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The golgi apparatus is present in perisynaptic, subependymal and perivascular processes of astrocytes and in processes of retinal Müller glia.

The Golgi apparatus (GA) of innervated rat and chicken skeletal muscle is present in a typical perinuclear location, and in subsynaptic areas where it disperses after denervation. It was suggested that the subsynaptic segments of the GA are linked with functions involved in the maturation and targeting of synaptic proteins. Similarly, the GA of rat myocardium is found in a perinuclear location and between myofibrils, adjacent to the T system of tubules. These findings raise the question whether the GA of polarized cells is present in a typical perinuclear location, for the performance of general "housekeeping" functions, and in distal areas, for the mediation of specialized functions. Astrocytes may contain GA within their long cytoplasmic processes which are difficult to identify in thin sections. To ensure the astrocytic origin of GA in otherwise unidentifiable small processes, we used transgenic mice expressing the rat MG160 medial Golgi sialoglycoprotein only in the GA of astrocytes, and visualized the GA with monoclonal antibody 10A8 (mAb10A8) which reacts only with rat MG160. Thus, we identified cisternae of the GA in distal perisynaptic and subependymal processes, in perivascular foot plates of cerebral astrocytes, and in processes of the Müller glia in the retina. A similar strategy may be adopted in future investigations aiming at the detection of elements of the GA in distal processes of neurons and oligodendrocytes. The functional implications of GA in perisynaptic astrocytic processes and other processes are unknown. However, the isolation and molecular characterization of the perisynaptic subset of astrocytic Golgi may be feasible, since others have purified the astrocytic glutamate transporter 1 (GLT1) from crude synaptosomal fractions in which astrocytic processes are probably unavoidable contaminants.

Animals↗

Subcellular distribution of K+-pNPPase (Na+,K+-ATPase) in the Golgi apparatus of developing rat cortical neurons.

The distribution of K+-pNPPase (Na+,K+-ATPase) activity in the compartments of the Golgi apparatus in neurons of the cerebral cortex of young and adult Wistar rats was studied by ultrastructural cytochemistry. In adult rats, mainly the cis-most cisterna was associated with reaction deposits. In 10- and especially in 15-day-old rats, not only the cis-cisternae, but the cis- and trans-Golgi, as well as components of the Golgi stack, also revealed K+-pNPPase activity. The dynamic changes of K+ -pNPPase localization in the compartments of the neuronal Golgi complexes were discussed with respect to the biochemical evidence concerning the building, assembly and processing of Na+,K+-ATPase as plasma membrane glycoprotein. It was suggested that the high activity in the Golgi complexes seen in 15-day-old rats has to be associated with the advancing myelinization in this period and the necessity of Na+,K+-ATPase equipment of nodes of Ranvier.

Animals↗

Cytochemical differentiation of the Golgi apparatus from GERL.

GERL exhibits a number of structural properties, such as its location at the trans face of the Golgi apparatus, thick limiting membranes with occasional coated regions, and close relationship to the ER, which are similar in all cells wehre it has been identified. Acid hydrolase activity and the formation of lysosomes, also considered characteristic of GERL, have been shown to be less consistent properties of GERL in some cells. Finally, GERL and the Golgi saccules of certain cells undergo significant changes in their cytochemical and structural properties in response to specific alterations in cellular activity. These variations are important for at least two reasons: first, they indicate the care required in differentiating GERL from the Golgi saccules based on limited cytochemical observations; second, and most important, they may yield information regarding the biogenetic and functional relationships between the ER, Golgi saccules, and GERL, such as the origin of GERL in various cells and the role of each organelle in the processing of lysosomal and secretory proteins.

Acid Phosphatase↗

Isolation and characterization of endoplasmic reticulum and Golgi apparatus from hepatocyte nodules in male wistar rats.

A method is described for the isolation of endoplasmic reticulum and Golgi apparatus from hyperplastic liver nodules produced by discontinuous feeding of 2-acetylaminofluorene to male Wistar rats. The procedure involves three centrifugation steps and permits the separation of these cell components and their subfractions from the same sample of liver tissue as little as 1 g, wet weight. The fractions have been characterized by chemical, enzymatic, and morphological techniques and were found to be as pure as preparations from normal tissue. Furthermore, some of the characteristic histochemical features of hyperplastic liver nodules have been quantitated by biochemical methods in the fractions. Glucose-6-phosphatase activity in the endoplasmic reticulum subfractions of nodules is approximately 15% of the corresponding value in normal livers, whereas the activity of reduced nicotinamide adenine dinucleotide phosphate: cytochrome c reductase is reduced to 85% of the normal activity. The amount of cytochrome P-450 in nodular membranes as measured by differential spectroscopy is 25% of the control, indicating a decreased Phase I activity in drug metabolism. A 5-fold increase in cytosolic glutathione S-transferase activity without change in the corresponding microsomal activity was detected in hepatocyte nodules in rat liver. The activity of gamma-glutamyltransferase is increased more than 20-fold in all membrane fractions prepared from nodular tissue. The cytosolic activity, which is very low in the normal liver, is similarly increased more than 20-fold. The membrane-associated gamma-glutamyltransferase seems to be an integral membrane protein which cannot be washed away from the membranes. Chemically, membranes from nodules have phospholipid and cholesterol:protein ratios as found in membranes from normal liver tissue. However, the composition of individual phospholipids is changed with a 2-fold increase in nodular phosphatidylinositol and a slight decrease in phosphatidylcholine content in nodular membranes. The amount of endoplasmic reticulum membranes is of the same magnitude as in normal liver, although the smooth-surfaced component constitutes almost 60% of the isolated endoplasmic reticulum marker enzymes in nodules, compared with only 32% in preparations from normal tissue. The albumin contents of nodular and normal microsomal and Golgi membrane preparations are similar, indicating a normal synthesis of albumin by nodular tissue.

2-Acetylaminofluorene↗

The history of the Golgi apparatus in neurones from its discovery in 1898 to electron microscopy.

We celebrate in 1998 the centenary of the discovery of the Golgi apparatus. Neurones have played a special role in the history of this cell organelle, primarily because the endocellular apparatus was discovered in nerve cells by Camillo Golgi when he was studying the cerebellum of an owl. In the following years, the apparatus of the nerve cell represented the prototype of this cell component which, however, was soon also detected in many other cell types. During the 1920s and 1930s the original idea of a Golgi endocellular "network" was refused and research was focused on the constituents and function of the organelle, using various tissues and techniques. However, the real existence of the apparatus was seriously questioned until the organelle was finally identified by electron microscopy in the mid-1950s. The studies performed on nerve cells during these decades is briefly reviewed here.

Animals↗

The putative tumor suppressors EXT1 and EXT2 form a stable complex that accumulates in the Golgi apparatus and catalyzes the synthesis of heparan sulfate.

Hereditary multiple exostoses, a dominantly inherited genetic disorder characterized by multiple cartilaginous tumors, is caused by mutations in members of the EXT gene family, EXT1 or EXT2. The proteins encoded by these genes, EXT1 and EXT2, are endoplasmic reticulum-localized type II transmembrane glycoproteins that possess or are tightly associated with glycosyltransferase activities involved in the polymerization of heparan sulfate. Here, by testing a cell line with a specific defect in EXT1 in in vivo and in vitro assays, we show that EXT2 does not harbor significant glycosyltransferase activity in the absence of EXT1. Instead, it appears that EXT1 and EXT2 form a hetero-oligomeric complex in vivo that leads to the accumulation of both proteins in the Golgi apparatus. Remarkably, the Golgi-localized EXT1/EXT2 complex possesses substantially higher glycosyltransferase activity than EXT1 or EXT2 alone, which suggests that the complex represents the biologically relevant form of the enzyme(s). These findings provide a rationale to explain how inherited mutations in either of the two EXT genes can cause loss of activity, resulting in hereditary multiple exostoses.

Amino Acid Sequence↗

Golgi apparatus of the motor neurons in patients with amyotrophic lateral sclerosis and in mice models of amyotrophic lateral sclerosis.

We examined the Golgi apparatus (GA) of motor neurons of patients with ALS and in mice models of ALS by immunohistological method using antiserum against MG160 and against components of the trans-Golgi network (TGN46). The GA of half of the remaining spinal cord motor neurons of patients with sporadic ALS showed fragmentation, where the GA were dispersed or fragmented into numerous small, isolated elements. The GA of Betz cells in sporadic ALS were fragmented similar to that of anterior horn cells, and the GA of spinal cord motor neurons of those with familial ALS and of those with ALS with basophilic inclusions were fragmented or diminished. The GA in the majority of the motor neurons contained Bunina bodies, basophilic inclusions and superoxide dismutase 1 (SOD1)-positive aggregates were fragmented. The motor neurons in transgenic mice expressing G93A mutation of the SOD1 gene showed the fragmentation of the GA months before the onset of paralysis. These findings suggest that the fragmentation of GA may be related to the neuronal degeneration in patients with ALS.

Amyotrophic Lateral Sclerosis↗

Novel consensus sequence for the Golgi apparatus casein kinase, revealed using proline-rich protein-1 (PRP1)-derived peptide substrates.

Previous studies have shown that the Golgi apparatus casein kinase (G-CK) recognizes phosphoacceptor sites specified by the triplet SXE/Sp, which is found in several phosphoproteins, besides casein itself. In the present study, we report that G-CK can phosphorylate, with comparable efficiency, sequences surrounding Ser-22 of salivary proline-rich protein-1 (PRP1), which do not conform to the SXE/Sp motif. By using a series of peptide substrates derived from the PRP1 Ser-22 site, we also have shown that the optimal consensus sequence recognized by G-CK in this case was SXQXX(D/E)3, where the acidic residues at positions n+5 to n+7 and, to a lesser extent, the glutamine residue at position n+2 are the critical determinants.

Amino Acid Sequence↗

Fragmentation of Golgi apparatus of nigral neurons with alpha-synuclein-positive inclusions in patients with Parkinson's disease.

We examined whether the Golgi apparatus (GA) is fragmented in nigral neurons in 18 cases with Parkinson's disease (PD) and in 8 control cases. The nigral neurons in cases with PD showed various degrees of Lewy pathology with alpha-synuclein immunohistochemistry, and we divided the neurons into three subtypes according to differences in alpha-synuclein immunoreactivity: (1) neurons without pale bodies or Lewy bodies, (2) neurons with pale bodies, and (3) neurons with Lewy bodies. In controls, we did not observe fragmented GA in nigral neurons by immunocytochemistry with an anti-TGN46 antibody. In PD, the GA was fragmented in 3% of the nigral neurons without inclusions, and in 5% of the neurons with Lewy bodies. In contrast, fragmented GA was noted in 19% of the neurons containing pale bodies. Since pale bodies represent early stages in the development of brainstem Lewy bodies, our results suggest that the cytotoxicity of alpha-synuclein-positive aggregates is reduced in the process of Lewy body formation.

Aged↗

Isolation of plasma membranes and Golgi apparatus from a single chicken liver homogenate.

Chicken liver plasma membranes, minimally contaminated with Golgi apparatus-derived vesicles, were prepared from a low-speed (400 g) pellet by means of flotation in isotonic Percoll solution, followed by a hypotonic wash and flotation in a discontinuous sucrose gradient. Based on the analysis of suitable marker enzymes, alkaline phosphatase and alkaline phosphodiesterase, two plasma membrane fractions were isolated with enrichments, depending on the equilibrium density and marker of 28-97 and with a total yield of 4-5%. Golgi apparatus fractions were prepared by flotation of microsomes, obtained from the same homogenate as the low-speed pellet, in a discontinuous sucrose gradient. The trans-Golgi marker galactosyltransferase was 27-fold enriched in a fraction of intermediate density (d=1.077-1.116 g/ml). Approximately 12% of galactosyltransferase was recovered in the membranes equilibrating d=1.031-1.148 g/ml. Contamination with plasma membrane fragments was low in the light (d=1.031-1.077 g/ml) and intermediate density Golgi vesicles. The isolation of purified plasma membranes and Golgi vesicles from one liver homogenate will enable future studies on receptor cycling between these cell organelles.

Alkaline Phosphatase↗

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

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.

Acetylglucosaminidase↗

Flow cytometric detection of the Golgi apparatus using antibodies to glycosyltransferases.

Intracellular glycosyltransferase protein expression can be assessed by flow cytometry. We report the detectability of the Golgi associated beta1,4 galactosyltransferase (GT) and alpha2,6 sialyltransferase (ST) upon permeabilization in Jurkat and EBV-JY cells representing a T- and B-lymphoid cell line, respectively. The method employs fixation with paraformaldehyde and permeabilization with saponin. It ensures reliable internalization of the antibody and little background staining and does not cause leakage of the antigen. We first applied monoclonal antibodies to GT for establishment of this method by flow cytometry. The obtained flow cytometric signal could be localized to the Golgi apparatus by confocal laser scanning microscopy. To exclude interference from possible cell-surface staining, measurements were carried out on non-permeabilized cells. No signal was found in Jurkat cells, while low but measurable ecto-galactosyltransferase was found on EBV-JY cells. F(ab)'2 fragments of polyclonal antisera to GT and ST were generated and shown to be useful for double indirect staining with the monoclonal antibody. The method described here permits relative assessment of Golgi glycosyltransferase expression in lymphoid cells by flow cytometry.

Animals↗

PtK1 cells contain a nondiffusible, dominant factor that makes the Golgi apparatus resistant to brefeldin A.

Brefeldin A (BFA) was shown in earlier studies of numerous cell types to inhibit secretion, induce enzymes of the Golgi stacks to redistribute into the ER, and to cause the Golgi cisternae to disappear. Here, we demonstrate that the PtK1 line of rat kangaroo kidney cells is resistant to BFA. The drug did not disrupt the morphology of the Golgi complex in PtK1 cells, as judged by immunofluorescence using antibodies to 58- (58K) and 110-kD (beta-COP) Golgi proteins, and by fluorescence microscopy of live cells labeled with C6-NBD-ceramide. In addition, BFA did not inhibit protein secretion, not alter the kinetics or extent of glycosylation of the vesicular stomatitis virus (VSV) glycoprotein (G-protein) in VSV-infected PtK1 cells. To explore the mechanism of resistance to BFA, PtK1 cells were fused with BFA-sensitive CV-1 cells that had been infected with a recombinant SV-40 strain containing the gene for VSV G-protein and, at various times following fusion, the cultures were exposed to BFA. Shortly after cell fusion, heterokaryons contained one Golgi complex associated with each nucleus. Golgi membranes derived from CV-1 cells were sensitive to BFA, whereas those of PtK1 origin were BFA resistant. A few hours after fusion, most heterokaryons contained a single, large Golgi apparatus that was resistant to BFA and contained CV-1 galactosyltransferase. In unfused cells that had been perforated using nitrocellulose filters, retention of beta-COP on the Golgi was optimal in the presence of cytosol, ATP, and GTP. In perforated cell models of the BFA-sensitive MA104 line, BFA caused beta-COP to be released from the Golgi complex in the presence of nucleotides, and either MA104 or PtK1 cytosol. In contrast, when perforated PtK1 cells were incubated with BFA, nucleotides, and cytosol from either cell type, beta-COP remained bound to the Golgi complex. We conclude that PtK1 cells contain a nondiffusible factor, which is located on or very close to the Golgi complex, and confers a dominant resistance to BFA. It is possible that this factor is homologous to the target of BFA in cells that are sensitive to the drug.

Animals↗

Morphogenesis and dynamics of the yeast Golgi apparatus.

A kinetic and morphometric study was conducted with the electron microscope to clarify the biogenesis and structural diversity of the Golgi apparatus in the yeast Saccharomyces cerevisiae. Secretion was synchronized by inhibiting protein synthesis and/or by subjecting thermosensitive secretory mutants to double temperature shifts. Five membrane-bounded structures disappeared or reappeared in an orderly manner at approximately the rate of secretory protein flow. 1) The first detectable post-ER intermediates were very short-lived clusters of small vesicles that appeared next to the endoplasmic reticulum (ER). 2) Their constituent small vesicles were rapidly bridged by membrane tubules in a SEC18-dependent manner, giving short-lived tubular clusters of small vesicles, analogous to mammalian vesicular-tubular clusters. 3) Fine and 4) large nodular networks (coated with the Golgi protein Sec7), and 5) secretory granules. Upon relieving a secretory block, each structure successively reappeared, seemingly by transformation of the previous one. When no secretory cargo was to be transported, these structures were not renewed. They disappeared more than five times faster than some Golgi enzymes such as Och1p, implying that the latter are recycled and perhaps partially retained. Retention could arise from intra-compartmental flow of cargo/carrier, hinted at by the varying calibers within a single nodular network.

Adenosine Triphosphatases↗

Protein transport from the endoplasmic reticulum to the Golgi apparatus.

As the first step of protein transport along the biosynthetic (secretory/exocytotic) pathway, transport from the endoplasmic reticulum (ER) to the Golgi apparatus has received much attention over the past several decades. The general structural organization underlying this transport process is becoming more defined. The major protein components participating in the budding, pre-docking, and docking/fusion events have been identified and their mechanistic aspects investigated. Conceptually, it is now clear that protein export from the ER is a selective process. Although much remains to be defined or refined, the general picture of this transport step has now emerged.

Animals↗

Cytochemical studies on golgi apparatus, GERL, and lysosomes in neurons of dorsal root ganglia in mice.

Cytochemically demonstrable thiamine pyrophosphatase activity is present in the innermost Golgi element in both small and large neurons of the dorsal root ganglia in CF1, C57 black, and C57 beige mice, thus resembling the neurons of rat dorsal root ganglia. The localization of acid phosphatase (EC 3.1.3.2) activity in the large neurons of dorsal root ganglia in these mice is also similar to that in rats; it is not demonstrable in Golgi elements but is present in GERL and in three types of lysosomes apparently derived from GERL. However, the small neurons of the mouse differ from those of the rat in showing acid phosphatase activity in all elements of the Golgi apparatus. In the mouse neurons the acid phosphatase activity of residual bodies is "latent," i.e., it is not demonstrable in well-preserved cells.

Acid Phosphatase↗

Efficient trafficking of ceramide from the endoplasmic reticulum to the Golgi apparatus requires a VAMP-associated protein-interacting FFAT motif of CERT.

Ceramide is synthesized at the endoplasmic reticulum (ER) and transported to the Golgi apparatus by CERT for its conversion to sphingomyelin in mammalian cells. CERT has a pleck-strin homology (PH) domain for Golgi targeting and a START domain catalyzing the intermembrane transfer of ceramide. The region between the two domains contains a short peptide motif designated FFAT, which is supposed to interact with the ER-resident proteins VAP-A and VAP-B. Both VAPs were actually co-immunoprecipitated with CERT, and the CERT/VAP interaction was abolished by mutations in the FFAT motif. These mutations did not affect the Golgi targeting activity of CERT. Whereas mutations of neither the FFAT motif nor the PH domain inhibited the ceramide transfer activity of CERT in a cell-free system, they impaired the ER-to-Golgi transport of ceramide in intact and in semi-intact cells at near endogenous expression levels. By contrast, when overexpressed, both the FFAT motif and the PH domain mutants of CERT substantially supported the transport of ceramide from the ER to the site where sphingomyelin is produced. These results suggest that the Golgi-targeting PH domain and ER-interacting FFAT motif of CERT spatially restrict the random ceramide transfer activity of the START domain in cells.

Amino Acid Motifs↗