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Tris inhibits both proteolytic and oligosaccharide processing occuring in the Golgi complex in primary culture rat hepatocytes

October 1988
Journal of Biological Chemistry 263(25):12576-83

DOI:10.1016/S0021-9258(18)37794-9

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PubMed

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CC BY 4.0

Authors:

Shigenori Ogata

Fukuoka University

Elizabeth Sumi Yamada

Federal University of Para

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Tris caused the distention of the Golgi cisternae in primary cultured rat hepatocytes and perturbed the functions occurring there. Proteolytic cleavage of precursors of both albumin and complement C3 was inhibited, whereas that of prohaptoglobin was not affected by Tris. These effects on the proteolytic cleavages resemble those of acidotropic amines (Oda, K., and Ikehara, Y. (1985) Eur. J. Biochem. 152, 605-609; Oda, K., Koriyama, Y., Yamada, E., and Ikehara, Y. (1986) Biochem. J. 240, 739-745). However, the effects of Tris significantly differed from acidotropic amines on the basis of its effects on the processing of N-linked oligosaccharides of glycoproteins. Both alpha 1-protease inhibitor and haptoglobin secreted from the Tris-treated cells were found to contain almost equal amounts of endo-beta-N-acetylglucosaminidase H-sensitive and -resistant oligosaccharides, whereas the glycoproteins from both the control and methylamine-treated cells were resistant to the enzyme. The endo-beta-N-acetylglucosaminidase-sensitive oligosaccharides were analyzed to be Man8-5GlcNAc by high resolution gel permeation chromatography, suggesting that trimming of alpha-mannose residues from the precursor Man9GlcNAc2 is incomplete in the Tris-treated cells. On the other hand, Tris did not significantly inhibit incorporation of radioactive monosaccharides (N-acetylglucosamine, galactose, and fucose) into the glycoproteins. However, two-dimensional gel electrophoresis in combination with neuraminidase digestion demonstrated that sialylation was markedly inhibited by Tris. Taken together, our results reveal that Tris inhibits not only the sialic acid addition which takes place in the trans Golgi region, but also the trimming step of high mannose-type oligosaccharides, which is thought to occur before glycoproteins reach the trans Golgi region.

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A class of low‐cost alternatives to kifunensine for increasing high mannose N‐linked glycosylation for monoclonal antibody production in Chinese hamster ovary cells

Article

Full-text available

Sep 2020
BIOTECHNOL PROGR

N‐linked glycosylation of therapeutic monoclonal antibodies is an important product quality attribute for drug safety and efficacy. An increase in the percent of high mannose N‐linked glycosylation may be required for drug efficacy or to match the glycosylation profile of the innovator drug during the development of a biosimilar. In this study, the addition of several chemical additives to a cell culture process resulted in high mannose N‐glycans on monoclonal antibodies produced by Chinese hamster ovary (CHO) cells without impacting cell culture performance. The additives, which include known mannosidase inhibitors (kifunensine and deoxymannojirimycin) as well as novel inhibitors (tris, bis–tris, and 1‐amino‐1‐methyl‐1,3‐propanediol), contain one similar molecular structure: 2‐amino‐1,3‐propanediol, commonly referred to as serinol. The shared chemical structure provides insight into the binding and inhibition of mannosidase in CHO cells. One of the novel inhibitors, tris, is safer compared to kifunensine, 35x as cost‐effective, and stable at room temperature. In addition, tris and bis–tris provide multiple low‐cost alternatives to kifunensine for manipulating glycosylation in monoclonal antibody production in a cell culture process with minimal impact to productivity or cell health.

Effect of novel quercetin pivaloyl ester on functions of adult rat microglia

Article

May 2015

The pathogenic mechanisms involved in the development of ageing-related neurodegenerative diseases can involve alterations of microglia, the brain counterpart of macrophages. These include microglial over-activation, replicative senescence, accumulation of autofluorescent lipofuscin and mitochondrial dysfunction. Substantial evidence suggests that dietary flavonoids are capable to modulate and probably revert the hyperactive and senescence phenotype of these cells. The present study assessed the effect of a novel semisynthetic flavonoid 3'-O-(3-chloropivaloyl)quercetin (CPQ) on the functions of adult rat microglia, isolated secondarily to the establishment of mixed glial cultures and compared it with the effect of the unmodified molecule, quercetin. CPQ suppressed NO release by lipopolysaccharide-stimulated cells more effectively than did quercetin. Unlike quercetin, CPQ inhibited the injury of cell viability due to oxidative challenge and suppressed senescence-associated β-galactosidase staining of microglia isolated from long-term mixed glial cultures. Both flavonoids tested protected the functions of microglia in response to inflammatory stimuli. Furthermore, both compounds protected the isolated microglia from adverse effects of HEPES-buffered media. This was followed by an increase of cell yields, improvement of lysosomal function, suppression of nuclear protein oxidation and inhibition of lipofuscin accumulation (at a slightly more profound effect of CPQ). In conclusion, our data support the experimental evidence suggesting beneficial effects of flavonoids in modulation of neuropathology- and ageing-related alterations of microglia. In this regard, the novel pivaloyl ester of quercetin might represent a new drug with improved potential against neurodegenerative diseases.

Calcium depletion blocks proteolytic cleavages of plasma protein precursors which occur at the Golgi and/or trans-Golgi network. Possible involvement of Ca(2+)-dependent Golgi endoproteases

Article

Full-text available

Sep 1992

Kimimitsu Oda

The effects of calcium depletion on the proteolytic cleavage and secretion of plasma protein precursors were investigated in primary cultured rat hepatocytes and HepG2 cells. When the cells were incubated with A23187, the calcium-specific ionophore, in a medium lacking CaCl2, precursors of serum albumin and the third and fourth components of complement, C3 and C4, respectively, were found to be released into the medium. The addition of ionomycin or EGTA to the medium inhibited the processing of pro-C3 as well. Blocking the secretory pathway either at the mixed endoplasmic reticulum/Golgi in the presence of brefeldin A or at the endoplasmic reticulum/tubular-vesicular structure at a reduced temperature caused accumulation of pro-C3 within hepatocytes or HepG2 cells, indicating that the cleavage of the precursor occurs at a later stage of the secretory pathway. Once the blockade was released by incubating the cells either in the brefeldin A-free medium or at 37 degrees C, the secretion of plasma proteins resumed, irrespective of the presence of A23187. However, the processing of pro-C3 was almost completely inhibited in the presence of A23187, with only the precursor being released into the medium, implying that a decline in Ca2+ levels within the cell modulates the activity of a Golgi endoprotease responsible for the cleavage of pro-C3. When incubated with isolated Golgi membranes, pro-C3 secreted from Ca(2+)-depleted cells was cleaved in vitro into their subunits in the presence of Ca2+ but not in its absence, pointing to the involvement of a Ca(2+)-dependent Golgi endoprotease in the processing of pro-C3. These results collectively suggest that calcium depletion blocks the proteolytic cleavages of plasma protein precursors presumably by exhausting a Ca2+ pool available to the Ca(2+)-dependent processing enzyme(s) located at the Golgi and/or trans-Golgi network.

Environmental Effects on Protein Glycosylation

Article

Jun 1990
Bio Tech

Cultured mammalian cells are being used to produce proteins for therapeutic and diagnostic use because of their ability to perform complex post-translational modifications, including glycosylation. The oligosaccharide moieties can play an important role in defining several biological properties of glycoproteins, including clearance rate, immunogenicity, and biological specific activity. There is a growing interest in defining the factors that influence glycosylation, including the cell culture environment. In this review we organize the published data from in vitro cell culture and tissue culture studies that demonstrate direct effects of the culture environment on N-linked glycosylation.

Evidence for posttranscriptional stimulation of monoclonal secretion by L-glutanime during slow hybridoma growth

Article

Mar 1992
J BIOTECHNOL

The addition of 5-40 mM L-glutamine to batch cultures of a murine hybridoma following the cessation of rapid growth significantly stimulated monoclonal antibody (mAb) synthesis and secretion per cell. Stimulation of mAb secretion following the cessation of rapid growth was also observed in response to addition of mitochondrial intermediates of glutamate oxidation and was not found to be the result of release of transiently stored mAb. Less than 1% of the secreted mAb was detected by ELISA in isolated hybridoma lysosomes. This stimulation was posttranscriptional and not the result of enhancement of levels of mAb mRNAs or stabilization of heavy (H) or light (L) chain encoding message. Sub-inhibitory levels of lysosomotrophic weak bases stimulated release of lysosomal contents but did not result in release of intact or partially degraded mAb. Inhibition of aspartic proteinase activity secreted by the hybridoma did not enhance mAb secretion even though a high level of mAb degrading proteinase activity was continuously secreted during both rapid and slow growth. These responses indicate that during slow growth, the addition of L-glutamine increases the availability of cellular ATP generated by mitochondrial respiration which stimulates some posttranscriptional step in the pathway of mAb secretion such as the rate of H or L chain translation, chain assembly, interorganelle transport or vesicular transport from the Golgi to the cell membrane.

Modulation of the carbohydrate moiety of thyroglobulin by thyrotropin and calcium in Fisher rat thyroid line-5 cells

Article

Full-text available

Feb 1992

Thyroglobulin secreted in the medium by Fisher rat thyroid line-5 (FRTL-5) cells cultured in the presence of thyroid stimulating hormone (TSH) shows a slower electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a higher density position in a CsCl gradient than thyroglobulin secreted by FRTL-5 cells cultured in the absence of TSH for 5-7 days. Such a TSH effect is much less or not evident when secreted thyroglobulin is digested with peptide N-glycohydrolase F or when intracellular thyroglobulin is compared. Intracellular thyroglobulin migrates faster than thyroglobulin secreted either in the presence or in the absence of TSH. Evaluation of the mannose and galactose content of thyroglobulin demonstrates that intracellular thyroglobulin has more mannose and less galactose than extracellular thyroglobulin; it also shows that TSH decreases the mannose content of thyroglobulin while increasing its galactose content. Bio-Gel P6 chromatography shows that TSH increases the complex type carbohydrate chains while decreasing the high mannose chains in the secreted thyroglobulin. High mannose type oligosaccharides were characterized by fast atom bombardment-mass spectrometry analysis. Treatment with the calcium ionophore A23187 (5 microM) of FRTL-5 cells cultured with or without TSH causes the appearance of a "fast" migrating form of thyroglobulinin in the culture medium. Bio-Gel P6 chromatography shows that A23187 causes a dramatic decrease of the complex carbohydrate chains of the secreted thyroglobulin.

Polymerization of secretory IgM in B lymphocytes is prevented by a prior targeting to a degradation pathway

Article

Full-text available

Jan 1993

B lymphocytes express on their surface a membrane form of IgM (mIgM), and synthesize but fail to secrete a secretory form of IgM (sIgM). Plasma cells shift to the exclusive synthesis and efficient secretion of sIgM. The sIgM in B cells differs from that in plasma cells in its pattern of assembly: in plasma cells, monomers of sIgM are assembled into polymers and only polymers are secreted; in B lymphocytes, monomeric sIgM is neither polymerized nor secreted and is degraded intracellularly. In this article we blocked the export of proteins from the endoplasmic reticulum at low temperatures or with energy poisons or brefeldin A, and localized the different assembly steps of mIgM and sIgM in the 38C B lymphocytes and of sIgM in the 38C-derived sIgM-secreting D2 hybridoma. In both cell lines, sIgM assembly into monomers was not affected, whereas polymerization of sIgM in D2 cells and monomer formation of mIgM in 38C cells were strongly inhibited. Moreover, probing with specific lectins revealed galactosylated monomers and polymers in D2 cells and galactosylated hemimer and monomers only of mIgM in 38C cells. In addition, when Golgi functions were hampered with Tris base, monomerization of mIgM and polymerization of sIgM were attenuated. These results indicate that polymerization of sIgM in D2 cells and monomerization of mIgM in 38C cells are post-endoplasmic reticulum events, occurring in or beyond the trans-Golgi galactosylation compartment. Since only polymers are secreted from D2 cells and only monomeric mIgM is displayed on the surface of 38C cells, partially assembled molecules may traverse the secretory pathway yet are restricted from the cell surface. Furthermore, monomeric sIgM in 38C cells is never galactosylated, thus it is degraded prior to the galactosylation compartment. We conclude that targeting of sIgM to degradation in 38C cells precedes its assembly site into polymers in D2 cells. This implies that degradation of sIgM does not result from the incompetence of 38C cells to polymerize. Rather, assembly of sIgM into polymers and their subsequent secretion are prevented in B lymphocytes by preceding targeting of monomeric sIgM to degradation.

Intracellular processing of apolipoprotein J precursor to mature heterodimer

Article

Full-text available

Jul 1991

Circulating apolipoprotein J (apoJ) is a 70 kDa glycoprotein comprised of disulfide-linked alpha and beta subunits derived from a single precursor. Post-translational modifications that occur prior to apoJ secretion were assessed, with specific focus on carbohydrate type, the timing of proteolytic cleavage, and the importance of glycosylation on the cleavage and secretion processes. ApoJ was initially resolved as a single chain, intracellular precursor of 58 kDa which contained N-linked oligosaccharide but no O-linked oligosaccharide. The precursor was converted to an intracellular 70 kDa glycoprotein, which became the major intracellular form of apoJ prior to secretion. Maturation of the 58 kDa precursor involved conversion of high-mannose carbohydrate to complex-type carbohydrate containing sialic acid, as well as intracellular cleavage to yield alpha and beta subunits. This cleavage event occurred at a late stage of carbohydrate modification, most likely in the trans-Golgi or a post-Golgi compartment. The maturation and secretion of apoJ occurred rapidly, with a half-time of 30-35 min. Tunicamycin treatment of cells resulted in an unglycosylated doublet comprised of one single chain and one cleaved form of apoJ. The unglycosylated apoJ species were secreted rapidly with a half-time of 20 min. Both cleavage and secretion were independent of glycosylation.

Balilomycin A1inhibits the targeting of lysosomal acid hydrolases in cultured hepatocytes

Article

Aug 1991

Effects of bafilomycin A1, an inhibitor of vacuolar H(+)-ATPase, on the synthesis and processing of cathepsin D and cathepsin H were investigated in primary cultured rat hepatocytes. Pulse-chase experiments showed that after being synthesized as procathepsin D and procathepsin H the precursors were converted into mature forms in the control cells as the chase time elapsed. However, in the presence of 5 x 10(-7) M of bafilomycin A1, both precursors were largely secreted into the medium and no mature forms were found within the cells. Thus bafilomycin A1 mimics lysosomotropic amines with regard to perturbation of the targeting of lysosomal acid hydrolases. In contrast, bafilomycin A1 was found not to inhibit processings of proalbumin and procomplement component 3, which are thought to occur at the acidic trans-Golgi, implying that the proteolytic event of the proproteins is not sensitive to an increase of intra-Golgi pH. The results suggest that bafilomycin A1 is useful as a pH-perturbant to study the role of acidity in living cells.

Brefeldin A arrests the intracellular transport of viral envelope proteins in primary cultured rat hepatocytes and HepG2 cells

Article

Feb 1990

We have studied the effect of brefeldin A (BFA) on the intracellular transport of the envelope proteins of vesicular stomatitis virus (VSV) and sindbis virus in primary cultured rat hepatocytes. BFA (2.5 micrograms/ml) inhibited not only the secretion of plasma proteins into the medium, but also the assembly of both G protein of VSV and E1 and E2 proteins (envelope proteins) of sindbis virus into respective virions. Concomitantly, both the acquisition of endo-beta-N-acetylglucosaminidase H resistance by the G protein and the proteolytic conversion of PE2 to E2 were found to be inhibited in the BFA-treated cells, suggesting that the intracellular transport of the envelope proteins was arrested in the endoplasmic reticulum. Such inhibitory effects of the drug were variable depending upon the culture conditions of the hepatocytes. In the 1-day-cultured cells, even in the presence of the drug, newly synthesized envelope proteins were assembled into the virions after a 3 h chase period, at the same time as secretion of plasma proteins into the medium resumes. In contrast, in 4-day-cultured hepatocytes, BFA continuously blocked the entry of the envelope proteins into the virions and the release of plasma proteins into the medium for at least 5 h. BFA also completely inhibited the exocytotic pathway in HepG2 cells. These results indicate that the duration time of the effect of BFA is different from one cell to another and may change depending upon the culture conditions of the cells.

Novel blockade by brefeldin A of intracellular transport of secretory proteins in cultured rat hepatocytes.

Article

Full-text available

Aug 1986

We examined the effect of brefeldin A, an antiviral antibiotic, on protein synthesis, intracellular processing, and secretion in primary culture of rat hepatocytes. The secretion was strongly blocked by the drug at 1 microgram/ml and higher concentrations, while the protein synthesis was maintained fairly well. Pulse-chase experiments with [35S]methionine demonstrated that brefeldin A completely blocked the proteolytic conversion of proalbumin to serum albumin up to 60 min of chase, although its conversion was observed as early as 20 min in the control cells. The drug also inhibited the terminal glycosylation of oligosaccharide chains of alpha 1-protease inhibitor and haptoglobin. These two modifications have been shown to occur at the trans region of the Golgi complex. The drug, however, had no effect on the proteolytic processing of the haptoglobin proform which takes place within the endoplasmic reticulum. Such an effect by brefeldin A is very similar with that induced by the carboxylic ionophore monensin. However, in contrast to evidence that monensin causes a delayed secretion of the unprocessed forms of these proteins, brefeldin A allowed the completely processed forms to be secreted after a prolonged accumulation of the unprocessed forms. Morphological observations demonstrated that the endoplasmic reticulum was markedly dilated by treatment with the drug at 10 micrograms/ml which continuously blocked the secretion. On the other hand, brefeldin A caused no inhibitory effect on the endocytic pathway as judged by cellular uptake and degradation of 125I-asialofetuin. These results indicate that brefeldin A is a unique agent which primarily impedes protein transport from the endoplasmic reticulum to the Golgi complex by a mechanism different from those considered for other secretion-blocking agents so far reported.

Immunoblotting Analysis of Plasma Protein Processing in the Secretory Pathway of Rat liver: Identification of Proteolytic Conversion Sites of Complement Pro-C3 and Prohaptoglobin1

Article

Oct 1986

Using an immunoblotting technique, we have studied the processing of plasma proteins in subcellular fractions of rat liver including rough and smooth micro-somes and the Golgi subtractions. Each subcellular fraction was directly subjected to SDS-polyacrylamide gel electrophoresis and analyzed by immunoblotting with antibodies against α1-protease inhibitor, haptoglobin, and the third component of complement (C3) in combination with ¹²⁵I-protein A or ¹²⁵I-rabbit anti-(goat IgG)-IgG. The results demonstrated that proteolytic processing of precursors of complement C3 and haptoglobin occurs in different compartments along the secretory pathway; conversion of prohaptoglobin takes place in the endoplasmic reticulum, while that of pro-C3 occurs in the Golgi complex. The processing in oligosaccharide chains of glycoproteins was also analyzed. The Golgi fraction was characterized by the presence of the mature 56 kDa α1-protease inhibitor, which was indistinguishable from the serum α1-protease inhibitor in SDS-polyacrylamide gel electrophoresis. In contrast, the immature 51 kDa form was the only form of α1-protease inhibitor found in the microsomal fraction. Similar results were obtained for the β subunit of haptoglobin; the immature 33 kDa form was detected in the microsomal fraction, while the mature 36 kDa form was found in the Golgi fraction. Taken together, these results identified the intracellular sites where these plasma proteins are modified by selective proteolysis and/or glycosylation.

Possible pathways for lysosomal enzyme delivery

Article

Dec 1985

H. J. Geuze

Immunogold double-labeling and ultrathin cryosections were used to compare the subcellular distribution of albumin, mannose 6-phosphate receptor (MPR), galactosyltransferase, and the lysosomal enzymes cathepsin D, beta-hexosaminidase, and alpha-glucosidase in Hep G2 cells. MPR and lysosomal enzymes were found throughout the stack of Golgi cisternae and in a trans-Golgi reticulum (TGR) of smooth-surfaced tubules with coated buds and vesicles. The trans-Golgi orientation of TGR was ascertained by the co-localization with galactosyltransferase. MPR was particularly abundant in TGR and CURL, the compartment of uncoupling receptors and ligands. Both TGR and CURL also contained lysosomal enzymes, but endogenous albumin was detected in TGR only. The coated buds on TGR tubules contained MPR, lysosomal enzymes, as well as albumin. MPR and lysosomal enzymes were also found in coated pits of the plasma membrane. CURL tubules seemed to give rise to smooth vesicles, often of the multivesicular body type. In CURL, the enzymes were found in the lumina of the smooth vesicles while MPR prevailed in the tubules. These observations suggest a role of CURL in transport of lysosomal enzymes to lysosomes. When the cells were treated with the lysosomotropic amine primaquine, binding of anti-MPR to the cells in culture was reduced by half. Immunocytochemistry showed that MPR accumulated in TGR, especially in coated buds. Since these buds contain endogenous albumin and lysosomal enzymes also, these data suggest that coated vesicles originating from TGR provide for a secretory route in Hep G2 cells and that this pathway is followed by the MPR system as well.

In vivo effect of colchicine on hepatic protein synthesis and on the conversion of proalbumin to serum albumin

Article

May 1978

C. M. Redman

Treatment of rats with 0.5-25 mumol/100 g body weight of colchicine for 1 h or more caused an inhibition of hepatic protein synthesis. This effect was not seen if animals were exposed to colchicine for less than 1 h. The delayed inhibition of protein synthesis affected both secretory and nonsecretory proteins. Treatment with colchicine (15 mumol/100 g) for 1 h or more caused the RNA content of membrane-bound polysomes to fall but did not change the polysomal profile of this fraction. By contrast, the total RNA content in the free polysome cell fraction was increased, and this was due to the presence of more ribosomal monomers and dimers. Electron microscope examination of the livers from rats treated for 3 h with colchicine showed an accumulation of secretory vesicles within the hepatocytes and a general distention of the endoplasmic reticulum. Administration of radioactive L-leucine to the rats led to an incorporation of radioactivity into two forms of intracellular albumin which were precipitable with antiserum to rat serum albumin but which were separable by diethylaminoethyl-cellulose chromatography. One form has arginine at the amino-terminal position and is proalbumin, and the other form, which more closely resembles serum albumin chromatographically, has glutamic acid at its amino terminus. Only proalbumin was found in rough and smooth endoplasmic reticulum fractions and in a Golgi cell fraction wich corresponds morphologically to mostly empty and partially filled secretory vesicles. However, in other Golgi cell fractions which were filled with secretory products, both radioactive proalbumin and serum albumin were found. This indicates that proalbumin is converted to serum albumin in these secretory vesicles just before exocytosis. Colchicine delayed the discharge of radioactive albumin from these filled secretory vesicles and caused an accumulation of both proalbumin and serum albumin within these cell fractions.

Effect of lysosomotropic amines on the secretory pathway and on the recycling of the asialoglycoprotein receptor in human hepatoma cells

Article

Aug 1985

Ger J Strous

We studied the intracellular transport of secretory and membrane proteins in the human hepatoma cell line HepG-2 infected with vesicular stomatitis virus. Cells were pulse-labeled in the presence of [35S]methionine and chased in the presence of the lysosomotropic agent primaquine. At a concentration of 0.3 mM primaquine effectively inhibited the secretion of albumin and, to a lesser extent, that of orosomucoid and transferrin. The drug also prevented the budding of virus particles at the cell surface. The intracellular transport to the Golgi complex of the membrane protein VSV-G was not affected by primaquine as it acquires resistance to endo-beta-N-acetylglucosaminidase H at the same rate as in control cells. Addition of primaquine at various times after the initiation of the chase period indicates that the effect of primaquine occurs just before secretion. In confirmation of the biochemical data, immunocytochemical localization of albumin in cells treated with NH4Cl demonstrated that albumin accumulated in vesicles at the trans side of the Golgi complex. The effect of primaquine on secretion was also compared with its effect on receptor recycling. The dose-response characteristics of the effect of primaquine on receptor recycling are identical to those of the effects on protein secretion and virus budding. These results indicate that both processes involve the same transport mechanism, and/or that they occur via at least one identical intracellular compartment.

Golgi membranes contain an electrogenic H+ pump in parallel to a chloride conductance

Article

Oct 1983

Jonathan N. Glickman

Rat liver Golgi vesicles were isolated by differential and density gradient centrifugation. A fraction enriched in galactosyl transferase and depleted in plasma membrane, mitochondrial, endoplasmic reticulum, and lysosomal markers was found to contain an ATP-dependent H+ pump. This proton pump was not inhibited by oligomycin but was sensitive to N-ethyl maleimide, which distinguishes it from the F0-F1 ATPase of mitochondria. GTP did not induce transport, unlike the lysosomal H+ pump. The pump was not dependent on the presence of potassium nor was it inhibited by vanadate, two of the characteristics of the gastric H+ ATPase. Addition of ATP generated a membrane potential that drove chloride uptake into the vesicles, suggesting that Golgi membranes contain a chloride conductance in parallel to an electrogenic proton pump. These results demonstrate that Golgi vesicles can form a pH difference and a membrane potential through the action of an electrogenic proton translocating ATPase.

Attachment of terminal N-acetylglucosamine to asparagine-linked oligosaccharides occurs in central cisternae of the golgi stack

Article

Feb 1985
CELL

W G Dunphy

Using monoclonal antibodies and electron microscopy, we have localized N-acetylglucosamine transferase I within the Golgi apparatus. This enzyme initiates the conversion of asparagine-linked oligosaccharides to the complex type. We have found that the enzyme is concentrated in the central (or medial ) cisternae of the Golgi stack. Cisternae at the cis and trans ends of the Golgi complex appear to lack this protein. These experiments establish a function for the medial portion of the Golgi and imply that the Golgi is partitioned into at least three biochemically and morphologically distinct cisternal compartments.

Initial glycosylation and acidic pH in the Golgi apparatus are required for multimerization of von Willebrand factor

Article

Apr 1986

Denisa D Wagner

Two conditions were identified that interfered with the complex polymerization process in biosynthesis of von Willebrand factor (vWf). Treatment of human umbilical vein endothelial cells with tunicamycin inhibited N-linked glycosylation of nascent vWf and the resulting pro-vWf monomers failed to dimerize. The single subunits accumulated in the endoplasmic reticulum and were neither processed further nor secreted. In the presence of a weak base (ammonium chloride or chloroquine), interdimer disulfide bond formation was inhibited in a dose-dependent manner. This process appeared therefore to be pH sensitive and likely to be initiated in the acidic trans-Golgi apparatus (Anderson, R. G. W., and R. K. Pathak, 1985, Cell, 40: 635-643). The weak base had no obvious effect on the other processing steps, i.e. dimerization, complex carbohydrate formation and sulfation, and produced only slight inhibition of prosequence cleavage. On the other hand, the weak base interfered with the targeting of newly synthesized vWf into Weibel-Palade bodies, with all of the vWf being secreted constitutively and none stored in the Weibel-Palade bodies. In summary, initial glycosylation of the nascent vWf protein and low pH in the trans-Golgi apparatus were important conditions for the successful polymerization of human vWf. Genetic defects disrupting any one of these conditions could result in the phenotype of von Willebrand disease.

Demonstration of an extensive trans-tubular network continuous with the Golgi apparatus stack that may function in glycosylation

Article

Nov 1985
CELL

Jürgen Roth

Sialyltransferase (Galβ1,4GlcNAc α2,6 sialyltransferase) was localized by immunoelectron microscopy in rat liver hepatocytes using affinity-purified antibodies. Immunoreactivity for sialyltransferase was found in the Golgi apparatus, where it was restricted to an interconnected system consisting of the trans-cisternae and the trans-tubular network. This region of the Golgi apparatus exhibited both TPPase and CMPase activity and was the intracellular site where sialic acid residues bound to glycoprotein were detected using the Limax flavus lectin. Sialyltransferase and sialic acid residues were not detected in medial and cis-cisternae of the Golgi apparatus. These findings suggest that in rat hepatocytes sialylation of N-linked glycoproteins occurs in the complex formed by the trans-cisternae and the trans-tubular network of Golgi apparatus.

Intercompartmental transport in the Golgi complex is a dissociative process: facile transfer of membrane protein between two Golgi populations

Article

Jul 1984

J. E. Rothman

The transfer of the vesicular stomatitis virus-encoded glycoprotein (G protein) between Golgi populations in fused cells (Rothman, J. E., L. J. Urbani, and R. Brands. 1984. J. Cell Biol. 99:248-259) is exploited here to study and to help define the compartmental organization of the Golgi stack and to characterize the mechanism of intercompartmental transport. We find that G protein that has just received its peripheral N-acetylglucosamine in the Golgi complex of one cell is efficiently transferred to the Golgi complex of another cell to receive galactose (Gal). Remarkably, this transport occurs at the same rate between these two compartments whether they are present in the same or different Golgi populations. Therefore, a dissociative (presumably vesicular) transport step moves G protein from one part of the Golgi in which N-acetylglucosamine is added to another in which Gal is added. Minutes later, upon receiving Gal, the same G protein molecules are very poorly transferred to an exogenous Golgi population after cell fusion. Therefore, once this intercompartmental transfer has already taken place (before fusion), it cannot take place again (after fusion); i.e., transport across the compartment boundary in the Golgi complex that separates the sites of N-acetylglucosamine and Gal incorporation is a vectorial process. We conclude that transfers between Golgi cisternae occur by a stochastic process in which transport vesicles budding from cisternae dissociate, can diffuse away, and then attach to and fuse with the appropriate target cisterna residing in the same or in a different stack, based on a biochemical pairing after a random encounter. Under these circumstances, a transported protein would almost always randomize among stacks with each intercisternal transfer; it would not progress systematically through a single stack. Altogether, our studies define three sequential compartments in the Golgi stack.

Tris inhibits both proteolytic and oligosaccharide processing occuring in the Golgi complex in primary culture rat hepatocytes

Abstract

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