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PubMed1998

Mapping the interaction between GRASP65 and GM130, components of a protein complex involved in the stacking of Golgi cisternae.

The nature of the complex containing GRASP65, a membrane protein involved in establishing the stacked structure of the Golgi apparatus, and GM130, a putative Golgi matrix protein and vesicle docking receptor, was investigated. Gel filtration revealed that GRASP65 and GM130 interact in detergent extracts of Golgi membranes under both interphase and mitotic conditions, and that this complex can bind to the vesicle docking protein p115. Using in vitro translation and site-directed mutagenesis in conjunction with immunoprecipitation, the binding site for GRASP65 on GM130 was mapped to the sequence xxNDxxxIMVI-COOH at the C-terminus of GM130, a region known to be required for its localization to the Golgi apparatus. The same approach was used to show that the binding site for GM130 on GRASP65 maps to amino acids 189-201, a region conserved in the mammalian and yeast proteins and reminiscent of PDZ domains. Using green fluorescent protein (GFP)-tagged reporter constructs, it was shown that one essential function of the interaction between GRASP65 and GM130 is in the correct targeting of the two proteins to the Golgi apparatus.

Amino Acid Sequence↗
PubMed1983

Intracellular transport and storage of secretory proteins in relation to cytodifferentiation in neoplastic pancreatic acinar cells.

The pancreatic acinar carcinoma established in rat by Reddy and Rao (1977, Science 198:78-80) demonstrates heterogeneity of cytodifferentiation ranging from cells containing abundant well-developed secretory granules to those with virtually none. We examined the synthesis intracellular transport and storage of secretory proteins in secretory granule-enriched (GEF) and secretory granule-deficient (GDF) subpopulations of neoplastic acinar cells separable by Percoll gradient centrifugation, to determine the secretory process in cells with distinctly different cytodifferentiation. The cells pulse-labeled with [3H]leucine for 3 min and chase incubated for up to 4 h were analyzed by quantitative electron microscope autoradiography. In GEF neoplastic cells, the results of grain counts and relative grain density estimates establish that the label moves successively from rough endoplasmic reticulum (RER) leads to the Golgi apparatus leads to post-Golgi vesicles (vacuoles or immature granules) leads to mature secretory granules, in a manner reminiscent of the secretory process in normal pancreatic acinar cells. The presence of approximately 40% of the label in association with secretory granules at 4 h postpulse indicates that GEF neoplastic cells retain (acquire) the essential regulatory controls of the secretory process. In GDF neoplastic acinar cells the drainage of label from RER is slower, but the peak label of approximately 20% in the Golgi apparatus is reached relatively rapidly (10 min postpulse). The movement of label from the Golgi to the post-Golgi vesicles is evident; further delineation of the secretory process in GDF neoplastic cells, however, was not possible due to lack of secretory granule differentiation. The movement of label from RER leads to the Golgi apparatus leads to the post-Golgi vesicles suggests that GDF neoplastic cells also synthesize secretory proteins, but to a lesser extent than the GEF cells. The reason(s) for the inability of GDF cells to concentrate and store exportable proteins remain to be elucidated.

Animals↗
PubMed1983

Biological effects of weightlessness and clinostatic conditions registered in cells of root meristem and cap of higher plants.

Research in cellular reproduction, differentiation and vital activity, i.e. processes underlying the development and functioning of organisms, plants included, is essential for solving fundamental and applied problems of space biology. Detailed anatomical analysis of roots of higher plants grown on board the Salyut 6 orbital research station show that under conditions of weightlessness for defined duration mitosis, cytokinesis and tissue differentiation in plant vegetative organs occur essentially normally. At the same time, certain rearrangements in the structural organization of cellular organelles--mainly the plastid apparatus, mitochondria, Golgi apparatus and nucleus--are established in the root meristem and cap of the experimental plants. This is evidence for considerable changes in cellular metabolism. The structural changes in the subcellular level arising under spaceflight conditions are partially absent in clinostat experiments designed to simulate weightlessness. Various clinostatic conditions have different influences on the cell structural and functional organization than does space flight. It is suggested that alterations of cellular metabolism under weightlessness and clinostatic conditions occur within existing genetic programs.

Arabidopsis↗
PubMed1991

Enzymology of intracellular membrane fusion.

A combination of cell-free biochemical and morphological studies has revealed that a coated bud-coated vesicle transport system shuttles newly synthesized proteins through the successive processing compartments of the Golgi apparatus. These "Golgi coated vesicles" operate in a manner formally analogous to the clathrin coated pit-coated vesicle system responsible for receptor-mediated endocytosis; however Golgi coated vesicles do not contain clathrin.

Biological Transport↗
PubMed2002

Immunopurification of Golgi vesicles by magnetic sorting.

We have designed a method that permits to isolate highly purified Golgi vesicles deprived of endoplasmic reticulum (ER), main contaminant of Golgi fractions. To this end, we prepared a rabbit polyclonal antibody against the cytosolic N-terminal oligopeptide of the enzyme heparan glucosaminyl N-deacetylase/N-sulphotransferase (HSST), a specific marker for Golgi apparatus. The Golgi localization of HSST was confirmed by indirect immunofluorescence microscopy. The antibody binding to Golgi vesicles was demonstrated by immunoelectronmicroscopy and allowed the immunopurification by magnetic sorting. Golgi vesicles subjected to purification by magnetic sorting showed the presence of HSST and p28, which is an integral membrane protein on the cis-Golgi also used as a specific Golgi marker. The purified material was devoid of calreticulin, a specific ER marker. This purification method will allow to improve studies requiring highly purified Golgi membranes such as identification of specific receptors and the electrophysiological characterization of Golgi membrane ion channels, which have been jeopardized up to now by ER membrane contamination.

Amidohydrolases↗
PubMed1989

Mitotic Golgi fragments in HeLa cells and their role in the reassembly pathway.

Immunoelectron microscopy and stereology were used to identify and quantitate Golgi fragments in metaphase HeLa cells and to study Golgi reassembly during telophase. On ultrathin frozen sections of metaphase cells, labeling for the Golgi marker protein, galactosyltransferase, was found over multivesicular Golgi clusters and free vesicles that were found mainly in the mitotic spindle region. The density of Golgi cluster membrane varied from cell to cell and was inversely related to the density of free vesicles in the spindle. There were thousands of free Golgi vesicles and they comprised a significant proportion of the total Golgi membrane. During telophase, the distribution of galactosyltransferase labeling shifted from free Golgi vesicles towards Golgi clusters and the population of free vesicles was depleted. The number of clusters was no more than in metaphase cells so the observed fourfold increase in membrane surface meant that individual clusters had increased in size. More than half of these had cisterna(e) and were located next to "buds" on the endoplasmic reticulum. Early in G1 the number of clusters dropped as they congregated in the juxtanuclear region and fused. These results show that fragmentation of the Golgi apparatus yields Golgi clusters and free vesicles and reassembly from these fragments is at least a two-step process: (a) growth of a limited number of dispersed clusters by accretion and fusion of vesicles to form cisternal clusters next to membranous "buds" on the endoplasmic reticulum; (b) congregation and fusion to form the interphase Golgi stack in the juxtanuclear region.

Cell Division↗
PubMed1968

Distribution of phosphatases in the golgi region and associated structures of the thoracic ganglionic neurons in the grasshopper, Melanoplus differentialis.

The neuronal perikarya of the grasshopper contain sudanophilic lipochondria which exhibit an affinity for vital dyes. These lipochondria are membrane-delimited and display acid phosphatase activity; hence they correspond to lysosomes. Unlike those of most vertebrates, these lysosomes also hydrolyze thiamine pyrophosphate and adenosine triphosphate. Like vertebrate lysosomal "dense bodies," they are electron-opaque and contain granular, vesicular, or lamellar material. Along with several types of smaller dense bodies, they are found in close spatial association with the Golgi apparatus. The Golgi complexes are frequently arranged in concentric configurations within which these dense bodies lie. Some of the smaller dense bodies often lie close to or in association with the periphery of dense multivesicular bodies. Further, bodies occur that display gradations in structure between these multivesicular bodies and the dense lysosomes. Acid phosphatase activity is present in the small as well as the larger dense bodies, in the multivesicular bodies, and in some of the Golgi saccules, associated vesicles, and fenestrated membranes; thiamine pyrophosphatase is found in both the dense bodies and parts of the Golgi complex. The close spatial association of these organelles, together with their enzymatic similarities, suggests the existence of a functional or developmental relationship between them.

Acid Phosphatase↗
PubMed1981

Organization and relationship of the axonal reticulum in the cell body of sympathetic ganglion cells.

Previous findings and results obtained with the zinc iodide-osmium tetroxide method and a method for demonstration of biogenic amines indicate that the so-called axonal smooth endoplasmic reticulum is directly related to inner elements of the Golgi apparatus. These Golgi elements provide a network for local catecholamine synthesis and/or storage in the cell body and in the axon terminals.

Animals↗
PubMed1978

Human malignant melanoma. Melanosomal polymorphism and the ultrastructural dopa reaction.

An ultrastructural dopa reaction, with glutaraldehyde--formaldehyde prefixation, was carried out on ten specimens of malignant melanoma showing a wide variation in melanosomal morphology. All tumours, but only a minority of tumour cells, contained reaction product. In all tumours the reaction product was distributed similarly in the Golgi apparatus and Golgi associated endoplasmic reticulum (GERL). However, differences were noted in its deposition in melanosomes. Whereas vacuolar and lamellar profiles sometimes contained reaction product, it was not seen in normal, granular and abortive melanosomes. Tumour cells without melanosomes were also seen to contain reaction product.

Dihydroxyphenylalanine↗
PubMed1981

Some findings on the cytochemistry of the thyroid follicle epithelial cell in rats and mice.

The fine structural localization of endogenous peroxidase was studied in follicle epithelial cells of the rat thyroid. The localization of thiamine pyrophosphatase (TPPase) and acid phosphatase (AcPase) as well as the result of long time osmium impregnation in thyroid follicle epithelial cells were examined in the mouse. All the subapical secretory granules are principally peroxidase-positive, while the reabsorbed colloid droplets are almost peroxidase-negative. In addition, the cisternae of the rough endoplasmic reticulum, the nuclear envelope, 1-3 lamellae on the trans-side of the Golgi apparatus, rigid lamellae, and small vesicles on the trans-side of the Golgi apparatus, and the peripheral part of the follicle lumen are usually positive for endogenous peroxidase activity. The Golgi lamellae of the cis-side are very weakly positive. The TPPase activity is localized in 1-3 lamellae and vesicles on the trans-side of the Golgi apparatus and sometimes in rigid lamellae, while the AcPase activity is in 1-3 lamellae and smooth or coated vesicles on the trans-side of the Golgi apparatus, rigid lamellae, lysosomes and some colloid droplets. The areas positive for both enzyme activities are fairly well overlapped. Functional heterogeneity in the Golgi lamellae is suggested by the fact that TPPase, AcPase and endogenous peroxidase activities are limited on the trans-side of the Golgi apparatus, while osmium tetroxide stains only the cisternae of 1-2 lamellae on the cis-side of the Golgi apparatus. From these findings, GERL can be considered a part of the Golgi apparatus and not independent from this organelle in the thyroid follicle epithelial cell.

Acid Phosphatase↗
PubMed1997

Effects of brefeldin A on autophagy in cultured rat fibroblasts.

Effects of brefeldin A on cellular autophagy were studied in cultured rat fibroblasts. Brefeldin A inhibits the activation and membrane-binding properties of most ADP-ribosylation factors and causes the redistribution of Golgi proteins into the endoplasmic reticulum. Immunofluorescence and enzyme cytochemical methods revealed the disappearance of the Golgi apparatus and trans-Golgi network during the brefeldin A incubation. The volume fractions of autophagic vacuoles increased about threefold in cells treated with brefeldin A for 4 h and about sixfold in serum-deprived cells as compared with controls. When cells were first treated with brefeldin A for 1 h and were then deprived of serum and treated with brefeldin A for 3 h, the volume fraction of autophagic vacuoles increased about 4.5-fold as compared with untreated cells. The results showed that brefeldin A is unable to prevent serum deprivation-induced accumulation of autophagic vacuoles and that brefeldin A even when acting alone increases the volume fraction of autophagic vacuoles. It was concluded that an intact Golgi apparatus and trans-Golgi network are not essential for the formation of autophagic vacuoles. It seems also probable that ADP-ribosylation factors are not needed when vacuoles are formed.

Acid Phosphatase↗
PubMed2003

A serine protease inhibitor prevents endoplasmic reticulum stress-induced cleavage but not transport of the membrane-bound transcription factor ATF6.

Mammalian cells express several transcription factors embedded in the endoplasmic reticulum (ER) as transmembrane proteins that are activated by proteolysis, and two types of these proteins have been extensively investigated. One type comprises the sterol regulatory element-binding proteins (SREBP-1 and SREBP-2). The other type comprises the activating transcription factors 6 (ATF6alpha and ATF6beta), which are activated in response to ER stress. It was shown previously that both SREBP and ATF6 are cleaved sequentially first by the Site-1 protease (serine protease) and then by the Site-2 protease (metalloprotease) (Ye, J., Rawson, R. B., Komuro, R., Chen, X., Dave, U. P., Prywes, R., Brown, M. S., and Goldstein, J. L. (2000) Mol. Cell 6, 1355-1364). In this study, we examined various protease inhibitors and found that 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), a serine protease inhibitor, prevented ER stress-induced cleavage of ATF6alpha and ATF6beta, resulting in inhibition of transcriptional induction of ATF6-target genes. AEBSF also inhibited production of the mature form of SREBP-2 that was induced in response to sterol depletion, and appeared to directly prevent cleavage of ATF6alpha and ATF6beta by inhibiting Site-1 protease. As the Site-1 protease is localized in the Golgi apparatus, both SREBP and ATF6 must relocate to the Golgi apparatus to be cleaved. We showed here that AEBSF treatment had little effect on ER stress-induced translocation of ATF6 from the ER to the Golgi apparatus, but blocked nuclear localization of ATF6. These results indicate that the transport of ATF6 from the ER to the Golgi apparatus and that from the Golgi apparatus to the nucleus are distinct steps that can be distinguished by treatment with AEBSF.

Activating Transcription Factor 6↗
PubMed2001

Regulation of Golgi structure and function by ARF-like protein 1 (Arl1).

Arl1 is a member of the ARF-like protein (Arl) subfamily of small GTPases. Nothing is known about the function of Arl1 except for the fact that it is essential for normal development in Drosophila and that it is associated with the Golgi apparatus. In this study, we first demonstrate that Arl1 is enriched at the trans side of the Golgi, marked by AP-1. Association of Arl1 with the Golgi is saturable in intact cells and depends on N-terminal myristoylation. Over-expression of Arl1(T31N), which is expected to be restricted to the GDP-bound form and thus function as a dominant-negative mutant, causes the disappearance of the Golgi apparatus (marked by Golgi SNARE GS28), suggesting that Arl1 is necessary for maintaining normal Golgi structure. Overexpression of Arl1(Q71L), a mutant restricted primarily to the activated GTP-bound form, causes an expansion of the Golgi apparatus with massive and stable Golgi association of COPI and AP-1 coats. Interestingly, Golgi ARFs also become stably associated with the expanded Golgi. Transport of the envelope protein of vesicular stomatitis virus (VSV-G) along the secretory pathway is arrested at the expanded Golgi upon expression of Arl1(Q71L). The structure of stacked cisternae of the Golgi is disrupted in cells expressing Arl1(Q71L), resulting in the transformation of the Golgi into an extensive vesicule-tubule network. In addition, the GTP form of Arl1 interacts with arfaptin-2/POR1 but not GGA1, both of which interact with GTP-restricted ARF1, suggesting that Arl1 and ARF1 share some common effectors in regulating cellular events. On the basis of these observations, we propose that one of the mechanisms for the cell to regulate the structure and function of the Golgi apparatus is through the action of Arl1.

ADP-Ribosylation Factors↗
PubMed1991

Determination of the intracellular sites and topology of glucosylceramide synthesis in rat liver.

We examined the intracellular site(s) and topology of glucosylceramide (GlcCer) synthesis in subcellular fractions from rat liver, using radioactive and fluorescent ceramide analogues as precursors, and compared these results with those obtained in our recent study of sphingomyelin (SM) synthesis in rat liver [Futerman, Stieger, Hubbard & Pagano (1990) J. Biol. Chem. 265, 8650-8657]. In contrast with SM synthesis, which occurs principally at the cis/medial Golgi apparatus, GlcCer synthesis was more widely distributed, with substantial amounts of synthesis detected in a heavy (cis/medial) Golgi-apparatus subfraction, a light smooth-vesicle fraction that is almost devoid of an endoplasmic-reticulum marker enzyme (glucose-6-phosphatase), and a heavy vesicle fraction. Furthermore, no GlcCer synthesis was detected in an enriched plasma-membrane fraction after accounting for contamination by Golgi-apparatus membranes. These results suggest that a significant amount of GlcCer may be synthesized in a pre- or early Golgi-apparatus compartment. Unlike SM synthesis, which occurs at the luminal surface of the Golgi apparatus, GlcCer synthesis appeared to occur at the cytosolic surface of intracellular membranes, since (i) limited proteolytic digestion of intact Golgi-apparatus vesicles almost completely inhibited GlcCer synthesis, and (ii) the extent of UDP-glucose translocation into the Golgi apparatus was insufficient to account for the amount of GlcCer synthesis measured. These findings imply that, after its synthesis, GlcCer must undergo transbilayer movement to the luminal surface to account for the known topology of higher-order glycosphingolipids within the Golgi apparatus and plasma membrane.

Animals↗
PubMed1991

Cell-free transfer of membrane lipids. Evidence for lipid processing.

A latent phospholipase A is concentrated in cis elements of rat liver Golgi apparatus, the presumed sites of fusion of the 50-70-nm transition vesicles formed from endoplasmic reticulum. As a result, conversion of transferred phospholipids to their corresponding lysoforms may provide an index of post transfer lipid processing in a corresponding reconstituted membrane transfer system. To label the phosphatidylcholine of transitional endoplasmic reticulum in vitro, [14C]CDP-choline and endogenous cytidyltransferases were used. In the reconstituted transfer system, the radiolabeled phosphatidylcholine was transferred via transition vesicles to Golgi apparatus immobilized on nitrocellulose strips in a time- and temperature-dependent process. Transfer was promoted by ATP and the ATP-dependent transfer was specific for cis Golgi apparatus elements as acceptor. Trans Golgi apparatus elements were ineffective as acceptors. Median Golgi apparatus elements were intermediate. A portion of the transferred phosphatidylcholine was converted subsequently to lysophosphatidylcholine also in a time- and ATP-dependent manner. The phospholipase A activity of the Golgi apparatus was more than 90% latent (active site located on the lumens of the Golgi apparatus membranes). Therefore, the lipid-containing vesicles derived from endoplasmic reticulum must have combined with cis Golgi apparatus membranes as the basis for Golgi apparatus-dependent phospholipase A processing of endoplasmic reticulum-derived phosphatidylcholine. Since the lipids were processed by phospholipase A in approximately the same proportion as occurs in situ, the findings offer evidence both for the specificity of the ATP-dependent component of cell-free lipid transfer from endoplasmic reticulum to Golgi apparatus and its fidelity to lipid transfer observed in vivo.

Adenosine Triphosphate↗
PubMed1980

Cytochemical demonstration of NADH-ferricyanide reductase and nucleoside diphosphatase activities in dictyosome-like structures of guinea pig spermatocytes.

Structures superficially resembling dictyosomes of Golgi apparatus are present in guinea pig spermatocytes and in spermatids in late stages of development. They coexist with Golgi apparatus. In this report, we demonstrate that the dictyosome-like structures (DLS) and Golgi apparatus share cytochemical "markers", inosine diphosphatase and thiamine pyrophosphatase. Additionally, the cytochemical marker for the mature face of conventional Golgi apparatus as well as most plasma membranes, a glutaraldehyde-resistant NADH-ferricyanide reductase, is present in DLS. The latter reaction is also given by the membranes of the acrosome and that portion of the conventional Golgi apparatus (i.e., the thick cisternae) presumed to be responsible for acrosome formation. A distinguishing feature between DLS and Golgi apparatus is in the distribution of reaction product. Golgi apparatus reaction product is concentrated toward one face of each dictyosome while DLS reaction product is usually randomly distributed across the stacks.

Acid Anhydride Hydrolases↗
PubMed1990

Alteration of intracellular traffic by monensin; mechanism, specificity and relationship to toxicity.

Monensin, a monovalent ion-selective ionophore, facilitates the transmembrane exchange of principally sodium ions for protons. The outer surface of the ionophore-ion complex is composed largely of nonpolar hydrocarbon, which imparts a high solubility to the complexes in nonpolar solvents. In biological systems, these complexes are freely soluble in the lipid components of membranes and, presumably, diffuse or shuttle through the membranes from one aqueous membrane interface to the other. The net effect for monensin is a trans-membrane exchange of sodium ions for protons. However, the interaction of an ionophore with biological membranes, and its ionophoric expression, is highly dependent on the biochemical configuration of the membrane itself. One apparent consequence of this exchange is the neutralization of acidic intracellular compartments such as the trans Golgi apparatus cisternae and associated elements, lysosomes, and certain endosomes. This is accompanied by a disruption of trans Golgi apparatus cisternae and of lysosome and acidic endosome function. At the same time, Golgi apparatus cisternae appear to swell, presumably due to osmotic uptake of water resulting from the inward movement of ions. Monensin effects on Golgi apparatus are observed in cells from a wide range of plant and animal species. The action of monensin is most often exerted on the trans half of the stacked cisternae, often near the point of exit of secretory vesicles at the trans face of the stacked cisternae, or, especially at low monensin concentrations or short exposure times, near the middle of the stacked cisternae. The effects of monensin are quite rapid in both animal and plant cells; i.e., changes in Golgi apparatus may be observed after only 2-5 min of exposure. It is implicit in these observations that the uptake of osmotically active cations is accompanied by a concomitant efflux of H+ and that a net influx of protons would be required to sustain the ionic exchange long enough to account for the swelling of cisternae observed in electron micrographs. In the Golgi apparatus, late processing events such as terminal glycosylation and proteolytic cleavages are most susceptible to inhibition by monensin. Yet, many incompletely processed molecules may still be secreted via yet poorly understood mechanisms that appear to bypass the Golgi apparatus. In endocytosis, monensin does not prevent internalization. However, intracellular degradation of internalized ligands may be prevented.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1983

A cytochemical study of acid phosphatase, thiamine pyrophosphatase and horseradish peroxidase in the Golgi-GERL complex of hepatoma ascites cells.

Data from studies of ascitic cells of Chang hepatoma have shown that acid phosphatase (ACPase) can be localized simultaneously within the trans portion of the Golgi apparatus and in tubules of the Golgi-endoplasmic reticulum-lysosome (GERL) system. Reaction products of thiamine pyrophosphatase (TPPase) were also present consistently within trans elements of the Golgi apparatus and within GERL tubules. These new findings indicate that a close physiological association may exist between the Golgi apparatus and GERL, a concept that is consistent with previous observations of fibroblasts. When horseradish peroxidase (PO) is injected intraperitoneally into ascites-bearing rats and the ascitic cells withdrawn at different time intervals, PO could be localized within vesicles and tubules in the GERL region but could not be detected within the Golgi apparatus. Bulk-phase endocytosis requires a long time and a high concentration of PO to occur. The presence of PO within GERL indicates that this organelle may play a role in transporting or processing of certain exogenous proteins.

Acid Phosphatase↗