Function Of Golgi Apparatus Research Articles

The functioning of the yeast Golgi apparatus requires an ER protein encoded by ANP1, a member of a new family of genes affecting the secretory pathway.

Mnt1p is an alpha 1.2-mannosyltransferase which resides in an early compartment of the Saccharomyces cerevisiae Golgi apparatus. We have shown that the signal-anchor region is sufficient, and the transmembrane domain necessary, for its normal Golgi localization. This is similar to the transmembrane domain-mediated retention of mammalian glycosyltransferases, and distinct from the tail-mediated recycling retention of certain mammalian and yeast trans-Golgi proteins. To examine the mechanism involved in transmembrane domain-mediated retention, we have isolated six classes of mutants which fail to retain Mnt1p-reporter fusions in the early Golgi. These mutants all show additional phenotypes which are consistent with alterations in Golgi function. We have called the mutant classes 'gem', for Golgi enzyme maintenance. GEM3 is identical to the previously cloned gene ANP1, and homologous to VAN1 and MNN9. Together, these define a new class of proteins involved in the organization and functioning of the secretory pathway. Interestingly, Anp1p is localized to the endoplasmic reticulum (ER), implying that some function of the ER is required to maintain a functional Golgi apparatus.

Histochemical and autoradiographic studies on elcatonin internalization and intracellular movement in osteoclasts.

The binding sites and chronologic localization of elcatonin (eCT) in osteoclasts were examined by autoradiography using [125I]elcatonin (125I-eCT). In addition to the structural changes induced by calcitonin (CT) reported so far, changes were also observed in the structure of Golgi apparatus. These changes continued until 48-72 h after incubation with eCT. Developed silver grains of 125I-eCT were localized into multinucleated osteoclasts and mononuclear cells that were ultrastructurally defined as "preosteoclasts." The silver grains located on plasma membranes of those cells and were then internalized; they accumulated, especially in the Golgi apparatus, and remained for 48-72 h. A few silver grains were also detected in lysosomes and small vesicles. The decrease in the number of silver grains in the Golgi apparatus accompanied the recovery of osteoclast structures--Golgi apparatus and then ruffled borders. These findings suggest that (1) CT especially inhibits the sorting function of Golgi apparatus in osteoclasts, resulting in prolonged retention of CT in this organelle. (2) The CT in Golgi apparatus may keep its activity and cause the prolonged effect of CT on osteoclast activity.

Inhibition of Golgi-apparatus function by brefeldin A in maize coleoptiles and its consequences on auxin-mediated growth, cell-wall extensibility and secretion of cell-wall proteins

Brefeldin A (BFA), a fungal metabolite causing dysfunction of the Golgi apparatus in plant and animal cells, was used to investigate the role of secretory processes at the plasma membrane in auxin-mediated elongation growth of maize (Zea mays L.) coleoptiles. In abraded coleoptile segments BFA produced, within less than 30 min, a decrease in the incorporation of [3H]leucine into tightly bound cell-wall proteins, accompanied by an increased incorporation into the intracellular pool of putative cell-wall glycoproteins. Total protein synthesis was not affected. Electron micrographs revealed striking morphological changes in dictyosomes (especially vesiculation of trans-cisternae), accumulation of Golgi vesicles and dilation of the endoplasmic reticulum. These effects are taken as indication that BFA interferes with the secretion of cell-wall components. Elongation growth of coleoptile segments in the presence and absence of auxin was inhibited by 80% in 20 mg·l−1 BFA. If BFA was applied to segments growing in the presence of auxin, maximum inhibition was reached after about 30 min, indicating that the growth response depends on an uninterrupted supply of a cell-wall or plasma-membrane component (“wall-loosening factor”) delivered by the secretory pathway. After its secretion, this factor has a rather short growth-effective life time. The inhibition of auxin-mediated growth by BFA was accompanied by an elimination of auxin-induced cell-wall extensibility and by an inhibition of auxin-induced proton excretion. Fusicoccin-induced proton excretion was similarly affected by BFA. It is concluded that both the wall-loosening process underlying elongation growth as well as proton excretion depend on an intact secretory pathway from the Golgi apparatus to the cell wall; however, a causal relationship between these processes is not warranted by the data.

Unique properties of cathepsin D purified from alcoholic cirrhotic liver

Abstract In order to show the possibility of the impaired function of Golgi apparatus in alcoholic cirrhotic liver, we studied the property of sugar chains on purified cathepsin D, which was an aspartic protease in lysosome. Alcoholic cirrhotic liver contained a large amount of cathepsin D which had no binding affinity for concanavalin A. On the other hand, no cathepsin D was detected in concanavalin A-unbound fractions from normal liver, viral cirrhotic liver and hepatoma tissues. In the treatment with exogenous hydrolases to cleave carbohydrate moieties, abnormal cathepsin D purified from alcoholic cirrhotic liver contained altered sugar chains. Since the processing of oligosaccharide moieties on lysosomal hydrolases are performed in Golgi apparatus, the alteration of sugar chains suggests that the abnormal intracellular processing is caused by the dysfunction of Golgi apparatus. Unique cathepsin D described here may support the hypothesis that the impaired function of Golgi apparatus is associated with the mechanism of alcoholic liver injury.

Brefeldin A blocks the response of cultured cells to cholera toxin. Implications for intracellular trafficking in toxin action

Cholera toxin (CT) consists of a pentameric B subunit which binds to ganglioside GM1 on the cell surface and an A subunit which activates adenylylcyclase. The latter process involves the reduction of A to the A1 peptide which ADP-ribosylates the stimulatory G protein, Gs of adenylylcyclase. There is a distinct lag phase between toxin binding and activation of adenylylcyclase. Little is known about the events during this lag including where A1 is generated and how it gains access to Gs on the cytoplasmic side of the plasma membrane. We explored the effects of several inhibitors of intracellular trafficking on the response of human SK-N-MC neurotumor and Caco-2 intestinal tumor cells to CT. Whereas chloroquine or monensin had little or no effect on CT stimulation of cyclic AMP accumulation, brefeldin A (BFA) totally inhibited the response to CT in a time- and dose-dependent and reversible manner. BFA was effective when added at the same time as CT and had an IC50 of 30 ng/ml. BFA did not alter cell surface GM1 as cells treated with BFA for 30 min bound as much 125I-CT as control cells. Furthermore, BFA inhibited CT stimulation of GM1-treated rat glioma C6 cells. BFA treatment did not affect beta-adrenergic agonist stimulation of cyclic AMP. In addition, adenylylcyclase was activated by A1 peptide and NAD+ to the same extent in membranes from control and BFA-treated cells, or when BFA was added directly to the assay. Whereas control cells generated small amounts of A1 from bound CT with time, no A1 was detected in BFA-treated cells. BFA treatment did not prevent the internalization of CT but did inhibit its degradation. BFA is known to disrupt the organization of the Golgi complex, resulting in inhibition of protein transport from the endoplasmic reticulum and redistribution of Golgi enzymes to the endoplasmic reticulum. BFA also prevents the formation of non-clathrin-coated vesicles from Golgi membranes and thus vesicular transport between Golgi cisternae. We confirmed that BFA caused the morphological disruption of the Golgi apparatus in Caco-2 cells. The data support a role for a functional Golgi apparatus with its associated vesicular routing in CT action.

Chronic ethanol consumption induces accumulation of proteins in the liver Golgi apparatus and decreases galactosyltransferase activity.

The effect of chronic ethanol consumption on labeled glycoprotein secretion and galactosyltransferase activity has been analyzed in cis- and trans-Golgi apparatus fractions isolated from rat liver. Rats were injected intraperitoneally with 3H-leucine, after different chase periods (30, 60, 180 min), and the radioactivity of the different subcellular fractions as well as of the isolated Golgi apparatus was measured. Chronic alcohol treatment induces an increase in liver weight as well as an enhancement of total liver protein. Ethanol treatment produces a significant accumulation of labeled proteins in isolated Golgi apparatus fractions after a 60- and 180-min chase. An accumulation of labeled proteins in the cytosolic fraction was observed only after 180 min. The alcohol treatment also induces a significant decrease in the activity of galactosyltransferase in both liver homogenate and Golgi apparatus fractions. These results suggest that an impairment of Golgi apparatus functions, including glycosylation and glycoprotein trafficking, could be one of the mechanisms involved in the accumulation of hepatic protein and thus in the pathogenesis of alcohol-induced injury in the liver of chronic ethanol-consuming animals.

Swelling response of golgi apparatus cisternae in cells treated with monensin is reduced by cell injury

The effect of mechanical stress on Golgi apparatus was examined in thin slices of rat liver. The findings should be of relevance both to electron microscopists who routinely mince tissue, and to biochemists who homogenize tissues to isolate membranous components. The swelling response of Golgi apparatus to monensin was used as an assay because the swelling response is distinct and is thought to result from a well-characterized metabolic process, namely the acidification of vesicles. The results showed that the swelling response was compromised by monensin as far away as 6–7 cells from a cut surface even though other aspects of cell ultrastructure were not altered from normal. The monensin-induced swelling response was also evaluated in isolated Golgi apparatus and found to be similar to that with tissue. Thus, mechanical stress such as commonly used to mince tissue or isolate tissue components, appears to markedly alter Golgi apparatus function compared to the situation in vivo. In this example, the altered response of Golgi apparatus to monensin indicated that some aspects associated with the ATP-dependant proton-pumping machinery of the transmost cisternae and trans Golgi network were compromised.

The effect of cicloxolone sodium on the replication of vesicular stomatitis virus in BSC-1 cells.

The effect of cicloxolone sodium (CCX) on the replication of vesicular stomatitis virus (VSV) was investigated. The drug was active during all stages of the virus replication cycle, indicating that it does not operate by the specific inhibition of any single essential virus gene product. The drug reduced the number of VSV particles assembled and released by 100- to 1000-fold. Infectious virus yield was reduced 1000- to 10000-fold, giving a 10-fold or greater increase in the particle/p.f.u. ratio. The reduced number of virus particles produced in the presence of CCX results from two superimposed effects: suppression of VSV secondary transcription and viral protein synthesis, and perturbation of virion assembly. The inhibition of VSV assembly is due to impairment of a Golgi apparatus function related to transport of VSV glycoprotein G to the cell surface, and is characterized by accumulation of viral G and M proteins within the cell. Incubation of VSV-infected cells in the presence of two glycosylation inhibitors, tunicamycin and monensin, similarly leads to intracellular accumulation of G and M proteins, suggesting a common mechanism of action affecting VSV virion assembly. The differential effect of CCX concentration on intracellular levels of the L, N and NS proteins was analysed. CCX also possesses a virucidal effect on mature infectious VSV particles in suspension, 300 microM reducing the VSV titre about 10-fold in 24 h at 4 degrees C or 37 degrees C. The mode of antiviral activity against VSV is compared with that against herpes simplex virus.

A Cytochemical Ultrastructural Study of the Lysosomal System of Different Species of Malaria Parasites

We have used ultrastructural techniques in different malarial species to demonstrate a lysosomal system. First, we have tried to localize acid phosphatase, a typical lysosomal label. Its activity was localized in the endoplasmic reticulum and in endocytic vesicles, and in dense-cored vesicles near the digestive vacuoles, especially in Plasmodium falciparum (FCR3 strain). Then, we have studied the different cellular compartments of the malarial parasite by the zinc iodide-osmium tetroxide technique that heavily contrasted the cellular compartments of the parasite. This experiment led to the observation of a profound rearrangement of the endoplasmic reticulum, especially in P. berghei. A very atypical but functional Golgi apparatus was demonstrated in all the growing stages of the parasite and lysosome-like vesicles were observed, showing a structure very similar to those of the coated vesicles of a true Golgi complex. The presence of these organelles are in favor of the existence of a lysosomal system and of the endogenicity of some enzymes involved hemoglobin degradation.

Endocytosis in plants

Endocytosis in animal cells has been heavily documented. Both fluid‐phase and receptor‐mediated modes of uptake have been frequently studied, and the endocytic pathway is well defined. This contrasts markedly with the situation in plants where our knowledge of this process is still rudimentary. Partly responsible for this situation has been the view, widely held among plant physiologists, that because of turgor, endocytosis cannot occur in plant cells. As discussed below, the case against endocytosis is no longer water‐tight.Endocytosis is a fact in protoplasts. It can be demonstrated with electron‐dense tracers as well as with membrane impermeant dye Lucifer Yellow CH. The latter has also been used with success on both suspension‐cultured and tissue cells of higher plants, suggesting that fluid‐phase endocytosis is also a feature of cells with walls. Through the application of fluorescently labelled elicitor molecules, which specifically bind to the cell surface of suspension‐cultured cells, it has also been possible to provide convincing evidence for the operation of receptor‐mediated endocytosis in plants. A number of studies on protoplasts and cells clearly indicate that endocytosis in plants can be mediated by coated pits in the plasma membrane. At least one of the organelles that lie on the endocytic pathway in plants has a structurally similar counterpart in animal cells: the multivesicular body. The first recipient of internalized molecules is the partially coated reticulum, although its relationship to the Golgi apparatus and Golgi function remain to be clarified. The final target for endocytosis in plants appears to be the vacuole.

Membrane structure and function

Metabolism of Xenobiotics by the Nuclear Envelope Nucleocytoplasmic mRNA Transport: Its Status in Current Subcellular Biology The Centrosome The Golgi Apparatus Ribosome Structure and Function: Localization of rRNA Index.

An ultrastructural study of cell plate modifications induced by 2,6-dichlorobenzonitrile in onion root meristems

The effect of 2,6-dichlorobenzonitrile on cytokinesis of meristematic cells of onion root during both treatment and recovery has been studied by electron microscopic techniques. 2,6-dichlorobenzonitrile interferes with cell plate formation in such a way that Golgi apparatus vesicles of treated cells appear to be different than controls and seem to coalesce as anomalous partial cell plates. During recovery, an apparently normal progression of cytokinesis is observed and abnormal portions of the cell plate are retained. Nuclear constrictions are observed frequently during recovery as a result of temporal alterations in cytokinesis. Our results show that 2,6-dichlorobenzo-nitrile induces anomalous and/or incomplete cell plates, which might be caused by an altered function of Golgi apparatus.

Expression of a plasma membrane proteolipid during differentiation of neuronal and glial cells in primary culture.

Plasma membrane proteolipid protein (PM-PLP) synthesis was examined in embryonic rat neurons and neonatal rat glial cells during differentiation in culture. Glial cultures were treated with 1 mM N6, O2, dibutyryl cyclic adenosine monophosphate (dbcAMP) following confluency to induce differentiation, which resulted in the elaboration of long cellular processes. However, no changes in the biosynthetic level of PM-PLP was observed during the differentiation of these cells. Neurons differentiated spontaneously in culture, forming cellular aggregates immediately following plating and elaborating a network of neurites over 7 days. The differentiation of neurons was accompanied by a seven-fold increase in PM-PLP synthesis with increases in biosynthetic increase in PM-PLP synthesis with increases in biosynthetic rate observed between days 1 and 3 and between days 3 and 7 in culture. Ultrastructural examination of neurons indicated that the Golgi apparatus was also developing during this period of time, with an increase in both the number of lamellae and generation of vesicles. The transport of PM-PLP to the plasma membrane was therefore examined in neurons at day 7 in culture by pulse labeling experiments with monensin and colchicine. Monensin (1 microM) was found to inhibit the appearance of radiolabeled PM-PLP in the plasma membrane by 63%, indicating that a functional Golgi apparatus is required for transport of PM-PLP to its target membrane. Colchicine (125 microM) also inhibited the appearance of newly synthesized PM-PLP in the plasma membrane by greater than 40%, suggesting that microtubules may also be required for PM-PLP transport to the plasma membrane.

Inhibition of Elongation in Pellia Setae by the Monovalent Ionophore Monensin

Segments 5 mm long, cut from young setae of the liverwort Pellia, elongated when floated on an aqueous solution. Growth rates were constant for at least 20 h and in the presence of auxin approached those of intact setae. Elongation was inhibited by 60% by the monovalent ionophore monensin at concentrations of 0.5 μM or greater. Monensin, however, did not inhibit preferentially elongation of the segments during the first 4 h of auxin-promoted growth. As a result, the percentages of inhibition were nearly constant at all concentrations of monensin tested over the range from 5 nM to 50 μM either with or without auxin. Blockage of Golgi apparatus function occurred within 30-60 min of monensin treatment. Inhibition was observed as a failure to produce normal secretory vesicles. In their place, accumulations of vacuoles presumed to represent swollen cisternal elements and secretory vesicles were found. Within the first 4 h; the monensin effects were partially reversible but not thereafter. After 4 h the segment...

Relationship between microtubules and Golgi apparatus in hepatocytes: a quantitative study during experimental nephrosis.

In many cell types, microtubules are preferentially associated with the Golgi apparatus. However, the existence of a functional link between these two organelles is still hypothetical. To gain insight into this question, the relationships between microtubules and the Golgi apparatus were studied in rat hepatocytes during experimental nephrosis induced by the aminonucleoside of puromycin. This condition is known to cause prolonged stimulation of plasma protein production by the hepatocytes. Rats were studied 2, 4, 5, 10 and 20 days after aminonucleoside injection. The amount of albumin was measured in serum and hepatic microsomes by laser immunonephelometry. The volume densities of microtubules around the Golgi apparatus and in the remaining cytoplasm were measured by ultrastructural morphometry. Changes of the Golgi apparatus were analysed by measuring the volume density of the whole organelle and the respective proportion of saccules and vesicles. Proteinuria began 5 days after aminonucleoside injection and was accompanied by a decrease in serum albumin and a rise in microsomal albumin. These changes were still more striking after 10 days, but protein and albumin levels were almost back to normal after 20 days. Concomitantly, the volume density of the microtubules increased significantly around the Golgi apparatus (32% after 10 days), and not in the remaining cytoplasm. The Golgi apparatus was enlarged (80% after 10 days) with a higher ratio of secretory vesicle to saccule volume densities. These results show that additional microtubules are present around the Golgi apparatus during the enhanced production of plasma proteins which occurs in nephrosis. They suggest that in hepatocytes, microtubules play a part in the Golgi apparatus function of plasma protein processing.

Biosynthesis and secretion of fibronectin in human melanoma cells.

The biosynthesis and secretion of cellular fibronectin from human melanoma cells have been investigated by pulse-chase/immunoprecipitation analysis. Melanoma cells synthesize endoglycosidase H (Endo H)-sensitive glycoprotein precursors of fibronectin glycoproteins which chase to an Endo H-resistant monomer with an apparent Mr of 240,000 (240 K). This molecule, which has a significantly higher molecular weight than normal plasma or cellular fibronectin, is rapidly secreted by melanoma cells, resulting in the secretion of 80% of newly synthesized fibronectin in 120 min, following a 10-min biosynthetic pulse. This active secretory process can be inhibited by brief exposure of melanoma cells to sodium monensin (10(-7) M), which also results in a modified fibronectin of lower apparent Mr. Monosaccharide-incorporation studies of melanoma fibronectin reveal that monensin significantly inhibits galactose and fucose incorporation into this glycoprotein, correlating with reported effects of monensin on Golgi apparatus functions. These studies indicate that this tumor-associated and biosynthetically altered cellular fibronectin is a rapidly secreted major N-linked glycoprotein of metastatic human melanoma cells.

Structure of rat liver golgi apparatus: Relationship to lipoprotein secretion

The Golgi apparatus of rat liver is a complex structure consisting of stacks of cisternae plus systems of attached tubules and secretory vesicles. The tubules are of two types: (1) extensions of the cisternal peripheries that interface with adjacent cisternal stacks as well as connect secretory vesicles with the central saccules, and (2) a specialized smooth endoplasmic reticulum of the Golgi apparatus zone (= “boulevard périphérique”) containing very low-density lipoprotein particles. The particles appear to migrate from their sites of origin beginning at or near transition regions between rough and smooth endoplasmic reticulum via the second system of smooth tubules which provide direct luminal continuity between cisternae of rough endoplasmic reticulum and the secretory vesicles. According to the model proposed, the process of vesicle formation and entry of secretory product are spatially separated, allowing a multiplicity of Golgi apparatus function in the segregation of secretory products.

Isoantigens A, B and H in urinary bladder carcinomas following radiotherapy.

ABH tissue isoantigens were measured by the Specific Red Cell Adherence (SRCA) test in 66 surgical specimens of urinary bladder, including 53 transitional cell carcinomas, 2 squamous cell carcinomas and 11 controls. The SRCA test was strongly positive in 10 of 11 controls. ABH isoantigens were absent or equivocally present in 68 percent of noninvasive carcinomas (stage 0) and in 65 percent of invasive carcinomas. Clinical histories revealed that all patients with invasive carcinoma who had strongly positive SRCA test results had received prior radiotherapy to the bladder region. None of the patients with invasive bladder carcinoma with negative or weakly positive SRCA tests had been radiated. Histopathology of tumors in both groups was similar. Results of this retrospective study support the hypothesis that radiation may induce differentiation in tumors, possibly through an enhancement of Golgi apparatus function. The SRCA test should not be used as a predictor of the biological behavior of future recurrences in patients with bladder carcinoma who have received therapeutic radiation since radiation may produce "false positive" SRCA test results.

The nature of the granules within sulcular epithelial cells.

An ultrastructural, cytochemical, autoradiographic and radioisotopic uptake study has been made of the sulcular epithelium in an attempt to explain the function of the prominent Golgi apparatus and the associated membrane bound granules which are found in the cytoplasm of the sulcular epithelial cells. Some of the granules appear to be lysosomal in nature in that they are acid phosphatase positive and in some instances they appear to release their hydrolytic enzymes into the intercellular space. The evidence presented suggests that many of the remainder of the granules are related to the production of materials which become incorporated into the extraneous cell surface coat of the epithelial cells. The radioisotope experiments suggest that the rate of synthesis of cell coat material is higher in the sulcular epithelium than it is in the oral epithelium

Isolation of germ cell Golgi apparatus from seminiferous tubules of rat testes.

Intact Golgi apparatus have been isolated with good purity from rat testis by a simplified sucrose gradient technique. The procedure is inherently selective in that most of the Golgi apparatus in the isolate are from germ cells in late spermatocyte or early spermatid development. No Sertoli cell Golgi apparatus are present in the fraction. Biochemical analyses showed that the enzyme N-acetylglucosamine galactosyltransferase is enhanced in the band containing Golgi apparatus. Because they are easy to isolate, and because they are involved with the formation of a specific internal cellular component (the acrosome), these Golgi apparatus will be useful objects for comparison with other kinds of Golgi apparatus and for other studies leading to a better understanding of the basic functioning of Golgi apparatus.