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PubMed2003

Major mutations in calf-1 and calf-2 domains of glycoprotein IIb in patients with Glanzmann thrombasthenia enable GPIIb/IIIa complex formation, but impair its transport from the endoplasmic reticulum to the Golgi apparatus.

The crystal structure of integrin alphavbeta3 comprises 3 regions of contact between alphav and beta3. The main contact on alphav is located in the beta-propeller while calf-1 and calf-2 domains contribute minor interfaces. Whether or not contacts between calf-1 and calf-2 domains of glycoprotein (GP) IIb (alphaIIb) and GPIIIa (beta3) play a role in GPIIb/IIIa complex formation has not been established. In this study we analyzed the effects of 2 naturally occurring mutations in calf-1 and calf-2 domains on GPIIb/IIIa complex formation, its processing, and transport to the cell membrane. The mutations investigated were a deletion-insertion in exon 25 located in calf-2 and an in-frame skipping of exon 20 located in calf-1. Mutated GPIIb cDNAs were cotransfected in baby hamster kidney cells with normal GPIIIa (beta3) cDNA. Analysis by flow cytometry failed to demonstrate detectable amounts of GPIIb or GPIIb/IIIa complex on the surface of cells transfected with each mutation, but immunohistochemical staining revealed their intracellular presence. GPIIb was mainly demonstrable as pro-GPIIb by immunoprecipitation of cell lysates expressing each mutation. Differential immunofluorescence staining of GPIIb and cellular organelles suggested that most altered complexes were located in the endoplasmic reticulum. Homology modeling of normal GPIIb based on the alphavbeta3 crystal structure revealed similar contacts between alphav and beta3 and between alphaIIb and beta3. Introduction of the mutations into the model yielded partial disruption of the normal contacts in the corresponding domains. These data suggest that despite partial disruption of calf-1 or calf-2 domain, GPIIb/IIIa complex is formed but its transport from the endoplasmic reticulum is impaired.

Animals↗
PubMed1985

UDP-GlcNAc:Glycoprotein GlcNAc-Transferase is Located in the Golgi Apparatus of Developing Bean Cotyledons.

The transport and accumulation of phytohemagglutinin in developing bean (Phaseolus vulgaris L.) cotyledons is accompanied by the transient presence of N-acetylglucosamine (GlcNAc) residues on the oligosaccharide sidechains of this glycoprotein. These peripheral GlcNAc residues can be distinguished from those in the chitobiose portion of the oligosaccharide sidechains by their sensitivity to removal by the exoglycosidase beta-N-acetylglucosaminidase. GlcNAc residues sensitive to removal by beta-N-acetylglucosaminidase are present not only on phytohemagglutinin, but also on other newly synthesized proteins. The enzyme UDPGlcNAc:glycoprotein GlcNAc-transferase which transfers GlcNAc residues to glycoproteins was first described by Davies and Delmer (Plant Physiol 1981 68: 284-291). The data presented here show that this enzyme is associated with the Golgi complex of developing cotyledons.

Journal Article↗
PubMed1972

Distribution and activation of the Golgi apparatus in geotropism.

We find a differential distribution of dictyosomes in the avascular tip cells of the oat (Avena sativa) coleoptile upon geostimulation. A differential activation (increased vesicle production) of the dictyosomes with respect to gravity also occurs, but only in the tip cells of the lower tissues. Similar differences in distribution and activation of dictyosomes occur also in cells subjacent to the avascular tip (1st and 5th millimeter from the apex) of both the upper and lower half-tissues. When only the outer epidermal cells below the apex are considered, the differential distribution and activation of dictyosomes occur only in the lower outer epidermis. The changes in distribution of dictyosomes begin at 6 minutes, or sooner, from the start of geostimulation, before the onset of geotropism. The kinetics of amyloplast sedimentation and Golgi movement do not appear to differ in the cells of the avascular tip. We suggest that the Golgi participates in, and possibly initiates, the differential elongation of cells of geotropically stimulated coleoptiles.

Journal Article↗
PubMed1992

Glycosylation of CD45: carbohydrate processing through Golgi apparatus is required for cell surface expression and protein stability.

The importance of glycosylation on cell surface expression, protein stability and function of the CD45 phosphotyrosine phosphatase leukocyte common antigen, has been studied in the K562 erythroleukemic cell line. Cell treatment with the N-glycosylation inhibitor tunicamycin generated unglycosylated CD45 polypeptides of 130 and 140 kDa. Immunofluorescence flow cytometry and Scatchard techniques revealed that CD45 cell surface expression was decreased in a time- and dose-dependent manner upon tunicamycin incubation. Moreover, a remarkable decrease in CD45 phosphatase activity was detected in tunicamycin-treated cells, which correlated with the diminished CD45 cell surface expression. Pulse-chase biosynthetic assays demonstrated that the fate of the unglycosylated CD45 proteins underwent a late non-lysosomal degradation, since it was not prevented either by monensin, an inhibitor of Golgi transport, or by the lysosomal function inhibitor chloroquine. These results demonstrate that intact glycosylation of CD45 molecules through Golgi compartment is required for stability and proper transport towards the plasma membrane of these phosphotyrosine phosphatase proteins.

Antigens, CD↗
PubMed1992

Evidence that globular Golgi clusters in mitotic HeLa cells are clustered tubular endosomes.

By conventional electron microscopy we observed in mitotic HeLa cells the structures termed Golgi clusters by Lucocq et al. (J. Cell Biol. 104, 865-874 (1987)) and interpreted by them as clusters of vesicular remnants of the Golgi apparatus. Golgi clusters consist of tubular and vesicular profiles about 50 nm in diameter, sometimes associated with larger 250 nm vesicles. When cultures of HeLa cells were incubated for 60 min or 120 min with medium containing high specific activity horseradish peroxidase (HRP) at 10 mg/ml we found that the membrane-bound compartments in the Golgi clusters in mitotic cells contained heavy deposits of HRP reaction product. Neither interphase nor mitotic HeLa cells contain an endogenous peroxidase activity. We concluded that Golgi clusters are an endocytic compartment and confirmed this by showing that Golgi clusters could be labeled with two other endocytic tracers--bovine serum albumin conjugated to colloidal gold and transferrin conjugated to HRP. When cultures were incubated with HRP for only 15 min most of the Golgi clusters in the mitotic cells were either unlabeled or consisted of a mixture of HRP-labeled and unlabeled profiles. Since during mitosis endocytosis is inhibited this was the expected result. When interphase HeLa cells were incubated with Brefeldin A (BFA), the Golgi apparatus disassembled and immunofluorescence microscopy showed that 1,4 beta galactosyltransferase had relocated to the endoplasmic reticulum. When cells in the presence of BFA and lacking the Golgi apparatus were allowed to endocytose HRP and then entered mitosis, typical HRP-labeled Golgi clusters were seen in the mitotic cells. It is therefore highly unlikely that these structures contain membrane derived from the Golgi cisternae that are sensitive to BFA, including in HeLa cells those containing galactosyltransferase. Finally, we found that interphase HeLa cells incubated with okadaic acid contain structures that are morphologically indistinguishable from Golgi clusters but can be labeled by endocytic tracer. Taken together, this evidence indicates that most, if not all, of the membrane-bound compartments in Golgi clusters are tubular early endosomes.

Brefeldin A↗
PubMed1982

The internal reticular apparatus of Camillo Golgi: a complex, heterogeneous organelle, enriched in acid, neutral, and alkaline phosphatases, and involved in glycosylation, secretion, membrane flow, lysosome formation, and intracellular digestion.

Its topography is one of the most characteristic features of the Golgi apparatus and the reticular nature of this organelle is evident in Golgi's first drawings, in light microscopic enzyme cytochemical preparations, and in high voltage electron micrographs of thick sections. Although individual components of the Golgi apparatus may differ in staining characteristics, morphology, contents, and enzymatic activities, they are integrated into a dynamic topographical and functional unit that is closely associated with the endoplasmic reticulum. Modulation of enzymatic activities and morphological and enzymatic heterogeneity are not surprising in an organelle that is the site of both synthetic and digestive events, including glycosylation, sulfation, formation of secretory granules and lysosomes, and the degradation of endocytized material.

Acid Phosphatase↗
PubMed1985

Acyltransferase and acid hydrolase activities of the abalone photoreceptor cell.

In order to study the synthesis and degradation processes of the photoreceptor membranes in the abalone, Nordotis discus, the localization of acyltransferase and acid hydrolase activities, respectively, were determined at the electron-microscopic level. Acyltransferase activity was localized on the cytoplasmic sides of thick (greater than 10 nm) membranes of the following organelles: a few cisternae at the trans (or concave) side of Golgi apparatus, Golgi and probably related vesicles, short tubules, curved pentalaminar disks and limiting membranes of the phagosomal multivesicular bodies; all organelles were scattered in the peri- to supranuclear cytoplasm. The phospholipids, which are major components of the photoreceptor membrane, are considered to be synthesized by these membranes. Acid phosphatase activity was localized in the lumina of Golgi cisternae and vesicles, lysosomes, and smaller multivesicular and related bodies, but not in multilamellar bodies. The matrices of the larger multivesicular bodies and of the pigment granule complexes showed arylsulfatase activity. Vesiculated and autophagocytosed photoreceptor microvilli seemed to be degraded by acid hydrolases, forming multivesicular and related bodies. Supporting cells also showed acyltransferase and acid hydrolase activities.

Acyltransferases↗
PubMed1994

Imaging of total intracellular calcium and calcium influx and efflux in individual resting and stimulated tumor mast cells using ion microscopy.

Ion microscopy was employed to investigate intracellular total calcium concentrations and calcium influx, and efflux in resting and antigen-stimulated tumor mast cells (RBL-2H3 cells). The nucleus, a perinuclear region which included the Golgi apparatus (Golgi region), and the remaining cytoplasm were spatially resolved with the Cameca IMS-3f ion microscope in cryogenically prepared cells. In resting cells the nucleus contained about 0.60 mM, the Golgi region about 1.2 mM, and the remaining cytoplasm about 1.0 mM total calcium. Antigen stimulation of rat basophilic leukemia cells resulted in a significant loading of calcium in all three cellular compartments. Antigen stimulation in the absence of extracellular calcium resulted in a significant loss of total calcium from all three intracellular compartments. Influx and efflux of calcium were measured simultaneously in resting and stimulated cells by using stable 44Ca in the extracellular solution, and by imaging mass 40 to determine the native intracellular calcium (40Ca) and mass 44 to localize the 44Ca that entered the cell from extracellular solution. After a 10-min incubation, 0.240 fmol of the total calcium per cell had been replaced with 44Ca, which amounts to about 33% of the total cell calcium. If antigen was present during this incubation there was an additional loss of 0.229 fmol of 40Ca and an added gain of 0.476 fmol of 44Ca per cell, which corresponds to a net increase in total intracellular calcium of 0.247 fmol.

Animals↗
PubMed1988

The subcellular localization of apomucin and nonreducing terminal N-acetylgalactosamine in porcine submaxillary glands.

Antibodies prepared against enzymatically deglycosylated porcine submaxillary gland mucin (apomucin), which were unreactive with native mucin and its partially deglycosylated derivatives, were used to immunolocalize apomucin in situ. Electron microscopy of sections of Lowicryl K4M-embedded tissue reacted successively with antibodies and protein A-gold complexes showed apomucin exclusively in mucous cells within the rough endoplasmic reticulum, transitional elements of the endoplasmic reticulum, and vesicles at the cis side of the Golgi apparatus. The Golgi apparatus, forming mucous droplets, and mucous droplets contained no apomucin. Although the rough endoplasmic reticulum contained most of the apomucin in mucous cells, some cisternae of the endoplasmic reticulum and the nuclear envelope were devoid of apomucin. Examination of tissue sections treated with the glycosidases used to prepare apomucin revealed immunolabel for apomucin throughout the secretory pathway. Colloidal gold coated with Helix pomatia lectin was used to detect nonreducing N-acetylgalactosamine residues. In mucin-producing cells lectin-gold was found in the mucous droplets, the forming mucous droplets, and throughout the Golgi apparatus but mostly in the cis portion of this organelle. In tissue sections reacted successively with lectin-gold and anti-apomucin/protein A-gold, both types of gold complex could be found in the cis side of the Golgi apparatus. These data indicate that the O-glycosylation of mucin is a posttranslational event that occurs in the Golgi apparatus and begins in the cis side of the Golgi apparatus.

Acetylgalactosamine↗
PubMed2001

Age-related changes in rat cerebellar basket cells: a quantitative study using unbiased stereological methods.

Cortical cerebellar basket cells are stable postmitotic cells; hence, they are liable to endure age-related changes. Since the cerebellum is a vital organ for the postural control, equilibrium and motor coordination, we aimed to determine the quantitative morphological changes in those interneurons with the ageing process, using unbiased techniques. Material from the cerebellar cortex (Crus I and Crus II) was collected from female rats aged 2, 6, 9, 12, 15, 18, 21 and 24 mo (5 animals per each age group), fixed by intracardiac perfusion, and processed for transmission electron microscopy, using conventional techniques. Serial semithin sections were obtained (5 blocks from each rat), enabling the determination of the number-weighted mean nuclear volume (by the nucleator method). On ultrathin sections, 25 cell profiles from each animal were photographed. The volume density of the nucleus, ground substance, mitochondria, Golgi apparatus (Golgi) and dense bodies (DB), and the mean surface density of the rough endoplasmic reticulum (RER) were determined, by point counting, using a morphometric grid. The mean total volumes of the soma and organelles and the mean total surface area of the RER [SN (RER)] were then calculated. The results were analysed with 1-way ANOVA; posthoc pairwise comparisons of group means were performed using the Newman-Keuls test. The relation between age and each of the parameters was studied by regression analysis. Significant age-related changes were observed for the mean volumes of the soma, ground substance, Golgi, DB, and SN (RER). Positive linear trends were found for the mean volumes of the ground substance, Golgi, and DB; a negative linear trend was found for the SN (RER). These results indicate that rat cerebellar basket cells endure important age-related changes. The significant decrease in the SN (RER) may be responsible for a reduction in the rate of protein synthesis. Additionally, it may be implicated in a cascade of events leading to cell damage due to the excitotoxic activity of glutamate, which could interfere in the functioning of the complex cerebellar neuronal network.

Aging↗
PubMed2006

Retrograde transport on the COG railway.

The conserved oligomeric Golgi (COG) complex is essential for establishing and/or maintaining the structure and function of the Golgi apparatus. The Golgi apparatus, in turn, has a central role in protein sorting and glycosylation within the eukaryotic secretory pathway. As a consequence, COG mutations can give rise to human genetic diseases known as congenital disorders of glycosylation. We review recent results from studies of yeast, worm, fly and mammalian COG that provide evidence that COG might function in retrograde vesicular trafficking within the Golgi apparatus. This hypothesis explains the impact of COG mutations by postulating that they impair the retrograde flow of resident Golgi proteins needed to maintain normal Golgi structure and function.

Adaptor Proteins, Vesicular Transport↗
PubMed1971

Isolation of a Golgi apparatus-rich fraction from rat liver. IV. Thiamine pyrophosphatase.

The thiamine pyrophosphatase (the enzyme [s] catalyzing the release of inorganic phosphate with thiamine pyrophosphate as the substrate) activities of Golgi apparatus-, plasma membrane-, endoplasmic reticulum-, and mitochondria-rich fractions from rat liver were compared at pH 8. Activity was concentrated in the Golgi apparatus fractions, which, on a protein basis, had a specific activity six to eight times that of the total homogenates or purified endoplasmic reticulum fractions. However, only 1-3% of the total activity was recovered in the Golgi apparatus fractions under conditions where 30-50% of the UDPgalactose:N-acetylglucosamine-galactosyl transferase activity was recovered. Considering both recovery of galactosyl transferase and fraction purity, we estimate that approximately 10% of the total thiamine pyrophosphatase activity of the liver was localized within the Golgi apparatus, with a specific activity of about ten times that of the total homogenate. Cytochemically, reaction product was found in the cisternae of the endoplasmic reticulum as well as in the Golgi apparatus. This is in contrast to results obtained in most other tissues, where reaction product was restricted to the Golgi apparatus. Thus, enzymes of rat liver catalyzing the hydrolysis of thiamine pyrophosphate, although concentrated in the Golgi apparatus, are widely distributed among other cell components in this tissue.

Animals↗
PubMed1976

Synthesis in vitro of intrinsic membrane proteins by free, membrane-bound, and Golgi apparatus-associated polyribosomes from rat liver.

A fraction of intrinsic membrane proteins was prepared from the major membranous cell components of rat liver by extraction of the membranes with KCl and deoxycholate. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the compositions of the intrinsic protein fractions from rough and endoplasmic reticulum, smooth endoplasmic reticulum. Golgi apparatus, plasma membrane, and nuclear envelope were similar to each other but distinct from that of mitochondria. Among endomembranes, differences were in the ratios of protein constituents plus a few protein bands of Golgi apparatus and plasma membranes not found in endoplasmic reticulum or nuclear envelope. The abilities of total rough endoplasmic reticulum, polysomes released from rough endoplasmic reticulum, and free polysomes to incorporate amino acids into the intrinsic protein fraction were tested in vitro. Polysomes bound to endoplasmic reticulum has the greatest capacity to synthesize proteins of this fraction as shown by co-purification of radioactive products and by immunoprecipitation. Although the majority of the radioactive products synthesized by bound polysomes were distinct from those synthesized by free polysomes, certain radioactive products synthesized by free polysomes also co-purified with intrinsic membrane proteins. The results show no absolute segregation between free and bound polysomes in the synthesis of intrinsic membrane proteins. However, the majority of these proteins appear to be synthesized by polysomes bound to the endoplasmic reticulum. Several intrinsic proteins found in plasma membranes do not appear in rough endoplasmic reticulum. To determine where these proteins were synthesized, the ability of other endomembrane components to support in vitro incorporation of [14C]leucine into protein was examined. In contrast to plasma membranes, isolated Golgi apparatus fractions did incorporate [14C]leucine to an extent greater than could be explained by contamination with rough endoplasmic reticulum. Golgi apparatus in situ and isolated from rat liver have polyribosomes associated with a zone of cytoplasm at the Golgi apparatus periphery occupied by tubules and vesicles. The polysomes are not directly attached to membranes as with rough endoplasmic reticulum and may represent a special class of "Golgi apparatus-associated" polysomes. The polysomes, when associated with Golgi apparatus membranes, incorporated amino acids in vitro. The products synthesized in vitro were analyzed by treatment with KCl and deoxycholate and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Certain proteins synthesized by the Golgi apparatus-associated polysomes remained insoluble after the treatment with KCl and deoxycholate. The proteins synthesized by the Golgi apparatus fraction had mobilities similar to proteins in plasma membranes which were absent from endoplasmic reticulum, and which were relatively minor components of Golgi apparatus...

Animals↗
PubMed1981

Inhibition of Golgi apparatus-mediated secretion by cinnamic acid derivatives.

Two test systems have been utilized for screening potential inhibitors of Golgi apparatus function. In one system we measured formation of external polysaccharide droplets by the outer cap cells of maize roots. The droplets, direct products of the Golgi apparatus, were quantitated by visual estimates of droplet size compared to known standards. In the second system, we monitored secretory granule formation in the parotid gland of the rat, an activity also mediated by the Golgi apparatus. Parotid gland slices were stimulated with epinephrine to achieve 90% release of granules. The ability of the Golgi apparatus to package new secretory granules was then tested by incubating degranulated gland slices in the presence of putative inhibitors followed by restimulation with epinephrine. The amounts of alpha-amylase or (3H)leucine labeled proteins secreted into the medium were used as measures of secretory granule formation. The phenolic acid cinnamic acid and a related compound scopoletin were found to show substantial inhibition of secretion in both test systems.

Animals↗