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PubMed2004

Protein localization in the plant Golgi apparatus and the trans-Golgi network.

This review presents plant-specific characteristics of the Golgi apparatus and discusses their impact on retention of membrane proteins in the Golgi or the trans-Golgi network (TGN). The plant Golgi consists of distinct stacks of cisternae that actively move throughout the cytoplasm. The Golgi apparatus is a very dynamic compartment and the site for maturation of N-linked glycans. It is also a factory for complex carbohydrates that are part of the cell wall. The TGN is believed to be the site from where vacuolar proteins are sorted by receptors towards each type of vacuole. To maintain the structure and specific features of the Golgi, resident proteins ought to be maintained in the proper Golgi cisternae or in the TGN. Two families of membrane proteins will be taken as examples for Golgi/TGN retention: (i) the enzymes involved in N-glycosylation processes and (ii) a vacuolar sorting receptor. Although the number of available plant proteins localized in Golgi/TGN is low, the basis of retention appears to be shared over all kingdoms and may result from pure retention and recycling mechanisms. In this review, we will summarize the characteristics of a plant Golgi and will discuss especially their consequences on on the study of this highly dynamic structure. We then choose membrane proteins with a single transmembrane domain to illustrate the signals and mechanisms involved in plants to localize and maintain proteins in the Golgi and the TGN.

Golgi Apparatus↗

PubMed2000

Inositol 1,4,5-trisphosphate but not ryanodine-receptor agonists induces calcium release from rat liver Golgi apparatus membrane vesicles.

We investigated the direct effect of inositol 1,4,5-trisphosphate (IP(3)) and ryanodine receptor agonists on Ca(2+) release from vesicles of a rat liver Golgi apparatus (GA) enriched fraction, which were actively loaded with (45)Ca(2+). Results in GA were compared with those obtained in a rat liver endoplasmic reticulum (ER) enriched fraction. The addition of IP(3) at concentrations ranging from 100 nm to 100 microm, in the presence of thapsigargin, a specific inhibitor of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPases, promoted a rapid decrease in the Ca(2+) content of GA vesicles. The amount of Ca(2+) released from the vesicles was a function of IP(3) concentration, reaching about 60% in both GA and ER fractions at 100 microm IP(3). Calcium release was inhibited by heparin, an antagonist of IP(3) receptors. Calcium exhibited a bell-shaped effect on IP(3)-dependent Ca(2+) released from GA vesicles: it activated Ca(2+) release at concentrations up to 1 microm, and inhibited it at higher concentrations. In contrast to that found in the endoplasmic reticulum fraction, none of the ryanodine receptor agonists tested (cyclic ADP-ribose, caffeine and ryanodine) significantly induced Ca(2+) release from GA fraction vesicles in the presence of thapsigargin. Our results indicate the presence of an IP(3)-sensitive Ca(2+) release mechanism in the Golgi apparatus membrane analogous to that of the ER. However, a Ca(2+) release mechanism sensitive to ryanodine receptor agonists like that of ER is not evident in the GA membrane.

Adenosine Diphosphate Ribose↗

PubMed2002

Identification and characterization of an insect homologue of the vertebrate Golgi apparatus protein 1 (MG-160/cysteine-rich fibroblast growth factor receptor/E-selectin ligand-1/latent transforming growth factor-beta complex protein-1) with a Golgi-specific monoclonal antibody.

A monoclonal antibody 14F10 was raised against Golgi fractions from Sf21 cells and selected as Golgi specific. Immunohistochemical stainings with the antibody localized the antigen in Golgi cisterns of the cells. The antigen was purified and shown to be a 130-K membrane protein with N-glycans and intrachain disulfide bonds. Amino acid sequencing of its peptide fragments revealed that the antigen contained homologous sequences to those encoded by CG7190 and CG7193 Drosophila melanogaster genes. No possible transmembrane domain existed in these deduced amino acid sequences, while one did in that encoded by CG7195, an adjacent gene to CG7193. Furthermore, 5' and 3' expression sequence tags of LD19434 had been mapped to CG7190 and a downstream region of CG7195, respectively. These findings supported that all of these genes actually composed a single gene, which encoded an orthologous protein to a vertebrate Golgi-resident protein, Golgi apparatus protein 1, also called cysteine-rich FGF receptor, E-selectin ligand-1, or latent TGF-beta complex protein-1. Our results suggested that the Golgi apparatus protein 1 played a critical role in the Golgi cisterns through the animal kingdom.

Amino Acid Sequence↗

PubMed2007

MUC16 in the lacrimal apparatus.

The aim of the present study was to determine the possible expression of the mucin MUC16 in the lacrimal apparatus. Expression and distribution of MUC16 in lacrimal gland, accessory lacrimal glands, and nasolacrimal ducts was monitored by RT-PCR and immunohistochemistry. MUC16 was expressed and detected in all tissues investigated. Comparable to conjunctiva and cornea it was membrane-anchored in accessory lacrimal glands whereas in lacrimal gland acinar cells and columnar cells of the nasolacrimal ducts it was stored in intracytoplasmic vesicles without membrane-association. Subepithelial serous glands of the nasolacrimal ducts revealed staining of the secretion product. Intracelluar production of MUC16 is present in lacrimal gland and epithelial cells of the nasolacrimal ducts but it is not clear whether this MUC16 is secreted. MUC16 seems to be shedded or secreted from the epithelial surface of subepithelial serous glands of the nasolacrimal ducts. Our results show that MUC16 is present in the whole lacrimal apparatus. Its distribution pattern suggests different physiological functions with regard to tear film physiology and tear outflow. Moreover, the results demonstrate the existence of so far not recognized qualitative differences in the secretion product of main lacrimal gland and accessory lacrimal glands (glands of Krause).

Aged↗

PubMedDate not supplied

Camillo Golgi and the discovery of the Golgi apparatus.

Camillo Golgi (1843-1926) was born at Corteno, near Brescia, in northern Italy. After graduating in Medicine at the ancient University of Pavia, the former seat of great scientists and naturalists, Golgi continued a long-standing Italian tradition by studying the histology of the nervous system. While working as a modest physician at Abbiategrasso, a small town near Pavia, he developed a silver-osmium technique, the "reazione nera" (black reaction), for which he was awarded the Nobel Prize in 1906. In the late 1890's, 25 years after the publication of his black reaction and while Professor of General Pathology in Pavia, Golgi noticed a fine internal network in only partially silver-osmium-blackened Purkinje cells. Following confirmation by his assistant Emilio Veratti, Golgi published the discovery, called the "apparato reticolare interno", in the Bollettino della Società medico-chirurgica di Pavia in 1898, which is now considered the birthday of the "Golgi apparatus". The discovery of the Golgi apparatus can be added to the long list of accidental discoveries. The man after whom it is named was not a cytologist engaged in studying the inner structure of the cell, but a pathologist searching to prove a neuroanatomical theory.

Eponyms↗

PubMedDate not supplied

Tubules of the trans Golgi apparatus visualized by immunoelectron microscopy.

Tubules constitute an integral part of the Golgi apparatus and have been shown to form a complex and dynamic network at its trans side. We have studied in detail structural features of the trans Golgi network and its relationship with the cisternal stack in thin sections of Lowicryl K4M embedded human absorptive enterocytes by immunolectron microscopy. Immunoreactive sites for alpha1,3 N-acetylgalactosaminyltransferase and blood group A substance were detectable throughout the cisternal stack and the entire trans Golgi network. Furthermore, the entire trans Golgi network was reactive for CMPase activity. Evidence for two kinds of tubules at the trans side of the Golgi apparatus was found: tubules that laterally connect adjacent and distant cisternal stacks, and others extending from central and lateral portions of trans cisternae to form the complex and extensive trans Golgi network. Trans cisternae showed often the peeling-off phenomenon and were continuous with the trans Golgi network. Both, trans cisternae and tubules of the trans Golgi network exhibited regionally buds and vesicles with a lace-like, non clathrin coat, previously reported by others in NRK cells, which contained glycoproteins with terminal N-acetylgalactosamine residues. These buds and vesicle are therefore involved in constitutive exocytosis.

Acid Phosphatase↗

PubMedDate not supplied

Endocytic routes to the Golgi apparatus.

The endocytic routes of labelled lectins as well as cationic ferritin were studied in cells with a regulated secretion, i.e. pancreatic beta cells, and in constitutively secreting cells, i.e. fibroblasts and HepG2 hepatoma cells, paying particular attention to routes into the Golgi apparatus. Considerable amounts of internalised molecules were taken up into the trans Golgi network (TGN) and into Golgi subcompartments in all three cell types as well as in secretory granules of the pancreatic beta cells. The internalised materials did not pass rapidly the TGN and Golgi stacks, but were still present hours after internalisation, being then particularly concentrated in TGN-elements and in the transmost Golgi cisterna. Endocytosed materials reached forming secretory granules present in the TGN. Further, direct fusion between endocytotic vesicles and mature secretory granules was observed. Golgi subcompartments as well as endocytic TGN containing endocytosed materials were in close apposition to specialised regions of the endoplasmic reticulum. The Golgi apparatus including its parts containing endocytosed materials were transformed into a tubular reticulum upon treatment with the fungal metabolite Brefeldin A. Rarely, internalised material was observed in the lumen of the endoplasmic reticulum, thus providing evidence for an endocytic plasma membrane to endoplasmic reticulum route.

Animals↗

PubMed2005

AtUTr2 is an Arabidopsis thaliana nucleotide sugar transporter located in the Golgi apparatus capable of transporting UDP-galactose.

The synthesis of noncellulosic polysaccharides and glycoproteins in the plant cell Golgi apparatus requires UDP-galactose as a substrate. We have cloned and characterized a nucleotide sugar transporter from Arabidopsis thaliana (L.) Heynh. named AtUTr2. Expression in tobacco and Saccharomyces cerevisiae and subsequent biochemical characterization indicate that AtUTr2 transports UDP-galactose, but not UDP-glucose, UDP-N-acetyl glucosamine, UDP-xylose, UDP-glucuronic acid, GDP-fucose or GDP-mannose. Experiments expressing an AtUTr2-GFP fusion protein in onion epidermal cells suggest that AtUTr2 is located in the Golgi apparatus. Finally, northern analysis indicates that the AtUTr2 transcript was more abundant in roots and calli although it was also present in other Arabidopsis organs but at lower levels. Therefore, AtUTr2 is a nucleotide sugar transporter capable of transporting UDP-galactose that may play an important role in the synthesis of galactose-containing glycoconjugates in Arabidopsis.

Amino Acid Sequence↗

PubMed2000

The Golgi apparatus of the scaly green flagellate Scherffelia dubia: uncoupling of glycoprotein and polysaccharide synthesis during flagellar regeneration.

The flagella of the green alga Scherffelia dubia are covered by scales which consist of acidic polysaccharides and glycoproteins. Experimental deflagellation results in the regeneration of flagella complete with scales. During flagellar regeneration, scales are newly synthesized in the Golgi apparatus, exocytosed and deposited on the growing flagella. Flagellar regeneration is dependent upon protein synthesis and N-glycosylation, as it is blocked by cycloheximide and partially inhibited by tunicamycin. Metabolic labeling with [35S]methionine/cysteine demonstrated that scale-associated proteins were not newly synthesized during flagellar regeneration, suggesting that the proteins deposited on regenerating flagella were drawn from a pool. Quantitative immunoelectron microscopy using a monospecific antibody directed against a scale-associated protein of 126 kDa (SAP126) revealed that the pool of SAP126 was primarily located at the plasma membrane, with minor labeling of the scale reticulum and trans-Golgi cisternae, both before deflagellation and during flagellar regeneration. Since SAP126 was sequestered during flagellar regeneration into secretory vesicles together with newly synthesized scales, it is concluded that the persistent presence of SAP126 in the trans-Golgi cisternae during scale biogenesis requires retrograde transport of the protein from the plasma membrane to the Golgi apparatus.

Animals↗

PubMed2004

The proprotein convertase furin colocalizes with caveolin-1 in the Golgi apparatus and endosomes of hepatocytes.

The significance of furin in the maturation and activation of a wide array of proproteins in the secretory pathway has been well demonstrated. However, despite efforts made to characterize the subcellular locations where furin activates its substrates, doubts on the proprotein-processing compartments still persist. Using in vivo gene delivery, together with high-resolution immunogold electron microscopy, we were able to assign precise subcellular locations to furin. In rat hepatocyte, the enzyme was found concentrated in the Golgi apparatus, along the basolateral (sinusoidal) plasma membrane and in underlying endosomes. An asymmetry was detected in respect to amounts of furin between the basolateral domain and the apical (canalicular) one, favoring the former. The asymmetric recycling of furin through the basolateral domain may be of high importance for the polarized secretion of processed bioactive compounds. Double immunogold labelings indicate that furin colocalizes with the caveolae/raft-marker caveolin-1 in the Golgi apparatus and in the basolateral endosomes. Furthermore, co-immunoprecipitation experiments show the possible interaction of caveolin-1 and furin. Our results suggest that, in addition to the Golgi, furin-/caveolin-1-containing endosomes could represent a compartment where furin processes its substrates at the basolateral domain of the hepatocyte.

Animals↗

PubMed2006

Autoantibodies to mitotic apparatus: association with other autoantibodies and their clinical significance.

The most important mitotic apparatus (MA) antigens are centrosome (CE), nuclear mitotic apparatus (NuMA-1, NuMA-2), midbody, and centromere F (CENP-F). We studied associations of anti-MA antibodies with other autoantibodies and their clinical significance. A total of 6270 patients were studied for the presence of anti-MA antibodies on HEp-2 cells. Sera positive for anti-MA were tested for anti-extractable nuclear antigens (ENA) antibodies. Anti-MA antibodies were detected in 56 (45 females and 11 males) of 6270 sera (0.9%). Of these 56, NuMA-1 was found in 23, NuMA-2 in 7, CE in 20, CENP-F in 5, and CENP-F/centrosome in 1 case. Anti-NuMA-1 were associated with anti-ENA antibodies (p < 0.001). Diagnoses were established in 43/56 patients: 22 connective tissue diseases, 7 infections, 6 autoimmune hepatitis, 3 vasculitis, 3 primary antiphospholipid syndrome, 1 malignancy, and 1 fever of unknown origin. The differential diagnosis of anti-NuMA-1-positive patients must include Sjögren's syndrome, while patients with anti-CE antibodies must be observed for HCV infection.

Adult↗

PubMed1984

Purification of a UTP:D-glucose-1-phosphate uridylyltransferase from Golgi apparatus of cat liver by affinity chromatography on UTP-agarose.

UDP-glucose pyrophosphorylase from Golgi apparatus solubilized by detergent has been purified 100-fold from microsomes by affinity chromatography on UTP-agarose. The purified enzyme has apparent Mr 270,000 and isoelectric pH 3.9 against 360,000 and 4.2 for soluble enzyme. According to these characteristics, UDP-glucose pyrophosphorylase from Golgi apparatus is different from cytosolic enzyme.

Animals↗

PubMed1988

Cytochemical study of the Golgi apparatus and related organelles of the secretory ameloblasts of rat molar tooth germs cultured with and without colchicine.

The Golgi apparatus and Golgi-associated endoplasmic reticulum lysosome (GERL) were examined in the ameloblasts with a cytochemical marker, osmium impregnation, and two enzyme markers, thiamine pyrophosphatase (TPPase) and acid phosphatase (ACPase). In control cultured germs, osmium deposit appeared in one to two immature side cisternae of Golgi stacks; TPPase activity was restricted in a few mature side cisternae and condensing vacuoles. ACPase activity existed in the GERL and, sometimes, in the mature side-cisternae and condensing vacuoles. These findings show that Golgi stacks of ameloblasts consist of several distinct compartments. In colchicine-treated tooth germs, there were morphological and cytochemical changes in both Golgi stacks and GERL. The Golgi apparatus was fragmented and its stacks were scattered throughout the supranuclear region. In some stacks, the number of osmium-positive cisternae was greater than normal; in others they were absent. TPPase and ACPase activity was absent or diminished. These findings suggest the importance of microtubules in the organization of Golgi complex and GERL in the secretory ameloblast.

Acid Phosphatase↗

PubMed1984

Evidence for coupling between transport of UDP-glucose and its synthesis by membrane-bound pyrophosphorylase in Golgi apparatus of cat liver.

Incubation of sealed vesicles of cat-liver Golgi apparatus with UDP[14C]glucose showed that the vesicles accumulated radioactivity. After Triton X-100 treatment or sonication of washed vesicles, soluble radiolabeled species were released and identified by paper chromatography as UDP[14C]glucose, [14C]glucose 1-phosphate and free glucose. In the incubation medium, UDPglucose was effectively protected by addition of dimercaptopropanol and UTP. Presence of glucose 1-phosphate and glucose within the vesicles most probably arose from luminal pyrophosphatase and phosphatase. A portion of the [14C]glucose moiety became covalently linked to endogenous acceptors. Uptake of UDPglucose was saturable and dependent on time and on the concentration of sugar nucleotide. Together, these results were consistent with a transport system for UDPglucose in Golgi vesicles. Furthermore, penetration rate was considerably higher with UDPglucose synthetized in situ from glucose 1-phosphate by membrane-bound pyrophosphorylase than from added UDPglucose: Vmax values were respectively 10 and 2 pmol/15 min per mg protein. This result allows the conclusion that a coupling between translocase and synthetase is involved in UDPglucose transport through Golgi apparatus membranes. The mechanism of this 'kinetic advantage' is discussed.

Animals↗

PubMed1985

UDPglucose-ceramide glucosyltransferase from porcine submaxillary glands is associated with the Golgi apparatus.

Subcellular distribution of pig submaxillary gland UDPglucose-ceramide glucosyltransferase (EC 2.4.1.80), the enzyme which catalyses the first step during the sequential addition of carbohydrate moieties for ganglioside biosynthesis, was studied. The results presented strongly suggest that in pig submaxillary gland, the transfer of glucose on endogenous or exogenous ceramides takes place in the Golgi apparatus: the specific activity of UDPglucose-ceramide glucosyltransferase increased in parallel with the activity of a known marker of the Golgi apparatus, UDPgalactose-ovomucoid galactosyltransferase. The specific activity of the glucosyltransferase was 18-times higher in the purified Golgi membranes than in the postnuclear supernatant and the yield was over 30%. An apparent Km of 22 microM for UDPglucose and 54 microM for ceramides was determined. Maximal glucosylation of endogenous ceramides was achieved at pH 6.5 in the presence of NADH (1 mM) as inhibitor of pyrophosphatases and with Mn2+ (5 mM). It was found that the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps) is an efficient activator for the glucosylation of exogenous ceramides.

Animals↗

PubMed1992

Pyrophosphate-induced acidification of trans cisternal elements of rat liver Golgi apparatus.

Trans cisternal elements of the Golgi apparatus from rat liver, identified by thiamin pyrophosphatase cytochemistry, were isolated by preparative free-flow electrophoresis and were found to undergo acidification as measured by a spectral shift in the absorbance of acridine orange. Acidification was supported not only by adenosine triphosphate (ATP) but nearly to the same degree by inorganic pyrophosphate (PPi). The proton gradients generated by either ATP or PPi were collapsed by addition of a neutral H+/K+ exchanger, nigericin, or the protonophore, carbonyl cyanide m-chlorophenylhydrazone, both at 1.5 microM. Both ATP hydrolysis and ATP-driven proton translocation as well as pyrophosphate hydrolysis and pyrophosphate-driven acidification were stimulated by chloride ions. However, ATP-dependent activities were optimum at pH 6.6, whereas pyrophosphate-dependent activities were optimum at pH 7.6. The Mg2+ optima also were different, being 0.5 mM with ATP and 5 mM with pyrophosphate. With both ATPase and especially pyrophosphatase activity, both by cytochemistry and analysis of free-flow electrophoresis fractions, hydrolysis was more evenly distributed across the Golgi apparatus stack than was either ATP- or PPi-induced inward transport of protons. Proton transport colocalized more closely with thiamin pyrophosphatase activity than did either pyrophosphatase or ATPase activity. ATP- and pyrophosphatase-dependent acidification were maximal in different electrophoretic fractions consistent with the operation of two distinct proton translocation activities, one driven by ATP and one driven by pyrophosphate.

Adenosine Triphosphatases↗

PubMed1986

Naturally occurring masked antibodies in murine sera recognize a component of the mitotic spindle apparatus.

Antibody activities previously masked in autoimmune MRL and normal Balb/c mice were expressed by briefly subjecting their sera to acidic (pH 2.0) or alkaline (pH 12.0) environments. An enzyme-linked immunosorbent assay (ELISA) revealed that these pH-expressable immunoglobulins reacted with specific nucleotide-BSA antigens (primarily 5'-AMP, -GMP, -TMP) but not with single (ss)- or double (ds)-stranded DNAs or with unconjugated BSA. ELISA analysis of pH-expressed antibodies purified via GMP-BSA/Sepharose indicated that they bound not only to the homologous hapten (GMP) but to AMP and TMP as well, i.e., anti-pAGT antibodies. Further, indirect immunofluorescent assays (IIF) with fixed HEp-2 cells demonstrated that purified anti-pAGT antibodies recognized an epitope within the mitotic spindle apparatus. These results document the existence of a previously undefined masked antibody population in murine sera with specificities directed toward certain nucleotides and a component of the mitotic spindle apparatus. Last, these anti-pAGT (or anti-mitotic spindle) antibodies are not restricted to murine systems inasmuch as they have been detected in every human serum (greater than 100 samples) examined thus far.

Animals↗

PubMed1983

Localization of vasoactive intestinal polypeptide- and avian pancreatic polypeptide-like immunoreactivity in the Golgi apparatus of peripheral neurons.

Using the indirect immunofluorescence technique of Coons and collaborators, vasoactive intestinal polypeptide (VIP)-like immunoreactivity (VIP-LI) and avian pancreatic polypeptide (APP)-like immunoreactivity (APP-LI) was observed in certain neurons of the peripheral nervous system of the cat. In the cell bodies in the cat sympathetic ganglia a strong to very strong VIP-LI or APP-LI was observed with a distribution resembling that of the Golgi apparatus. In addition, a weaker immunoreactivity was seen diffusely in the cytoplasm. After photography, the sections processed for immunohistochemistry were stained with the thiamine pyrophosphatase technique of Novikoff and Goldfischer. The latter technique is assumed to be a specific marker for the Golgi complex. In all cases it was found that the strong peptide immunoreactivity and the thiamine pyrophosphatase activity had an identical distribution. Thus, one pool of these peptides appears to be localized to the Golgi apparatus. Yet another pool is localized to other components, such as vesicles.

Animals↗