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Results for “GOLGI APPARATUS”

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PubMed2005

The ZIP7 gene (Slc39a7) encodes a zinc transporter involved in zinc homeostasis of the Golgi apparatus.

It has been suggested that ZIP7 (Ke4, Slc39a7) belongs to the ZIP family of zinc transporters. Transient expression of the V5-tagged human ZIP7 fusion protein in CHO cells led to elevation of the cytoplasmic zinc level. However, the precise function of ZIP7 in cellular zinc homeostasis is not clear. Here we report that the ZIP7 gene is ubiquitously expressed in human and mouse tissues. The endogenous ZIP7 was associated with the Golgi apparatus and was capable of transporting zinc from the Golgi apparatus into the cytoplasm of the cell. Moreover, by using the yeast mutant strain Deltazrt3 that was defective in release of stored zinc from vacuoles, we found that ZIP7 was able to decrease the level of accumulated zinc and in the meantime to increase the nuclear/cytoplasmic labile zinc level in the ZIP7-expressing zrt3 mutant. We showed that the protein expression of ZIP7 was repressed under zinc-rich condition, whereas there were no effects of zinc on ZIP7 gene expression and intracellular localization. Neither did zinc deficiency affect the intracellular distribution of ZIP7 in mammalian cells. Our study demonstrates that ZIP7 is a functional zinc transporter that acts by transporting zinc from the Golgi apparatus to the cytoplasm of the cell.

Animals↗

PubMed2005

Cytosolic phospholipase A2-alpha mediates endothelial cell proliferation and is inactivated by association with the Golgi apparatus.

Arachidonic acid and its metabolites are implicated in regulating endothelial cell proliferation. Cytosolic phospholipase A2-alpha (cPLA2alpha) is responsible for receptor-mediated arachidonic acid evolution. We tested the hypothesis that cPLA2alpha activity is linked to endothelial cell proliferation. The specific cPLA2alpha inhibitor, pyrrolidine-1, inhibited umbilical vein endothelial cell (HUVEC) proliferation in a dose-dependent manner. Exogenous arachidonic acid addition reversed this inhibitory effect. Inhibition of sPLA2 did not affect HUVEC proliferation. The levels of cPLA2alpha did not differ between subconfluent and confluent cultures of cells. However, using fluorescence microscopy we observed a novel, confluence-dependent redistribution of cPLA2alpha to the distal Golgi apparatus in HUVECs. Association of cPLA2alpha with the Golgi was linked to the proliferative status of HUVECs. When associated with the Golgi apparatus, cPLA2alpha activity was seen to be 87% inhibited. Relocation of cPLA2alpha to the cytoplasm and nucleus, and cPLA2alpha enzyme activity were required for cell cycle entry upon mechanical wounding of confluent monolayers. Thus, cPLA2alpha activity and function in controlling endothelial cell proliferation is regulated by reversible association with the Golgi apparatus.

Cell Proliferation↗

PubMed1988

Immunolocalization of blood group A gene specified alpha 1,3N-acetylgalactosaminyltransferase and blood group A substance in the trans-tubular network of the Golgi apparatus and mucus of intestinal goblet cells.

The subcellular distribution of blood group A gene specified alpha 1,3N-acetylgalactosaminyltransferase and its product was studied in human intestinal goblet cells by immunoelectron microscopy. The O-glycosylation step yielding blood group A-active glycoconjugates occurred in the trans region of the Golgi apparatus as indicated by the presence of immunolabel for both antigens. In the Golgi apparatus, immunoreactive alpha 1,3N-acetylgalactosaminyltransferase was detectable in trans cisternae and in the trans-tubular network which was found to be continuous with the cisternal stack and exhibited acid phosphatase activity. This demonstrates that in intestinal goblet cells (i) the trans-tubular network does not constitute a compartment distinct from trans cisternae, and (ii) structures corresponding to GERL are structurally and functionally part of the Golgi apparatus. In addition to immunolabel for transferase at the inner surface of the cisternal membranes, luminally located immunolabel indicating the presence of free, not membrane-associated transferase became first detectable in the trans-tubular network and early forming mucus droplets contained therein. Further, the content of mature mucus droplets as well as the extracellular mucus layer were labeled. Absence of immunolabel in blood group 0 subjects lacking the blood group A gene specified transferase and the apparent non-reactivity of the antibodies with carbohydrate epitopes indicates that free alpha 1,3N-acetylgalactosaminyltransferase is present in mucus droplets and becomes secreted by intestinal goblet cells.

ABO Blood-Group System↗

PubMed1984

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

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

Animals↗

PubMed1989

Differential association of rat liver heparan sulfate proteoglycans in membranes of the Golgi apparatus and the plasma membrane.

Heparan sulfate proteoglycans (HSPG) of rat liver are associated with the plasma membrane in a hydrophobic intrinsic and a hydrophilic extrinsic form. We were interested in determining whether or not these two forms could be detected in the Golgi apparatus, the subcellular site of addition of oligosaccharides and sulfate to HSPG. In vivo and in vitro radiolabeled HSPG from rat liver Golgi apparatus membranes could only be solubilized with detergents that disrupt the membrane lipid bilayer, suggesting that they are solely associated via hydrophobic interactions. Both forms of HSPG were detected in plasma membranes of rat liver and isolated rat hepatocytes. The detergent-solubilized HSPG bound to octyl-Sepharose columns, whereas the hydrophilic form did not; this latter form, however, was released from the membrane by heparin. The hydrophobic anchor of HSPG in the Golgi and plasma membranes was insensitive to treatment with phosphatidylinositol-specific phospholipase C under conditions in which alkaline phosphatase was sensitive; this suggests that the hydrophobic anchor of HSPG is the core protein itself. Preliminary experiments suggest that the subcellular site of processing of the hydrophobic to the hydrophilic form of HSPG is the plasma membrane. A specific processing activity, probably a protease of the plasma membrane not present in serum or the endoplasmic reticulum membrane, converted hydrophobic HSPG of the Golgi membrane to the hydrophilic form. In addition, pulse-chase experiments with [35S]Na2SO4 in rats demonstrated that at short times, the bulk of the radiolabeled cellular HSPG was in the Golgi apparatus; later on, the bulk of the radioactivity was found in the plasma membrane, the only subcellular site where the hydrophilic form of HSPG was detected.

Animals↗

PubMed2003

Shape and position of the sarcoplasmic reticulum and the Golgi apparatus in the sole plate and remaining subsarcolemmal muscle region of the mouse using imidazole-osmium staining.

By means of thin (< or =150 nm) and thick (>150 nm) sections, the shape and position of the sarcoplasmic reticulum and of the Golgi apparatus in the sole plate and in the remaining subsarcolemmal sarcoplasmic region were investigated. For this purpose the membranes were stained by means of imidazole-osmium postfixation and unstained sections analyzed under the electron microscope. Both in the sarcoplasma of the sole plate and around the muscle fiber nuclei, a network of tubules is visible after imidazole-osmium staining which can be identified as the sarcoplasmic reticulum solely on the basis of its contacts with the perinuclear cistern and the cisterns of the triads. Findings in literature on the position of the Golgi apparatus are confirmed and similar spatial relationships and vesiculations between the perinuclear cisterns and the Golgi apparatus of the sole plate nuclei and the other subsarcolemmal fiber nuclei are also demonstrated using this new staining method.

Animals↗

PubMed2001

Cholesterol depletion by methyl-beta-cyclodextrin blocks cholera toxin transport from endosomes to the Golgi apparatus in hippocampal neurons.

We recently demonstrated that although cholera toxin (CT) is found in detergent-insoluble domains/rafts at the cell surface of cultured hippocampal neurons, it is internalized via a raft-independent mechanism. Thus, cholesterol depletion by methyl-beta-cyclodextrin (MbetaCD) did not affect the rate of CT internalization from the plasma membrane, but did affect the rate of CT degradation, which occurs in lysosomes. In the current study, we analyze which step of CT intracellular transport is inhibited by MbetaCD. Whereas pre-incubation with MbetaCD completely blocked CT degradation, it had no effect on the degradation of wheat germ agglutinin (WGA) or bovine serum albumin (BSA), which are internalized by receptor-mediated and fluid phase endocytosis, respectively. Brefeldin A also completely blocked CT degradation but had no effect on WGA or BSA degradation. In contrast, MbetaCD did not affect CT degradation, or CT-mediated cAMP generation, when added to neurons after CT had been transported to the Golgi apparatus. We conclude that CT transport from endosomes to the Golgi apparatus is cholesterol-dependent, whereas CT transport from the Golgi apparatus to lysosomes is cholesterol-independent.

Anti-Bacterial Agents↗

PubMed1989

The GTP-binding protein Ypt1 is required for transport in vitro: the Golgi apparatus is defective in ypt1 mutants.

The YPT1 gene encodes a raslike, GTP-binding protein that is essential for growth of yeast cells. We show here that mutations in the ypt1 gene disrupt transport of carboxypeptidase Y to the vacuole in vivo and transport of pro-alpha-factor to a site of extensive glycosylation in the Golgi apparatus in vitro. Two different ypt1 mutations result in loss of function of the Golgi complex without affecting the activity of the endoplasmic reticulum or soluble components required for in vitro transport. The function of the mutant Golgi apparatus can be restored by preincubation with wild-type cytosol. The transport defect observed in vitro cannot be overcome by addition of Ca++ to the reaction mixture. We have also established genetic interactions between ypt1 and a subset of the other genes required for transport to and through the Golgi apparatus.

Alleles↗

PubMed1988

ATP-dependent fusion of liposomes with the Golgi apparatus of perforated cells.

We examined the interactions of lipid vesicles (liposomes) labeled with various fluorescent markers with the intracellular membranes of semi-intact ("perforated") fibroblasts. When incubations were performed in the presence of an ATP-regenerating system, both vesicle lipids and entrapped water soluble markers were transferred to the Golgi apparatus of treated cells, indicative of membrane fusion. Fusion occurred using unilamellar vesicles 30-80 nm in diameter and composed of phosphatidylcholine alone, but was inhibited when equimolar amounts of either phosphatidylserine or sphingomyelin were present in the vesicles. Lipid vesicle-Golgi membrane fusion was also inhibited by pretreatment of the perforated cells with N-ethylmaleimide. These findings suggest that lipid vesicles may be useful for delivery of labeled lipids, macromolecules, and dyes to the Golgi apparatus, and for modeling the interactions of transport vesicles with the Golgi apparatus.

Adenosine Triphosphate↗

PubMed1992

Changes of the Golgi apparatus and lysosomes during decidualization in mice.

The Golgi apparatus of the endometrial stromal cells of pregnant mice increases in size simultaneously with the differentiation of stromal cells into decidual cells. The activity of acid phosphatase in this organelle increases during this stage. On the other hand, the involuting decidual cells show morphological and cytochemical signs of Golgi regression (dilated cisternae, lack of enzymatic activity) together with the finding of numerous, pleomorphic lysosomes that have intense cytochemical label. These results confirm morphological data suggesting that decidual cell death occurs by autophagic degeneration.

Acid Phosphatase↗

PubMed2003

PMR1/SPCA Ca2+ pumps and the role of the Golgi apparatus as a Ca2+ store.

Besides the well-known sarco/endoplasmic-reticulum Ca(2+)-transport ATPases (SERCA), animal cells contain a much less characterized P-type Ca(2+)-transport ATPase: the PMR1/SPCA Ca(2+)/Mn(2+)-transport ATPase. SPCA is mainly targeted to the Golgi apparatus. Phylogenetic analysis indicates that it might be more closely related to a putative ancestral Ca(2+) pump than SERCA. SPCA supplies the Golgi apparatus, and possibly other more distal compartments of the secretory pathway, with the Ca(2+) and Mn(2+) necessary for the production and processing of secretory proteins. In the lactating mammary gland, SPCA appears to be the primary pump responsible for supplementing the milk with high (60-100 mM) Ca(2+). It could also play a role in detoxification of cells overloaded with Mn(2+). Mutations in the human gene encoding the SPCA pump ( ATP2C1) result in Hailey-Hailey disease, a keratinocyte disorder characterized by incomplete cell adhesion. Recent observations show that the Golgi apparatus can function as a Ca(2+) store, which can be involved in setting up cytosolic Ca(2+) oscillations.

ATP-Binding Cassette Transporters↗

PubMed1996

Uncleaved env gp160 of human immunodeficiency virus type 1 is degraded within the Golgi apparatus but not lysosomes in COS-1 cells.

The fate of newly synthesized human immunodeficiency virus type 1 env gp160 was examined in COS-1 cells. The results of morphological chase experiments involving cycloheximide demonstrated that gp160 was retained in the Golgi apparatus for longer than the half-life of the molecule. The degradation of gp160 was insensitive to both bafilomycin A1 and leupeptin (< 0.2 mM), which block lysosomal proteolysis. However, degradation was effectively suppressed by leupeptin at higher concentrations, maximally at 1.7 mM. Furthermore, undegraded gp160 was accumulated in the Golgi apparatus, but was not detected in lysosomes. These results indicate that in COS-1 cells gp160 is not degraded in lysosomes, but rather that degradation takes place in the Golgi apparatus.

Animals↗

PubMed1985

Translocation of UDP-N-acetylglucosamine into vesicles derived from rat liver rough endoplasmic reticulum and Golgi apparatus.

A mixture of UDP-N-acetylglucosamine labeled with different radioisotopes in the uridine and glucosamine was used to show that the intact sugar nucleotide was translocated across the membrane of vesicles derived from rat liver rough endoplasmic reticulum (RER) and Golgi apparatus. Translocation was dependent on temperature, saturable at high concentrations of sugar nucleotide, and inhibited by treatment of vesicles with proteases, suggesting protein carrier mediated transport. Translocation of UDP-GlcNAc by RER-derived vesicles appeared to be specific since these vesicles were unable to translocate UDP-galactose, in contrast to those derived from the Golgi apparatus. Preliminary results suggest that the mechanism of UDP-GlcNAc translocation into RER-derived vesicles is via a coupled exchange with lumenal nucleoside monophosphate. This is similar to the recently postulated mechanism for translocation of sugar nucleotides into vesicles derived from the Golgi apparatus.

Animals↗

PubMed2005

The steady-state distribution of glycosyltransferases between the Golgi apparatus and the endoplasmic reticulum is approximately 90:10.

Several lines of evidence support a novel model for Golgi protein residency in which these proteins cycle between the Golgi apparatus and the endoplasmic reticulum (ER). However, to preserve the functional distinction between the two organelles, this pool of ER-resident Golgi enzymes must be small. We quantified the distribution for two Golgi glycosyltransferases in HeLa cells to test this prediction. We reasoned that best-practice, quantitative solutions would come from treating images as data arrays rather than pictures. Using deconvolution and computer calculated organellar boundaries, the Golgi fraction for both endogenous beta1,4-galactosyltransferase and UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferase 2 fused with green fluorescent protein (GFP) was 91% by fluorescence microscopy. Immunogold labeling followed by electron microscopy and model analysis yielded a similar value. Values reflect steady-state conditions, as inclusion of a protein synthesis inhibitor had no effect. These data strongly suggest that the fluorescence of a GFP chimera with an organellar protein can be a valid indicator of protein distribution and more generally that fluorescent microscopy can provide a valid, rapid approach for protein quantification. In conclusion, we find the ER pool of cycling Golgi glycosyltransferases is small and approximately 1/100 the concentration found in the Golgi apparatus.

Endoplasmic Reticulum↗

PubMed2001

Assembly of very low density lipoproteins in mouse liver: evidence of heterogeneity of particle density in the Golgi apparatus.

The assembly of very low density lipoproteins (VLDL) by hepatocytes is believed to occur via a two-step process. The first step is the formation of a dense phospholipid and protein-rich particle that is believed to be converted to VLDL by the addition of bulk triglyceride in a second step. Previous studies in our laboratory led us to hypothesize a third assembly step that occurs in route to or in the Golgi apparatus. To investigate this hypothesis, nascent lipoproteins were recovered from Golgi apparatus-rich fractions isolated from mouse liver. The Golgi fractions were enriched 125-fold in galactosyltransferase and contained lipoprotein particles averaging approximately 35 nm in diameter. These lipoproteins were separated by ultracentrifugation into two fractions: d < 1.006 g/ml and d1.006;-1.210 g/ml. The d < 1.006 g/ml fraction contained apolipoprotein B-100 (apoB-100), apoB-48, and apoE, while the d1.006;-1.210 g/ml fraction contained these three apoproteins as well as apoA-I and apoA-IV. Both fractions contained a 21-kDa protein that was isolated and sequenced and identified as major urinary protein. Approximately 50% of the apoB was recovered with the denser fraction. To determine if these small, dense lipoproteins were secreted without further addition of lipid, mice were injected with Triton WR1339 and [(3)H]leucine, and the secretion of apoB-100 and apoB-48 into serum VLDL (d < 1.006 g/ml) and d1.006;-1.210 g/ml fractions was monitored over a 2-h period. More than 80% of the newly synthesized apoB-48 and nearly 100% of the apoB-100 were secreted with VLDL. These studies provide the first characterization of nascent lipoproteins recovered from the Golgi apparatus of mouse liver. We conclude that these nascent hepatic Golgi lipoproteins represent a heterogeneous population of particles including VLDL as well as a population of small, dense lipoproteins. The finding of the latter particles, coupled with the demonstration that the primary secretory product of mouse liver is VLDL, suggests that lipid may be added to nascent lipoproteins within the Golgi apparatus.

Animals↗

PubMed1998

Ceramide transport from endoplasmic reticulum to Golgi apparatus is not vesicle-mediated.

Ceramide (Cer) transfer from the endoplasmic reticulum (ER) to the Golgi apparatus was measured under conditions that block vesicle-mediated protein transfer. This was done either in intact cells by reducing the incubation temperature to 15 degreesC, or in streptolysin O-permeabilized cells by manipulating the intracellular environment. In both cases, Cer transfer was not inhibited, as demonstrated by the biosynthesis of ceramide monohexosides and sphingomyelin (SM) de novo from metabolically (with [14C]serine) labelled Cer. This assay is based on the knowledge that Cer is synthesized, starting from serine and palmitoyl-CoA, at the ER, whereas glycosphingolipids and SM are synthesized in the (early) Golgi apparatus. Formation of [14C]glycosphingolipids and [14C]SM was observed under conditions that block vesicle-mediated vesicular stomatitis virus glycoprotein transport. These results indicate that [14C]Cer is transferred from ER to Golgi by a non-vesicular mechanism.

Adenosine Triphosphate↗

PubMed1982

Golgi apparatus and acid phosphatase in mammary gland and kidney cells under the influence of diethylstilbestrol (DES).

It is commonly accepted that hormonal disturbances in experimental animals produce pathological changes in several organs namely in mammary glands and kidney. Taking into consideration the close relationship between acid phosphatase and Golgi apparatus, the present study was undertaken, to study the relationship in kidney and mammary gland cells under the influence of DES. The observation indicate, that acid phosphatase and Golgi apparatus were localized in the cytoplasm of mammary gland and kidney cells. It has also been found a parallel change in the activity of acid phosphatase and the degree of development of Golgi apparatus after DES administration.

Acid Phosphatase↗

PubMed1996

Role of the Golgi apparatus in the phosphorylation of apolipoprotein B.

Rat hepatic Golgi apparatus-rich fractions were utilized in an in vitro phosphorylation system containing [gamma-32P]ATP to investigate the phosphorylation of apolipoproteins (apo) B48 and B100. Our results demonstrate that the Golgi apparatus contains a kinase(s) that phosphorylates both apoB48 and apoB100 as well as 290- and 460-kDa proteins recognized by antibody to apoB. We refer to the latter proteins as apoB57 and apoB90, respectively. Phosphorylations in the presence of Triton X-100, which increases the permeability of the membranes, or alamethicin, an ionophore that facilitates transmembrane diffusion of ATP, indicate that the active site of the kinase is on the luminal side of the membranes. However, studies with EDTA and EGTA, which are inhibitory to the kinase, suggest binding sites for Mg2+ and perhaps Ca2+ on the cytosolic membrane face. Phosphorylation of apoB was not stimulated by cAMP nor inhibited by protein kinase inhibitor peptide (5-24), indicating that cAMP dependent protein kinase was not involved in the phosphorylation process. Sodium carbonate treatment of the phosphorylated fraction, which permits separation of membrane and luminal contents, revealed that phosphorylated apoB90 and apoB57 are associated primarily with the membrane, whereas phosphorylated apoB48 is found in luminal contents as well as with the membranes. Phosphorylated apoB100 was found primarily with the membrane fraction. No evidence was found for phosphorylation of apoB in rough endoplasmic reticulum fractions. These studies demonstrate the importance of the Golgi apparatus as a subcellular site for the phosphorylation of apoB and suggest that apoB phosphorylation may be important in the assembly and secretion of apoB-containing lipoproteins.

Adenosine Triphosphate↗