortical Collecting Duct Principal Cells

2001 ◽  
Vol 12 (2) ◽  
pp. 255-264 ◽  
Author(s):  
Sandrine Gonin ◽  
Georges Deschênes ◽  
Frank Roger ◽  
Marcelle Bens ◽  
Pierre-Yves Martin ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Cell Surface ◽  
Atpase Activity ◽  
Cultured Cells ◽  
Collecting Duct ◽  
Brefeldin A ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Cortical Collecting Duct ◽  
Expression And Activity

Cyclic AMP (cAMP) stimulates the transport of Na+ and Na,K-ATPase activity in the renal cortical collecting duct (CCD). The aim of this study was to investigate the mechanism whereby cAMP stimulates the Na,K-ATPase activity in microdissected rat CCDs and cultured mouse mpkCCDc14 collecting duct cells. db-cAMP (10−3 M) stimulated by 2-fold the activity of Na,K-ATPase from rat CCDs as well as the ouabain-sensitive component of 86Rb+ uptake by rat CCDs (1.7-fold) and cultured mouse CCD cells (1.5-fold). Pretreatment of rat CCDs with saponin increased the total Na,K-ATPase activity without further stimulation by db-cAMP. Western blotting performed after a biotinylation procedure revealed that db-cAMP increased the amount of Na,K-ATPase at the cell surface in both intact rat CCDs (1.7-fold) and cultured cells (1.3-fold), and that this increase was not related to changes in Na,K-ATPase internalization. Brefeldin A and low temperature (20°C) prevented both the db-cAMP-dependent increase in cell surface expression and activity of Na,K-ATPase in both intact rat CCDs and cultured cells. Pretreatment with the intracellular Ca2+chelator bis-(o-aminophenoxy)-N,N,N′,N′-tetraacetic acid also blunted the increment in cell surface expression and activity of Na,K-ATPase caused by db-cAMP. In conclusion, these results strongly suggest that the cAMP-dependent stimulation of Na,K-ATPase activity in CCD results from the translocation of active pump units from an intracellular compartment to the plasma membrane.

scholarly journals Increasing the expression of calcium-permeable TRPC3 and TRPC7 channels enhances constitutive secretion

2008 ◽  
Vol 413 (3) ◽  
pp. 437-446 ◽  
Author(s):  
Verna Lavender ◽  
Setareh Chong ◽  
Katherine Ralphs ◽  
Adrian J. Wolstenholme ◽  
Barbara J. Reaves
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Transient Receptor Potential ◽  
Calcium Influx ◽  
Brefeldin A ◽  
Receptor Potential ◽  
Fold Increase ◽  
Extracellular Medium ◽  
Additional Function ◽  
Golgi Stack

The hTRPC [human TRPC (canonical transient receptor potential)] family of non-selective cation channels is proposed to mediate calcium influx across the plasma membrane via PLC (phospholipase C)-coupled receptors. Heterologously expressed hTRPC3 and hTRPC7 have been localized at the cell surface; however, a large intracellular component has also been noted but not characterized. In the present study, we have investigated the intracellular pool in COS-7 cells and have shown co-localization with markers for both the TGN (trans-Golgi network) and the cis-Golgi cisternae by immunofluorescence microscopy. Addition of BFA (Brefeldin A) to cells expressing hTRPC3 or hTRPC7 resulted in the redistribution of the Golgi component to the endoplasmic reticulum, indicating that this pool is present in both the Golgi stack and the TGN. Expression of either TRPC3 or TRPC7, but not TRPC1 or the cell surface marker CD8, resulted in a 2–4-fold increase in secreted alkaline phosphatase in the extracellular medium. Based on these results, we propose that an additional function of these members of the hTRPC family may be to enhance secretion either by affecting transport through the Golgi stack or by increasing fusion at the plasma membrane.

scholarly journals Scattered Golgi Elements during Microtubule Disruption Are Initially Enriched in Trans-Golgi Proteins

1998 ◽  
Vol 9 (1) ◽  
pp. 191-207 ◽  
Author(s):  
Wei Yang ◽  
Brian Storrie
Keyword(s):  
Endoplasmic Reticulum ◽  
Polyclonal Antibody ◽  
Time Course ◽  
Brefeldin A ◽  
Vero Cells ◽  
Protein Distribution ◽  
Microtubule Depolymerization ◽  
Drug Induced ◽  
Golgi Stack ◽  
Microtubule Disruption

We have addressed the question of whether or not Golgi fragmentation, as exemplified by that occurring during drug-induced microtubule depolymerization, is accompanied by the separation of Golgi subcompartments one from another. Scattering kinetics of Golgi subcompartments during microtubule disassembly and reassembly following reversible nocodazole exposure was inferred from multimarker analysis of protein distribution. Stably expressed α-2,6-sialyltransferase andN-acetylglucosaminyltransferase-I (NAGT-I), both C-terminally tagged with the myc epitope, provided markers for thetrans-Golgi/trans-Golgi network (TGN) and medial-Golgi, respectively, in Vero cells. Using immunogold labeling, the chimeric proteins were polarized within the Golgi stack. Total cellular distributions of recombinant proteins were assessed by immunofluorescence (anti-myc monoclonal antibody) with respect to the endogenous protein, β-1,4-galactosyltransferase (GalT,trans-Golgi/TGN, polyclonal antibody). ERGIC-53 served as a marker for the intermediate compartment). In HeLa cells, distribution of endogenous GalT was compared with transfected rat α-mannosidase II (medial-Golgi, polyclonal antibody). After a 1-h nocodazole treatment, Vero α-2,6-sialyltransferase and GalT were found in scattered cytoplasmic patches that increased in number over time. Initially these structures were often negative for NAGT-I, but over a two- to threefold slower time course, NAGT-I colocalized with α-2,6-sialyltransferase and GalT. Scattered Golgi elements were located in proximity to ERGIC-53-positive structures. Similartrans-first scattering kinetics was seen with the HeLa GalT/α-mannosidase II pairing. Following nocodazole removal, all cisternal markers accumulated at the same rate in a juxtanuclear Golgi. Accumulation of cisternal proteins in scattered Golgi elements was not blocked by microinjected GTPγS at a concentration sufficient to inhibit secretory processes. Redistribution of Golgi proteins from endoplasmic reticulum to scattered structures following brefeldin A removal in the presence of nocodazole was not blocked by GTPγS. We conclude that Golgi subcompartments can separate one from the other. We discuss how direct trafficking of Golgi proteins from the TGN/trans-Golgi to endoplasmic reticulum may explain the observed trans-first scattering of Golgi transferases in response to microtubule depolymerization.

scholarly journals Post-Golgi membrane traffic: brefeldin A inhibits export from distal Golgi compartments to the cell surface but not recycling.

1992 ◽  
Vol 118 (2) ◽  
pp. 267-283 ◽  
Author(s):  
S G Miller ◽  
L Carnell ◽  
H H Moore
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Brefeldin A ◽  
Endocytic Pathway ◽  
Golgi Membrane ◽  
Granule Formation ◽  
Secretogranin Ii ◽  
Golgi System

Recent studies using the fungal metabolite brefeldin A (BFA) have provided important insights into the dynamics and the organization of the ER/Golgi membrane system. Here we examined the effect of BFA on the functional integrity of the distal part of the secretory pathway, i.e., transport between trans-Golgi cisternae and the cell surface. To assay export via the constitutive pathway, we followed the movement of vesicular stomatitis virus (VSV) G glycoprotein that had been accumulated in the trans-Golgi network (TGN) by incubation of infected BHK-21 cells at 20 degrees C. Addition of BFA rapidly and reversibly inhibited cell surface transport of G protein. The block to secretion was not due to redistribution of externalized G protein to internal pools. It was also not due to collapse of TGN to the ER, since VSV G protein blocked in treated cells resided in compartments that were distinct from the ER/Golgi system. Similar effects were found with a bulk-flow marker: BFA blocked constitutive secretion of glycosaminoglycan chains that had been synthesized and sulfated in the trans-Golgi cisternae. To examine export via the regulated secretory pathway, we assayed secretion of [35S]SO4 labeled secretogranin II from PC12 cells, a marker that has been used to study secretory granule budding from the TGN (Tooze, S. A., U. Weiss, and W. B. Huttner. 1990. Nature [Lond.]. 347:207-208). BFA potently inhibited secretion of sulfated secretogranin II induced by K+ depolarization. Inhibition was at the level of granule formation, since BFA had no effect on regulated secretion from preformed granules. Taken together, the results suggest that BFA blocks export via both the constitutive and the regulated pathways. In contrast, endocytosis and recycling of VSV G protein were not blocked by BFA, consistent with previous studies that endocytosis is unaffected (Misumi, Y., Y. Misumi, K. Miki, A Takatsuki, G. Tamura, and Y. Ikehara. 1986. J. Biol. Chem. 261:11398-11403). These and earlier results suggest that the exo/endocytic pathway of mammalian cells consist of two similar but distinct endomembrane systems: an ER/Golgi system and a post-Golgi system. BFA prevents forward transport without affecting return traffic in both systems.

scholarly journals Endothelin-converting enzyme-like 1 (ECEL1) is present both in the plasma membrane and in the endoplasmic reticulum

2004 ◽  
Vol 380 (3) ◽  
pp. 881-888 ◽  
Author(s):  
Alexandre BENOIT ◽  
Miguel Angel VARGAS ◽  
Luc DesGROSEILLERS ◽  
Guy BOILEAU
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Cultured Cells ◽  
Peptide Hormone ◽  
Similar Distribution ◽  
Cell Fractionation ◽  
Converting Enzyme ◽  
Confocal Immunofluorescence Microscopy ◽  
Endothelin Converting Enzyme ◽  
Cytosolic Domains

Enzymes of the M13 family of zinc-containing endopeptidases are recognized as important regulators of neuropeptide and peptide hormone activity. Peptidases of this family are type II integral-membrane proteins characterized by short cytosolic domains and large extracellular domains containing the active site. The M13 family has, at present, seven members, including ECEL1 (endothelin-converting enzyme-like 1), one of the newest members. ECEL1 is expressed predominantly in the central nervous system. It has been proposed that the enzyme has a role in the nervous regulation of the respiratory system. No physiological substrate has been identified yet. To better understand the function(s) of this enzyme, we have expressed human ECEL1 in cultured cells and monitored its biosynthesis and subcellular localization. Immunoblot and cell-surface biotinylation analysis of transfected cells expressing ECEL1 showed that only a fraction of the protein travelled to the cell surface, while most of the enzyme was present in an intracellular compartment identified by confocal immunofluorescence microscopy and cell fractionation as the ER (endoplasmic reticulum). Pulse–chase experiments showed that ER-localized ECEL1 was stable, with a half-life of more than 3 h. Endogenous ECEL1 from mouse pituitary gland had a similar distribution between the cell surface and the ER. Finally, using domain-swapping experiments with neprilysin, another member of the M13 family, we showed that localization of ECEL1 to the ER requires both the transmembrane and cytoplasmic domains. It thus appears that ECEL1 may have functions both at the cell surface and in the ER.

The immunogenicity of VP7, a rotavirus antigen resident in the endoplasmic reticulum, is enhanced by cell surface expression.

1990 ◽  
Vol 64 (10) ◽  
pp. 4776-4783 ◽  
Author(s):  
M E Andrew ◽  
D B Boyle ◽  
P L Whitfeld ◽  
L J Lockett ◽  
I D Anthony ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Surface Expression ◽  
Cell Surface Expression

scholarly journals Inhibition by brefeldin A of protein secretion from the apical cell surface of Madin-Darby canine kidney cells.

1991 ◽  
Vol 266 (27) ◽  
pp. 17729-17732 ◽  
Author(s):  
S.H. Low ◽  
S.H. Wong ◽  
B.L. Tang ◽  
P. Tan ◽  
V.N. Subramaniam ◽  
...  
Keyword(s):  
Cell Surface ◽  
Protein Secretion ◽  
Apical Cell ◽  
Brefeldin A ◽  
Kidney Cells ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

scholarly journals Degradation of aggrecan precursors within a specialized subcompartment of the chicken chondrocyte endoplasmic reticulum

1996 ◽  
Vol 316 (2) ◽  
pp. 487-495 ◽  
Author(s):  
Manuel ALONSO ◽  
Josefina HIDALGO ◽  
Linda HENDRICKS ◽  
Angel VELASCO
Keyword(s):  
Endoplasmic Reticulum ◽  
Protease Inhibitors ◽  
Degradation Rate ◽  
Redox State ◽  
Brefeldin A ◽  
Lag Phase ◽  
Cysteine Protease Inhibitors ◽  
Immunofluorescence Analysis ◽  
Smooth Membrane ◽  
Membrane Bound

Chicken chondrocytes in culture synthesize aggrecan proteoglycan as a 370 kDa precursor that is glycosylated and secreted into the medium with a half-life of 30 min. In metabolic studies the 370 kDa precursor was shown to render a degradation intermediate of 190 kDa, which appeared with no measurable lag phase; it was dependent on temperature (> 20 °C) and inhibited by certain serine and serine/cysteine protease inhibitors such as leupeptin and PMSF. By contrast, degradation was unaffected by treatment of the cells with brefeldin A or with lysosomotropic agents. Aggrecan precursors were detected by immunofluorescence analysis within a subcompartment of the endoplasmic reticulum (ER), previously characterized as a smooth-membrane-bound subregion [Vertel, Velasco, LaFrance, Walters and Kaczman-Daniel (1989) J. Cell Biol. 109, 1827–1836]. Analysis of the subcellular fractions derived from chondrocytes indicated that the degradation intermediate was concentrated in the ER subcompartment. Degradation was dependent on the Ca2+ concentration and the redox state in the ER. Treatment of the cells with agents or conditions that alter the degradation rate of aggrecan precursors, such as protease inhibitors, decreased temperature or dithiothreitol, also modified the retention of these molecules in the ER subcompartment, as seen by immunofluorescence. These results indicate that a fraction of the 370 kDa aggrecan precursor is targeted to a smooth ER subcompartment where it undergoes degradation.

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