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PubMed2004

Calcium release from the Golgi apparatus and the endoplasmic reticulum in HeLa cells stably expressing targeted aequorin to these compartments.

Extracellular agonists mobilize Ca2+ from SERCA-comprising intracellular Ca2+ stores located in both the Golgi apparatus and the endoplasmic reticulum. Ca2+ release from both these compartments was studied in HeLa cells stably expressing the luminescent Ca2+ indicator aequorin specifically targeted to these compartments. Changes in lumenal [Ca2+] as detected by the aequorin measurements were correlated with parallel changes in total Ca2+ content of the stores. The latencies and initial rates of Ca2+ release from the Golgi apparatus and the endoplasmic reticulum were quite similar. However, maximal Ca2+ release measured with Golgi-targeted aequorin terminated faster than that from the endoplasmic reticulum. The rate and extent of Ca2+ depletion from both compartments correlated well with the peak amplitude of the cytosolic [Ca2+] rise. Time-course experiments further revealed that the peak of the cytosolic Ca2+ response occurred before the lumenal [Ca2+] reached its lowest level. We conclude that both the Golgi apparatus and the endoplasmic reticulum contribute to the rise in cytosolic [Ca2+] upon agonist stimulation, but the kinetics of the Ca2+ release are different.

Aequorin↗
PubMed1989

Formation of secretion granules in the Golgi apparatus of plasma cells in the rat.

The three-dimensional structure of the components of the Golgi apparatus was analyzed in plasma cells of rat duodenum. The spheroidal juxtanuclear Golgi apparatus was formed by a continuous ribbonlike structure composed of the following stacked elements. On the cis-face of the Golgi stack, there was a tubular membranous network referred to as the cis-element and/or a slightly dilated saccule perforated with small pores. The two or three subjacent saccules, which showed few pores, were slightly dilated and contained a fluffy granulofilamentous material. They were also perforated in register by cavities or wells containing 80-nm vesicles. The next one or two underlying elements were fenestrated saccules showing flattened portions as well as distended portions containing a homogeneous material denser than that seen in the overlying saccules. The last two or three elements of the stack showed a partially separated or "peeling off" configuration. These last elements consisted of prosecretory granules attached to flattened, empty-looking saccules showing buds at their surface; detached, more-or-less fenestrated, flattened saccules; and shrivelled residual trans-tubular networks. In the trans-region of the stack, in addition to numerous small vesicles, short membranous tubules, detached prosecretory granules, and denser fully formed secretion granules were also seen. These images were interpreted to indicate that secretory material present in the trans-saccules flows toward the dilated portions which become prosecretory granules. The trans-most elements seemingly peel off the stack to yield prosecretory granules and fragmenting trans-tubular networks.

Animals↗
PubMed1992

Beta-amyloid precursor protein localization in the Golgi apparatus in neurons and oligodendrocytes. An immunocytochemical structural and ultrastructural study in normal and axotomized neurons.

We have used a polyclonal antibody raised against a synthetic peptide from the carboxyl terminal of the beta-amyloid precursor protein (APP) to examine the cellular and subcellular localization of this protein in the rat brain. Light and electron microscopic immunocytochemical techniques were used. Immunoreactivity was found throughout the brain in all the neurons examined as well as in oligodendrocytes. At the light microscopic level, a perinuclear filamentous distribution was seen in neurons, suggesting a concentration of the protein to the Golgi apparatus. Axotomy of motor neurons of the facial nucleus produced a decrease in choline acetyltransferase (ChAT) activity and an increase in the perineuronal microglial nucleoside diphosphatase (NDPase)-positive cells in addition to a hypertrophy of the GFAP immunoreactive astrocytes. On the other hand, increased APP-like immunoreactivity all over the neuronal cell bodies accompanied by a dispersion ('rete dispersion') of the Golgi apparatus labeling was demonstrated. In contrast, reactive microglia and hypertrophic astrocytes in axotomized facial nucleus were not immunolabeled. Oligodendrocytes showed a punctate APP immunoreactivity corresponding to the Golgi apparatus in both operated and control facial nucleus. This was further demonstrated by electron microscopic immunolabeling. These results show that the main localization of the C-terminal containing forms of the APP in the rat brain is the Golgi apparatus in both neurons and oligodendroglia and further supporting the secretory nature of these proteins. The increased synthesis of this protein after axotomy is suggestive of a role of the APPs in growth and/or regeneration.

Alzheimer Disease↗
PubMed1998

Mammalian Golgi apparatus UDP-N-acetylglucosamine transporter: molecular cloning by phenotypic correction of a yeast mutant.

Transporters in the Golgi apparatus membrane translocate nucleotide sugars from the cytosol into the Golgi lumen before these can be substrates for the glycosylation of proteins, lipids, and proteoglycans. We have cloned the mammalian Golgi membrane transporter for uridine diphosphate-N-acetylglucosamine by phenotypic correction with cDNA from MDCK cells of a recently characterized Kluyveromyces lactis mutant deficient in Golgi transport of the above nucleotide sugar. Phenotypically corrected transformants were separated from mutants in a fluorescent-activated cell sorter after labeling of K. lactis cells with fluorescein isothiocyanate (FITC) conjugated to Griffonia simplicifolia II lectin, which binds terminal N-acetylglucosamine. A 2-kb DNA fragment was found to restore the wild-type cell lectin binding phenotype, which reverted to the mutant one upon loss of the plasmid. The DNA fragment contained an ORF encoding a hydrophobic, multitransmembrane spanning protein of 326 aa that had only 22% amino acid sequence identity with the corresponding transporter from K. lactis but showed 53% amino acid sequence identity to the mammalian UDP-galactose transporters and 40% to the CMP-sialic acid transporter. Golgi vesicles from the transformant regained their ability to transport UDP-GlcNAc in an assay in vitro. The above results demonstrate that the mammalian Golgi UDP-GlcNAc transporter gene has all of the necessary information for the protein to be expressed and targeted functionally to the Golgi apparatus of yeast and that two proteins with very different amino acid sequences may transport the same solute within the same Golgi membrane.

Amino Acid Sequence↗
PubMed1999

Characterization of a novel giant scaffolding protein, CG-NAP, that anchors multiple signaling enzymes to centrosome and the golgi apparatus.

A novel 450-kDa coiled-coil protein, CG-NAP (centrosome and Golgi localized PKN-associated protein), was identified as a protein that interacted with the regulatory region of the protein kinase PKN, having a catalytic domain homologous to that of protein kinase C. CG-NAP contains two sets of putative RII (regulatory subunit of protein kinase A)-binding motif. Indeed, CG-NAP tightly bound to RIIalpha in HeLa cells. Furthermore, CG-NAP was coimmunoprecipitated with the catalytic subunit of protein phosphatase 2A (PP2A), when one of the B subunit of PP2A (PR130) was exogenously expressed in COS7 cells. CG-NAP also interacted with the catalytic subunit of protein phosphatase 1 in HeLa cells. Immunofluorescence analysis of HeLa cells revealed that CG-NAP was localized to centrosome throughout the cell cycle, the midbody at telophase, and the Golgi apparatus at interphase, where a certain population of PKN and RIIalpha were found to be accumulated. These data indicate that CG-NAP serves as a novel scaffolding protein that assembles several protein kinases and phosphatases on centrosome and the Golgi apparatus, where physiological events, such as cell cycle progression and intracellular membrane traffic, may be regulated by phosphorylation state of specific protein substrates.

A Kinase Anchor Proteins↗
PubMed1987

Golgi apparatus cisternae of monensin-treated cells accumulate in the cytoplasm of liver slices.

Protein transport via the endoplasmic reticulum Golgi apparatus-cell surface export route was blocked when slices (6-15 cells thick) of livers of 10-day-old rats were incubated with 1 microM monensin. Production of secretory vesicles by Golgi apparatus was reduced or eliminated and, in their place, swollen cisternae accumulated in the cytoplasm at the trans Golgi apparatus face. The swelling response was restricted to the six external cell layers of the liver slices, and the number of cells showing the response was little increased by either a greater concentration of monensin or by longer times of incubation. When monensin was added post-chase to the slices, flux of radioactive proteins to the cell surface was inhibited by about 80% as determined from standard pulse-chase analyses with isolated cell fractions. Radioactive proteins accumulated in both endoplasmic reticulum and Golgi apparatus and in a fraction that may contain monensin-blocked Golgi apparatus cisternae released from the stack. The latter fraction was characterized by galactosyltransferase/thiamine pyrophosphatase ratios similar to those of Golgi apparatus from control slices. The use of monensin with the tissue slice system may provide an opportunity for the cells to accumulate monensin-blocked Golgi apparatus cisternae in sufficient quantities to permit their isolation and purification by conventional cell fractionation methods.

Animals↗
PubMed2003

Phospholipase Cgamma activates Ras on the Golgi apparatus by means of RasGRP1.

Ras proteins regulate cellular growth and differentiation, and are mutated in 30% of cancers. We have shown recently that Ras is activated on and transmits signals from the Golgi apparatus as well as the plasma membrane but the mechanism of compartmentalized signalling was not determined. Here we show that, in response to Src-dependent activation of phospholipase Cgamma1, the Ras guanine nucleotide exchange factor RasGRP1 translocated to the Golgi where it activated Ras. Whereas Ca(2+) positively regulated Ras on the Golgi apparatus through RasGRP1, the same second messenger negatively regulated Ras on the plasma membrane by means of the Ras GTPase-activating protein CAPRI. Ras activation after T-cell receptor stimulation in Jurkat cells, rich in RasGRP1, was limited to the Golgi apparatus through the action of CAPRI, demonstrating unambiguously a physiological role for Ras on Golgi. Activation of Ras on Golgi also induced differentiation of PC12 cells, transformed fibroblasts and mediated radioresistance. Thus, activation of Ras on Golgi has important biological consequences and proceeds through a pathway distinct from the one that activates Ras on the plasma membrane.

Animals↗
PubMed1983

Ultrastructural studies of myeloma cells: observations concerning the Golgi apparatus and intermediate-size filaments.

Studies on the ultrastructure of tumor cells of 22 multiple myeloma patients revealed hitherto unreported observations concerning the Golgi apparatus and intermediate-size filaments. Various abnormalities of the Golgi apparatus including displacements of the Golgi stacks, dilatations of cisternae, and massive accumulations of vesicles as well as vacuoles in the Golgi region were found in myeloma cells of 11 patients. Intermediate-size filaments forming a fine intracytoplasmic network were apparent in myeloma cells of all 22 patients. In five patients, in addition to this architecture, intermediate-size filaments were gathered into thick bundles which were primarily but not exclusively located in the perinuclear region.

Cytoskeleton↗
PubMed1993

A soluble secretory protein is first concentrated in the endoplasmic reticulum before transfer to the Golgi apparatus.

A soluble secretory protein is usually present at a much higher concentration in the Golgi apparatus than in the endoplasmic reticulum (ER) inside eukaryotic secretory cells in the steady state. We show by immunoelectron microscopic experiments with the soluble secretory protein serum albumin, inside Hep-G2 human hepatoma cells in culture, that the secretory protein is first concentrated at isolated sites within the ER before it is transferred to the cis face of the Golgi apparatus. This is contrary to expectations of the bulk-flow hypothesis of ER-to-Golgi transfer, and it suggests the involvement of concentration and transfer mechanisms within the ER that have not previously been recognized.

Carcinoma, Hepatocellular↗
PubMed1984

Tridimensional architecture of the Golgi apparatus in the atrial muscle cell of the rat.

The tridimensional structure of the Golgi apparatus of atrial muscle cells has been studied in thin and thick sections with low- and high-voltage electron microscopes. Cardiac tissue was inpregnated with osmium, stained to demonstrate phosphate activity (i.e., nicotinamide adenine dinucleotide phosphatase or NADPase; thiamine pyrophosphatase or TPPase; cytidine monophosphatase or CMPase) or postfixed and stained with potassium ferrocyanide-reduced osmium. At low power, in thick (3-10 micron) sections, the cisosmiophilic element and the NADPase- and the TPPase-positive saccules each appeared as a continuous irregular ribbon that formed, at the two poles of the nucleus, two conical masses connected to each other by beltlike bands encircling the nucleus. At higher magnifications, the continuous Golgi apparatus showed saccular regions along its length connected by short intersaccular tubular regions. In the saccular regions, the following five superimposed elements formed a stack: (1) the cis-osmiophilic network of anastomosed tubules; (2) a chromophobic, dilated saccule perforated with numerous pores; (3) a thin NADPase-positive saccule showing few pores; (4) a thin TPPase-positive saccule perforated with numerous minute pores; and (5) a CMPase-positive transelement that showed saccular and tubular regions and was often partly separated from the overlying saccule. In the intersaccular tubular regions, membranous tubules connected and bridged saccules of two adjacent saccular regions. Secretory granules usually appeared in this region as dilations of the tubules connected to all elements of the Golgi stack except the cis-osmiophilic element.

Animals↗
PubMed1991

Localization in the Golgi apparatus of rat liver UDP-Gal:glucosylceramide beta 1----4galactosyltransferase.

The presence and subcellular localization of UDP-Gal:glucosylceramide beta 1----4galactosyltransferase (GalT-2) was investigated in rat liver. For this purpose, purified Golgi apparatus, endoplasmic reticulum, and plasma membrane fractions were prepared from the liver and used as the enzyme source for detecting GalT-2. A pure Golgi apparatus, highly enriched in many glycosyltransferases, was the only fraction where GalT-2 was measurable. The reaction product formation rate under appropriate assay conditions, which requires high detergent concentration and Mn2+, was low but comparable with that of other glycosyltransferases. The product formation was stimulated by exogenously added acceptor GlcCer, donor UDP-Gal, and Golgi protein. The reaction product was a single spot that was identified by chromatographic behavior, sensitivity to beta-galactosidase, and permethylation studies as Gal beta 1----4Glc beta 1----1'Cer (lactosylceramide). A metabolic experiment, performed by determining the glycosphingolipids which became radioactive in the above subcellular fractions prepared from the liver of animals treated with glucose-labeled glucosylceramide, further indicated that the in vivo glycosylation of glucosylceramide takes place in the Golgi apparatus.

Animals↗
PubMed1999

Genetic evidence for ATP-dependent endoplasmic reticulum-to-Golgi apparatus trafficking of ceramide for sphingomyelin synthesis in Chinese hamster ovary cells.

LY-A strain is a Chinese hamster ovary cell mutant resistant to sphingomyelin (SM)-directed cytolysin and has a defect in de novo SM synthesis. Metabolic labeling experiments with radioactive serine, sphingosine, and choline showed that LY-A cells were defective in synthesis of SM from these precursors, but not syntheses of ceramide (Cer), glycosphingolipids, or phosphatidylcholine, indicating a specific defect in the conversion of Cer to SM in LY-A cells. In vitro experiments showed that the specific defect of SM formation in LY-A cells was not due to alterations in enzymatic activities responsible for SM synthesis or degradation. When cells were treated with brefeldin A, which causes fusion of the Golgi apparatus with the endoplasmic reticulum (ER), de novo SM synthesis in LY-A cells was restored to the wild-type level. Pulse-chase experiments with a fluorescent Cer analogue, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-pentanoyl)-D-erythro-sphingosine (C5-DMB-Cer), revealed that in wild-type cells C5-DMB-Cer was redistributed from intracellular membranes to the Golgi apparatus in an intracellular ATP-dependent manner, and that LY-A cells were defective in the energy-dependent redistribution of C5-DMB-Cer. Under ATP-depleted conditions, conversion of C5-DMB-Cer to C5-DMB-SM and of [3H]sphingosine to [3H]SM in wild-type cells decreased to the levels in LY-A cells, which were not affected by ATP depletion. ER-to-Golgi apparatus trafficking of glycosylphosphatidylinositol-anchored or membrane-spanning proteins in LY-A cells appeared to be normal. These results indicate that the predominant pathway of ER-to-Golgi apparatus trafficking of Cer for de novo SM synthesis is ATP dependent and that this pathway is almost completely impaired in LY-A cells. In addition, the specific defect of SM synthesis in LY-A cells suggests different pathways of Cer transport for glycosphingolipids versus SM synthesis.

Adenosine Triphosphate↗
PubMed1997

Formation of a whorl-like autophagosome by Golgi apparatus engulfing a ribosome-containing vacuole in corpora allata of the cockroach Diploptera punctata.

Previously, we showed that synchronous autophagy occurred in the corpora allata during the declining phase of juvenile hormone synthesis in mated adult females of the cockroach Diploptera punctata. At the onset of this process, conspicuous whorl-like autophagosomes were often observed. Each consisted of a ribosome-containing vacuole at the center surrounded by about eight layers of double membranes. Labeling the cis-Golgi cisternae with hot osmium and histochemical detection of thiamine pyrophosphatase in the trans-Golgi cisternae were used to trace the autophagic process. We conclude that the membranes surrounding the whorl-like autophagosome are derived from the entire Golgi apparatus with its cis-cisternae forming the outermost layer and the trans-cisternae the innermost layer. The engulfing process is initiated through direct contact between trans-cisternae of the Golgi apparatus and the isolation membrane of the ribosome-containing vacuole. Formation of a whorl-like autophagosome appears to be a highly regulated process specific to ribosome-containing vacuoles since it occurs only on day 5 and the Golgi apparatus does not recognize membranes of other types of autophagic vesicles or of functional organelles. This novel behavior of the Golgi apparatus in the corpora allata should play an important role in the long-term inhibition of juvenile hormone synthesis (e.g., during gestation) since it results in the depletion of cellular machinery for juvenile hormone synthesis.

Journal Article↗
PubMed1977

Glycosylation of apoproteins of rat very low density lipoproteins during transit through the hepatic Golgi apparatus.

The glycosylation of apo very low density lipoproteins (apo-VLDL) in vivo was studied by following the incorporation of [14C]glucosamine into several groups of apoproteins of VLDL isolated from hepatic Golgi fractions and from serum of sucrose-fed, colchicine-treated rats. Simultaneous incorporation of [3H]leucine was used to quantitate the apoproteins following separation by polyacrylamide gel electrophoresis. Experimental conditions were selected so that the 14C:3H ratio in the apoproteins permitted estimations of the extent of glycosylation by glucosamine and its metabolites. A rapidly decreasing 14C:3H ratio was noted in serum apo-VLDL for the first 30 min after administration of the isotopically labelled precursors, followed by stabilization of the ratio. These data are consistent with the glycosylation of a preformed pool of apo-VLDL, probably apo-B. Glucosamine was progressively incorporated into apo-VLDL during transition from the forming face of the Golgi apparatus to the secretory vesicles, as indicated by an increasing 14C:3H ratio. On the other hand, the ratio of the rapidly migrating apoproteins of VLDL, corresponding to the apo-C-II and apo-C-III, showed the opposite trend, as did total apo high density lipoprotein (apo-HDL) and the rapidly migrating bands of apo-HDL. Division of the rapidly migrating apoproteins of VLDL into upper bands (probably apo-C-II and apo-C-III-0) and lower bands (probably apo-C-III-3) resulted in a 14C:3H ratio near zero in the upper band apoproteins, consistent with the absence of carbohydrates. The lower band showed a rising 14C:3H ratio during transition through the Golgi apparatus, suggesting increased glycosylation, The decreasing 14C:3H ratio in the rapidly migrating proteins is therefore due to the acquisition of apo-C-II and apo-C-III-0 by VLDL during passage from the forming face to the secretory vesicles of the Golgi apparatus.

Animals↗
PubMed1987

Intracellular distribution of yolk droplets, lipid bodies and Golgi apparatus in the chick neuroepithelial cells during neurulation.

Vitelline and lipidic inclusions which are present in the neuroepithelial cells during chick embryo neurulation show a typical intracellular localization in the apical zone of the cell. In the same cellular zone the Golgi apparatus can be seen during the successive stages of neurulation. These patterns of inclusion and organelle polarity during chick embryo neurulation may be related to active consumption of the reserves contained in inclusions during this morphogenetic process. Such an active consumption would imply a close relationship between the vitelline and lipidic inclusions and the Golgi apparatus. On the other hand, the apical position of the Golgi apparatus in the neuroepithelial cells reveals the remarkable apicobasal polarity of these cells which remains unchanged during chick embryo neurulation.

Animals↗
PubMed1995

[Corticosteroid responsive inflammatory myopathy with autoantibody against Golgi apparatus].

A 51-year-old woman of progressive myopathy predominantly involving proximal groups of limb-muscle was reported. Serum enzymes originating from the skeletal muscle always remained within normal range, and a positive autoantibody against Golgi apparatus and the SS-A (Ro) antibody in serum were noted. In muscle biopsy performed twice, extensive degenerating/regenerating fibers and sparce inflammatory cells between connective tissue elements in light microscopy, and disrupted lamellar structure of Golgi apparatus in electron microscopy were observed. Corticosteroid therapy was markedly effective. In the present case of a "so-called" limb-girdle syndrome, humoral autoantibodies, especially against Golgi apparatus, could induce a metabolic disturbance in the muscle fiber.

Autoantibodies↗
PubMed2005

Cisternal rab proteins regulate Golgi apparatus redistribution in response to hypotonic stress.

We show that a physiological role of the extensively studied cisternal Golgi rab protein, rab6, is modulation of Golgi apparatus response to stress. Taking exposure of cells to hypotonic media as the best-known example of mammalian Golgi stress response, we found that hypotonic-induced tubule extension from the Golgi apparatus was sensitive to GDP-rab6a expression. Similarly, we found that Golgi tubulation induced by brefeldin A, a known microtubule-dependent process, was inhibited by GDP-restricted rab6a, rab6a', and rab33b, the most commonly studied cisternal rab proteins. These GDP-rab levels were sufficient to inhibit rab-induced redistribution of Golgi glycosyltransferases into the endoplasmic reticulum (ER), also a microtubule-dependent process, and to depress Golgi membrane association of the GTP-conformer of rab6. Nocodazole-induced Golgi scattering, a microtubule-independent process, also was inhibited by GDP-rab6a expression. In comparison, we found similar GDP-rab expression levels had little inhibitory effect on another microtubule-independent process, constitutive recycling of Golgi resident proteins to the ER. We conclude that Golgi cisternal rabs, and in particular rab6a, are regulators of the Golgi response to stress and presumably the molecular targets of stress-activated signaling pathway(s). Moreover, we conclude that rab6a can regulate select microtubule-independent processes as well as microtubule-dependent processes.

Base Sequence↗
PubMed2000

The small GTPase Rab6B, a novel Rab6 subfamily member, is cell-type specifically expressed and localised to the Golgi apparatus.

Members of the Rab subfamily of small GTPases play an important role in the regulation of intracellular transport routes. Rab6A has been shown to be a regulator of membrane traffic from the Golgi apparatus towards the endoplasmic reticulum (ER). Here, we report on the identification of a Rab6 isoform, termed Rab6B. The corresponding full-length cDNA was isolated from a Caco-2 cell library. The deduced amino acid sequence showed 91% identity with the Rab6A protein and revealed that sequence divergence is dispersed over a large region of the COOH-terminal domain. Rab6B is encoded by an independent gene which is located on chromosome 3 region q21-q23. In contrast to Rab6A whose expression is ubiquitous, northern blot analysis, immunohistochemistry, and immunofluorescence demonstrated that Rab6B is expressed in a tissue and cell-type specific manner. Rab6B is predominantly expressed in brain and the neuroblastoma cell line SK-N-SH. In brain, Rab6B was found to be specifically expressed in microglia, pericytes and Purkinje cells. Endogenous Rab6B localises to the Golgi apparatus and to ERGIC-53-positive vesicles. Comparable studies between Rab6A and Rab6B revealed distinct biochemical and cellular properties. Rab6B displayed lower GTP-binding activities and in overexpression studies, the protein is distributed over Golgi and ER membranes, whereas Rab6A is more restricted to the Golgi apparatus. Since the GTP-bound form of Rab6B (Rab6B Q72L) does interact with all known Rab6A effectors, including Rabkinesin-6, the results suggest a cell-type specific role for Rab6B in retrograde membrane traffic at the level of the Golgi complex.

Animals↗