scholarly journals Glucose removal from N-linked oligosaccharides is required for efficient maturation of certain secretory glycoproteins from the rough endoplasmic reticulum to the Golgi complex.

1984 ◽  
Vol 98 (5) ◽  
pp. 1720-1729 ◽  
Author(s):  
H F Lodish ◽  
N Kong
Keyword(s):  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Gradient Centrifugation ◽  
Specific Inhibitor ◽  
Secretory Proteins ◽  
Secretory Glycoproteins ◽  
Alpha 1 Antitrypsin ◽  
Human Hepatoma Hepg2 Cells ◽  
Transport Receptor ◽  
Hepatoma Hepg2

1- Deoxynojirimycin is a specific inhibitor of glucosidases I and II, the first enzymes that process N-linked oligosaccharides after their transfer to polypeptides in the rough endoplasmic reticulum. In a pulse-chase experiment, 1- deoxynojirimycin greatly reduced the rate of secretion of alpha 1-antitrypsin and alpha 1-antichymotrypsin by human hepatoma HepG2 cells, but had marginal effects on secretion of the glycoproteins C3 and transferrin, or of albumin. As judged by equilibrium gradient centrifugation, 1- deoxynojirimycin caused alpha 1-antitrypsin and alpha 1-antichymotrypsin to accumulate in the rough endoplasmic reticulum. The oligosaccharides on cell-associated alpha 1-antitrypsin and alpha 1-antichymotrypsin synthesized in the presence of 1- deoxynojirimycin , remained sensitive to Endoglycosidase H and most likely had the structure Glu1- 3Man9GlcNAc2 . Tunicamycin, an antibiotic that inhibits addition of N-linked oligosaccharide units to glycoproteins, had a similar differential effect on secretion of these proteins. Swainsonine , an inhibitor of the Golgi enzyme alpha-mannosidase II, had no effect on the rates of protein secretion, although the proteins were in this case secreted with an abnormal N-linked, partially complex, oligosaccharide. We conclude that the movement of alpha 1-antitrypsin and alpha 1-antichymotrypsin from the rough endoplasmic reticulum to the Golgi requires that the N-linked oligosaccharides be processed to at least the Man9GlcNAc2 form; possibly this oligosaccharide forms part of the recognition site of a transport receptor for certain secretory proteins.

scholarly journals A vesicular intermediate in the transport of hepatoma secretory proteins from the rough endoplasmic reticulum to the Golgi complex.

1987 ◽  
Vol 104 (2) ◽  
pp. 221-230 ◽  
Author(s):  
H F Lodish ◽  
N Kong ◽  
S Hirani ◽  
J Rasmussen
Keyword(s):  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Supernatant Fraction ◽  
Secretory Proteins ◽  
Golgi Vesicles ◽  
Intermediate Fraction ◽  
Intermediate Compartment ◽  
Alpha 1 Antitrypsin ◽  
Endoglycosidase H

We have identified a vesicle fraction that contains alpha 1-antitrypsin and other human HepG2 hepatoma secretory proteins en route from the rough endoplasmic reticulum (RER) to the cis face of the Golgi complex. [35S]Methionine pulse-labeled cells were chased for various periods of time, and then a postnuclear supernatant fraction was resolved on a shallow sucrose-D2O gradient. This intermediate fraction has a density lighter than RER or Golgi vesicles. Most alpha 1-antitrypsin in this fraction (P1) bears N-linked oligosaccharides of composition similar to that of alpha 1-antitrypsin within the RER; mainly Man8GlcNac2 with lesser amounts of Man7GlcNac2 and Man9GlcNac2; this suggests that the protein has not yet reacted with alpha-mannosidase-I on the cis face of the Golgi complex. This light vesicle species is the first post-ER fraction to be filled by labeled alpha 1-antitrypsin after a short chase, and newly made secretory proteins enter this compartment in proportion to their rate of exit from the RER and their rate of secretion from the cells: alpha 1-antitrypsin and albumin faster than preC3 and alpha 1-antichymotrypsin, faster, in turn, then transferrin. Deoxynojirimycin, a drug that blocks removal of glucose residues from alpha 1-antitrypsin in the RER and blocks its intracellular maturation, also blocks its appearance in this intermediate compartment. Upon further chase of the cells, we detect sequential maturation of alpha 1-antitrypsin to two other intracellular forms: first, P2, a form that has the same gel mobility as P1 but that bears an endoglycosidase H-resistant oligosaccharide and is found in a compartment--probably the medial Golgi complex--of density higher than that of the intermediate that contains P1; and second, the mature sialylated form of alpha 1-antitrypsin.

scholarly journals Proteoglycan biosynthesis in chondrocytes: protein A-gold localization of proteoglycan protein core and chondroitin sulfate within Golgi subcompartments.

1985 ◽  
Vol 101 (6) ◽  
pp. 2355-2365 ◽  
Author(s):  
A Ratcliffe ◽  
P R Fryer ◽  
T E Hardingham
Keyword(s):  
Endoplasmic Reticulum ◽  
Chondroitin Sulfate ◽  
Rough Endoplasmic Reticulum ◽  
Intracellular Localization ◽  
Protein A ◽  
Keratan Sulfate ◽  
Glycosaminoglycan Synthesis ◽  
Protein Core ◽  
Late Event ◽  
Secretory Glycoproteins

The intracellular pathway of cartilage proteoglycan biosynthesis was investigated in isolated chondrocytes using a protein A-gold electron microscopy immunolocalization procedure. Proteoglycans contain a protein core to which chondroitin sulfate and keratan sulfate chains and oligosaccharides are added in posttranslational processing. Specific antibodies have been used in this study to determine separately the distribution of the protein core and chondroitin sulfate components. In normal chondrocytes, proteoglycan protein core was readily localized only in smooth-membraned vesicles which co-labeled with ricin, indicating them to be galactose-rich medial/trans-Golgi cisternae, whereas there was only a low level of labeling in the rough endoplasmic reticulum. Chondroitin sulfate was also localized in medial/trans-Golgi cisternae of control chondrocytes but was not detected in other cellular compartments. In cells treated with monensin (up to 1.0 microM), which strongly inhibits proteoglycan secretion (Burditt, L.J., A. Ratcliffe, P. R. Fryer, and T. Hardingham, 1985, Biochim. Biophys. Acta., 844:247-255), there was greatly increased intracellular localization of proteoglycan protein core in both ricin-positive vesicles, and in ricin-negative vesicles (derived from cis-Golgi stacks) and in the distended rough endoplasmic reticulum. Chondroitin sulfate also increased in abundance after monensin treatment, but continued to be localized only in ricin-positive vesicles. The results suggested that the synthesis of chondroitin sulfate on proteoglycan only occurs in medial/trans-Golgi cisternae as a late event in proteoglycan biosynthesis. This also suggests that glycosaminoglycan synthesis on proteoglycans takes place in a compartment in common with events in the biosynthesis of both O-linked and N-linked oligosaccharides on other secretory glycoproteins.

scholarly journals Pathogenesis of Placentitis in the Goat Inoculated with Brucella abortus. II. Ultrastructural Studies

1986 ◽  
Vol 23 (3) ◽  
pp. 227-239 ◽  
Author(s):  
T. D. Anderson ◽  
N. F. Cheville ◽  
V. P. Meador
Keyword(s):  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Brucella Abortus ◽  
Basement Membranes ◽  
Secretory Proteins ◽  
Chorionic Villi ◽  
Uterine Arteries ◽  
Ultrastructural Studies ◽  
Large Numbers ◽  
Late Stages

Pregnant goats were inoculated intravenously or in uterine arteries with Brucella abortus, and tissues from the uterus and placenta were examined by electron microscopy. Identification of B. abortus in placentae was with antibody-coated colloidal gold. B. abortus was first seen in phagosomes of erythrophagocytic trophoblasts and in the rough endoplasmic reticulum of chorioallantoic trophoblasts. Subsequently, trophoblast necrosis and ulceration of chorioallantoic membranes were present. Coincidently, B. abortus was present in the lumen of placental capillaries. In late stages of infection, placental vasculitis was present, and placentomal trophoblasts were separated from maternal syncytial epithelium. In lesions with vasculitis, large numbers of B. abortus were in connective tissue of chorionic villi. Within the placentome, trophoblasts that lined chorionic villi contained no intracellular bacteria and were separated from B. abortus by intact basement membranes. These results suggest that bacteremic B. abortus is endocytosed by erythrophagocytic trophoblasts and that B. abortus replicates in the rough endoplasmic reticulum of chorioallantoic trophoblasts. Replication of brucellae in trophoblastic rough endoplasmic reticulum is unique; we believe that B. abortus may utilize endoplasmic reticulum for synthesis and glycosylation of bacterial membrane proteins or that B. abortus catabolizes trophoblast secretory proteins.

scholarly journals Release of soluble resident as well as secretory proteins from HepG2 cells by partial permeabilization of rough-endoplasmic-reticulum membranes

1989 ◽  
Vol 257 (1) ◽  
pp. 159-163 ◽  
Author(s):  
G J Strous ◽  
P Van Kerkhof
Keyword(s):  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Hepg2 Cells ◽  
Integral Membrane Protein ◽  
Membrane Skeleton ◽  
Secretory Proteins ◽  
Low Concentrations ◽  
Specific Proteins ◽  
Selective Retention ◽  
Vesicular Stomatitis

Secretory proteins migrate from the rough endoplasmic reticulum (ER) to the Golgi complex at different rates. Selective retention of specific proteins to rough-ER membrane constituents could explain this phenomenon. We have permeabilized HepG2 cells with low concentrations of saponin. Release of newly synthesized proteins was studied after brief labelling in the presence of [35S]methionine. The efflux of several secretory proteins was studied at various saponin concentrations; a 2-fold higher saponin concentration was required to release transferrin compared with that required to release albumin and orosomucoid. Glucosidase II, a soluble resident protein of the ER, is released at the same saponin concentration as albumin. Saponin did not destroy the membrane skeleton structure; at the concentrations used, the integral membrane protein G of vesicular-stomatitis virus remained fully associated with the cells.

scholarly journals Hepatic and Extrahepatic Sources and Manifestations in Endoplasmic Reticulum Storage Diseases

2021 ◽  
Vol 22 (11) ◽  
pp. 5778
Author(s):  
Francesco Callea ◽  
Paola Francalanci ◽  
Isabella Giovannoni
Keyword(s):  
Endoplasmic Reticulum ◽  
Acute Phase ◽  
Rough Endoplasmic Reticulum ◽  
Storage Disease ◽  
Acute Phase Reactant ◽  
Storage Diseases ◽  
Phase Reactant ◽  
Alpha 1 Antitrypsin ◽  
Encoding Genes ◽  
Liver Storage

Alpha-1-antitrypsin (AAT) and fibrinogen are secretory acute phase reactant proteins. Circulating AAT and fibrinogen are synthesized exclusively in the liver. Mutations in the encoding genes result in conformational abnormalities of the two molecules that aggregate within the rough endoplasmic reticulum (RER) instead of being regularly exported. That results in AAT-deficiency (AATD) and in hereditary hypofibrinogenemia with hepatic storage (HHHS). The association of plasma deficiency and liver storage identifies a new group of pathologies: endoplasmic reticulum storage disease (ERSD).

scholarly journals Two fractions of rough endoplasmic reticulum from rat liver. I. Recovery of rapidly sedimenting endoplasmic reticulum in association with mitochondria.

1977 ◽  
Vol 72 (3) ◽  
pp. 714-725 ◽  
Author(s):  
G C Shore ◽  
J R Tata
Keyword(s):  
Endoplasmic Reticulum ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Rat Liver ◽  
Rough Endoplasmic Reticulum ◽  
Structural Damage ◽  
Gradient Centrifugation ◽  
Mitochondrial Protein ◽  
Enzyme Analysis ◽  
Marker Enzyme

Low-speed centrifugation (640 g) of rat liver homogenates, prepared with a standard ionic medium, yielded a pellet from which a rapidly sedimenting fraction of rough endoplasmic reticulum (RSER) was recovered free of nuclei. This fraction contained 20-25% of cellular RNA and approximately 30% of total glucose-6-phosphatase (ER marker) activity. A major portion of total cytochrome c oxidase (mitochondrial marker) activity was also recovered in this fraction, with the remainder sedimenting between 640 and 6,000 g. Evidence is provided which indicates that RSER may be intimately associated with mitochondria. Complete dissociation of ER from mitochondria in the RSER fraction required very harsh conditions. Sucrose density gradient centrifugation analysis revealed that 95% dissociation could be achieved when the RSER fraction was first resuspended in buffer containing 500 mM KCl and 20 mM EDTA, and subjected to shearing. Excluding KCl, EDTA, or shearing from the procedure resulted in incomplete separation. Both electron microscopy and marker enzyme analysis of mitochondria purified by this procedure indicated that some structural damage and leakage of proteins from matrix and intermembrane compartments had occurred. Nevertheless, when mitochondria from RSER and postnuclear 6,000-g pellet fractions were purified in this way fromanimals injected with [35S]methionine +/- cycloheximide, mitochondria from the postnuclear 6,000-g pellet were found to incorporate approximately two times more cytoplasmically synthesized radioactive protein per milligram mitochondrial protein (or per unit cytochrome c oxidase activity) than did mitochondria from the RSER fraction. Mitochondria-RSER associations, therefore, do not appear to facilitate enhanced incorporation of mitochondrial proteins which are newly synthesized in the cytoplasm.

Sign in / Sign up

Export Citation Format

Share Document