Four secretory proteins synthesized by hepatocytes are transported from endoplasmic reticulum to Golgi complex at different rates (original) (raw)
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Journal of Biological Chemistry, 1992
The abbreviations used are: ER, endoplasmic reticulum; dMM, deoxymannojirimycin; Endo H, endoglycosidase H; GC, Golgi complex; PBS, phosphate-buffered saline; PS, permeabilization solution; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid. MATERIALS AND METHODS Chemicals-Antibodies against human albumin and haptoglobin, which cross-react with the corresponding rat proteins (Fries and Lindstrom, 1986), were from Dakopatts, Denmark. (35S]Methionine 2760 This is an Open Access article under the CC BY license.
The Journal of Cell Biology, 1970
A comparative biochemical and radioautographic in vivo study was performed to identify the site of synthesis and route of migration of albumin in the parenchymal liver cell after labeling with leucine-14C or leucine-3H via the portal vein. Free cytoplasmic ribosomes, membrane-bound ribosomes, rough- and smooth-surfaced microsomes, and Golgi membranes were isolated. The purity of the Golgi fraction was examined morphologically and biochemically. After administration of leucine-14C, labeled albumin was extracted, and the sequence of transport was followed from one fraction to the other. Approximately 2 min after the intravenous injection, bound ribosomes displayed a maximal rate of leucine-14C incorporation into albumin. 4 min later, a peak was reached for rough microsomes. Corresponding maximal activities for smooth microsomes were recorded at 15 min, and for the Golgi apparatus at ∼20 min. The relative amount of albumin, calculated on a membrane protein basis, was higher in the Golg...
Journal of Cell Biology, 1988
We used a conjugate of transferrin and horseradish peroxidase (Tf/HRP) to label the intracellular transferrin receptor route in the human hepatoma cell line HepG2. The recycling kinetics of [125I]Tf/HRP were similar to those of unmodified [125I]Tf, implying identical routes for both ligands. 3,3'Diaminobenzidine (DAB)-cytochemistry was performed on post-nuclear supernatants of homogenates of cells which were incubated with both Tf/HRP and [125I]Tf, and caused two different effects: (a) the equilibrium density of [125I]Tf containing microsomes in a Percoll density gradient was increased, and (b) the amount of immunoprecipitable [125I]Tf from density-shifted lysed microsomes was only 20% of that of nonDAB treated microsomes. The whole biosynthetic route of alpha 1-antitrypsin (AT), a typical secretory glycoprotein in HepG2 cells, was labeled during a 60-min incubation with [35S]methionine. DAB cytochemistry was performed on post-nuclear supernatants of homogenates of cells which w...
Accumulation of unglycosylated liver secretory glycoproteins in the rough endoplasmic reticulum
Biochemical and Biophysical Research Communications, 1989
Previous studies in this laboratory (1) have shown that tunicamycin-treatment inhibits the secretion of three secretory glycoproteins-c~-macroglobulin ceruloplasmin and c~ 1-protease inhibitor in human 2 ' ' hepatoma (Hep G2) cell cultures. In the present study, we have investigated (i) their site of accumulation within the endoplasmic reticulum/Golgi pathway, and (ii) the solubility characteristics of these unglycosylated proteins. Using percoll density gradient centrifugation, we found that tunicamycintreatment markedly inhibited the transport of 0~2-macroglobulin, ceruloplasmin and c~ 1-Pr°tease inhibitor from the rough endoplasmic reticulum. However, there was no detectable changes in their solubility properties as both the glycosylated and unglycosylated species were associated with the 100,000 xg supernatant fraction following disruption of the microsomal fraction (i) with 0.2% Triton X-100 and (ii) by repeated freeze-thaw cycles. Also no evidence of protein aggregation was detected by liquid chromatography of the unglycosylated proteins on Bio-Gel A-1.5 column. ® 1989 Ao~demic Press, ~nc. The function of the carbohydrate moieties of glycoproteins is currently an area of intense investigation (2-8). Among the more established functions include (i) maintenance of physiochemical properties of glycoproteins such as solubility and tertiary conformation, (ii) proteolytic processing and stabilization against proteolysis, (iii) mediation of biologic activity, and (iv) intracellular sorting and pinocytic uptake of lysosomal hydrolases. Recent studies have suggested the direct involvement of N-linked oligosaccharides in the intracellular transport of glycoproteins (7, 9-13). Bauer and coworkers (1) recently reported that the intraceilular transport of three N-linked secretory glycoproteins, c~ 1-protease inhibitor, 0~2-macroglobulin and ceruloplasmin, was inhibited in Tintreated human hepatoma cells (Hep G2); whereas, the intracellular transport of others such as fibronectin, c~-fetoprotein, plasminogen, transferrin and fibrinogen were not affected. Previous studies on the mechanism of Tm-inhibition of deglycosylated protein transport showed that the inhibition was due to decreased solubUity with concomitant intraceilular accumulation of aggregated proteins (14-17). The present study was undertaken to determine (i) whether variability in the effect of Tm on the intracellular ~Presentaddress:BethlsraelHospitai,
The Journal of Cell Biology, 1972
These studies compare the secretory pathways of newly formed rat serum glycoproteins and albumin by studying their submicrosomal localization at early times after the beginning of their synthesis and also by determining the submicrosomal site of incorporation of N-acetylglucosamine, mannose, galactose, and leucine into protein . N-acetylglucosamine, mannose, and galactose were only incorporated in vitro into proteins from membraneattached polysomes and not into proteins from free polysomes . Mannose incorporation occurred in the rough endoplasmic reticulum, was stimulated by puromycin but not by cycloheximide, and 90% of the mannose-labeled protein was bound to the membranes .
The Journal of Cell Biology
Preparations enriched with plasmalemmal, outer mitochondrial, or Golgi complex membranes from rat liver were subfractionated by isopycnic centrifugation, without or after treatment with digitonin, to establish the subcellular distribution of a variety of enzymes . The typical plasmalemmal enzymes 5'-nucleotidase, alkaline phosphodiesterase I, and alkaline phosphatase were markedly shifted by digitonin toward higher densities in all three preparations. Three glycosyltransferases, highly purified in the Golgi fraction, were moderately shifted by digitonin in both this Golgi complex preparation and the microsomal fraction . The outer mitochondrial membrane marker, monoamine oxidase, was not affected by digitonin in the outer mitochondrial membrane preparation, in agreement with its behavior in microsomes . With the exception of NADH cytochrome c reductase (which was concentrated in the outer mitochondrial membrane preparation), typical microsomal enzymes (glucose-6-phosphatase, esterase, and NADPH cytochrome c reductase) displayed low specific activities in the three preparations ; except for part of the glucose-6-phosphatase activity in the plasma membrane preparation, their density distributions were insensitive to digitonin, as they were in microsomes. The influence of digitonin on equilibrium densities was correlated with its morphological effects. Digitonin induced pseudofenestrations in plasma membranes. In Golgi and outer mitochondrial membrane preparations, a few similarly altered membranes were detected in subfractions enriched with 5'-nucleotidase and alkaline phosphodiesterase I . The alterations of Golgi membranes were less obvious and seemingly restricted to some elements in the Golgi preparation . No morphological modification was detected in digitonin-treated outer mitochondrial membranes. These results indicate that each enzyme is associated with the same membrane entity in all membrane preparations and support the view that there is little overlap in the enzymatic equipment of the various types of cytomembranes.
European Journal of Biochemistry, 1980
A method for the subcellular fractionation of rat liver using whole homogenates of rat liver and analytical sucrose density gradient centrifugation is presented. The distributions in thk sucrose gradients of marker enzymes for all organelles have been determined for control homogenates and for homogenates prepared in the presence of selective membrane perturbants. This technique is not subject to potential loss of information inherent in the use of postnuclear supernatants as starting material for fractionation experiments. Particular attention has been paid to the distributions of putative plasma membrane marker enzymes, up to 50% of which may be found in the nuclear pellet. y-Glutamyltransferase has been found to be entirely plasma membrane in location but has a different distribution pattern when compared with other plasma membrane markers. Particulate alkaline phosphatase and alkaline phosphodiesterase are shown to have bimodal distribution, one peak of which is coincident with 5'-nucleotidase. The other peak is coincident with that of the golgi marker, galactosyltransferase, but the membrane structure containing these activities shows characteristics of plasma membrane rather than golgi apparatus.
Journal of Cell Biology, 1991
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 c...