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At least 1,333 records · Page 74Linked to original sources
PubMed2000

Synaptic proteins in rat taste bud cells: appearance in the Golgi apparatus and relationship to alpha-gustducin and the Lewis(b) and A antigens.

Taste receptor cells are continuously replaced during the life of the animal, but many of their sensory axons respond primarily to stimuli belonging to a single taste quality. This suggests that a newly arising taste cell must form a synapse with an appropriate sensory axon, requiring cell recognition that is likely to be mediated by surface markers. As an approach to studying this process, we attempted to locate synapses by immunolabeling taste buds of rats for proteins involved in neurotransmitter release. In taste bud cells of vallate papillae and nasoincisor ducts, double-labeling experiments showed that syntaxin-1, SNAP-25, synaptobrevin, and synaptophysin colocalized with the Golgi marker beta COP in elongated cytoplasmic compartments that extended from the perinuclear region into apical and basal processes of the cells. Labeled cells were spindle-shaped, identifying them as light cells. Syntaxin-1 appeared only in taste cells, but SNAP-25, synaptobrevin, and synaptophysin were also seen in nerve fibers. The synaptic vesicle glycoprotein SV2 appeared only in nerve fibers. Taste cells of fungiform papillae did not show immunoreactivity for presynaptic proteins or Golgi markers, but axonal labeling was similar to that in other regions. Taste cells with alpha-gustducin could express either presynaptic proteins or the carbohydrate blood group antigen Lewis(b), but not both. Therefore, Lewis(b) and presynaptic proteins are not expressed during the same period in the life of a taste bud cell. Most taste cells expressing syntaxin-1 (82%) also expressed the A blood group antigen, whether or not they expressed alpha-gustducin.

Animals↗
PubMed1979

Effects of ethanol on liver microtubules and Golgi apparatus. Possible role in altered hepatic secretion of plasma proteins.

A combined morphometric and biochemical approach was used to study the effects of alcohol on hepatic microtubules. Feeding of ethanol-containing diets to rats decreased the amount of polymerized tubulin and the number of visible microtubules, and it also produced morphologic alterations of this organelle. These microtubular alterations were reproduced in isolated hepatocytes after incubation with ethanol. Pyrazole, an inhibitor of alcohol dehydrogenase, prevented these effects. Two oxidation products of ethanol, acetaldehyde and acetate, in concentrations likely to occur "in vivo" also reproduced the effects of ethanol on microtubules. The alterations of microtubules, induced by either ethanol or its metabolites, were associated with significant increases in the volume and surface densities of the Golgi complex. Moreover, both the microtubular and the Golgi alterations were prevented by inhibiting the oxidation of ethanol with pyrazole, an inhibitor of alcohol dehydrogenase. These findings indicate that ethanol oxidation alters liver microtubules; as a likely result, delayed secretion of export proteins ensues with early retention in the Golgi vesicles. After chronic alcohol feeding, there was also a significant increase in the volume of the cytoplasm, which could reflect retention of soluble proteins and water.

Animals↗
PubMed1990

ERD1, a yeast gene required for the retention of luminal endoplasmic reticulum proteins, affects glycoprotein processing in the Golgi apparatus.

We have previously shown that the C-terminal sequence HDEL acts as a retention signal for luminal endoplasmic reticulum (ER) proteins in Saccharomyces cerevisiae, and that it is possible to isolate mutants that fail to retain an invertase fusion protein bearing this signal. Analysis of many such mutants defines two genes, ERD1 and ERD2. Cells lacking the ERD1 gene secrete the endogenous ER protein, BiP. Under normal growth conditions, the rate of secretion is equivalent to the rate at which wild-type cells secrete a modified form of BiP that lacks the HDEL signal altogether. Thus, erd1 cells show a profound disruption of the retention system. The mutant cells have no gross abnormality of their intracellular membrane system, but show defects in the Golgi-dependent modification of glycoproteins. We suggest that sorting of luminal ER proteins normally occurs in the Golgi, and that the function of ERD1 is required for the correct interaction of an HDEL receptor with its ligands. The sequence of ERD1 predicts a membrane protein with several transmembrane domains, a conclusion supported by analysis of ERD1-SUC2 fusion proteins.

Amino Acid Sequence↗
PubMed2004

Toll-like receptor 2 (TLR2) and TLR4 are present inside human dendritic cells, associated with microtubules and the Golgi apparatus but are not detectable on the cell surface: integrity of microtubules is required for interleukin-12 production in response to internalized bacteria.

The activation of dendritic cells (DCs) by microbes is mediated by pattern recognition receptors including the Toll-like receptors (TLR). Bacterial lipopolysaccharide acts via TLR4 whereas peptidoglycan and lipoprotein responses are mediated by TLR2. It is generally accepted that TLR binding to microbes occurs at the cell surface but this has not been directly demonstrated for human DCs. We show here that TLR2 and TLR4 are expressed inside DCs in an abundant tubulovesicular pattern with a focus of intense staining adjacent to the nucleus. In contrast, there was no detectable expression on the cell surface. TLR2 and TLR4 were readily found both intracellularly and on the surface of monocytes. They were shown to be closely associated with the Golgi complex and colocalized with alpha-tubulin, displaying a high focal concentration at the microtubule organizing centre. Alignment of TLR2 and TLR4 with microtubules was observed, suggesting that microtubules serve as transport tracks for TLR vesicles. Depolymerization of the microtubule network disrupted the intracellular expression of TLR2 and TLR4 and profoundly inhibited interleukin-12 (IL-12) production in response to Neisseria meningitidis but did not prevent phagocytosis. These data are consistent with the bacterial signalling through TLR2 and TLR4 required for IL-12 production occurring inside DCs after phagocytosis.

Cell Membrane↗
PubMed1996

Signal-mediated sorting of membrane proteins between the endoplasmic reticulum and the golgi apparatus.

Each organelle of the secretory pathway is required to selectively allow transit of newly synthesized secretory and plasma membrane proteins and also to maintain a unique set of resident proteins that define its structural and functional properties. In the case of the endoplasmic reticulum (ER), residency is achieved in two ways: (a) prevention of residents from entering newly forming transport vesicles and (b) retrieval of those residents that escape. The latter mechanism is directed by discrete retrieval motifs: Soluble proteins have a H/KDEL sequence at their carboxy-terminus; membrane proteins have a dibasic motif, either di-lysine or di-arginine, located close to the terminus of their cytoplasmic domain. Recently it was found that di-lysine motifs bind the complex of cytosolic coat proteins, COP I, and that this interaction functions in the retrieval of proteins from the Golgi to the ER. Also discussed are the potential roles this interaction may have in vesicular trafficking.

Animals↗
PubMed1999

Assembly of heteromeric connexons in guinea-pig liver en route to the Golgi apparatus, plasma membrane and gap junctions.

Guinea-pig liver gap junctions are constructed from approximately equal amounts of connexins 26 and 32. The assembly of these connexins into connexon hemichannels and gap junctions was studied using antibodies specific to each connexin. Intracellular membranes were shown to contain low amounts of connexin 26 relative to connexin 32 in contrast to the equal connexin ratios detected in lateral plasma membranes and gap junctions. Assembly of gap junctions requires oligomerization of connexins into connexons that may be homomeric or heteromeric. Immunoprecipitation using antibodies to connexins 26 and 32 showed that liver gap junctions were heteromeric. A chemical cross-linking procedure showed that connexons solubilized from guinea-pig liver gap junctions were constructed of hexameric assemblies of connexin subunits. The intracellular site of oligomerization of connexins was investigated by velocity sedimentation in sucrose-detergent gradients. Oligomers of connexins 26 and 32 were extensively present in Golgi membranes and oligomeric intermediates, especially of connexin 26, were detected in the endoplasmic reticulum-Golgi intermediate subcellular fraction. Two intracellular trafficking pathways that may account for the delivery of connexin 26 to the plasma membrane and explain the heteromeric nature of liver gap junctions are discussed.

Animals↗
PubMed1998

Localization of ADP-ribosylation factor domain protein 1 (ARD1) in lysosomes and Golgi apparatus.

ADP-ribosylation factor domain protein 1 (ARD1) is a member of the ADP ribosylation factor (ARF) family of guanine nucleotide-binding proteins that differs from other ARFs by the presence of a 46-kDa amino-terminal extension which acts as a GTPase-activating protein (GAP) for its ARF domain. Similar to ARF GAPs, the GAP domain of ARD1 contains a zinc finger motif and arginine residues that are critical for activity. It differs from other ARF GAPs in its covalent association with the GTP-binding domain and its specificity for the ARF domain of ARD1. ARFs are presumed to play a key role in the formation of intracellular transport vesicles and in their movement from one compartment to another. We report here that ARD1 overexpressed in cells, as a fusion or nonfusion protein, is localized in vesicular structures that are concentrated mainly in the perinuclear region, but are found also throughout the cytosol. Microscopic colocalization and subcellular fractionation studies showed that ARD1 was associated with the Golgi complex and lysosomal structures. ARD1 expressed as a green fluorescent fusion protein was initially associated with the Golgi network and subsequently localized to lysosomes. Lysosomal and Golgi membranes isolated from human liver by immunoaffinity contained native ARD1. Localization to these organelles, therefore, did not appear to be a result of overexpression. These observations suggest that the ARF-related protein ARD1 may play a role in the formation or function of lysosomes and in protein trafficking between Golgi and lysosomes.

3T3 Cells↗
PubMed2004

Coronin 7, the mammalian POD-1 homologue, localizes to the Golgi apparatus.

Coronins constitute an evolutionary conserved family of WD-repeat actin-binding proteins. Their primary function is thought to be regulating the actin cytoskeleton. Apart from that, several coronins were indirectly shown to participate in vesicular transport, establishment of cell polarity and cytokinesis. Here, we report a novel mammalian protein, coronin 7 (crn7), which is significantly different from other mammalian coronins in its domain architecture. Crn7 possesses two stretches of WD repeats in contrast to the other coronins only having one. The protein is expressed throughout the mouse embryogenesis and is strongly upregulated in brain and developing structures of the immune system in the course of development. In adult animals, both crn7 mRNA and protein are abundantly present in most organs, with significantly higher amounts in brain, kidney, thymus and spleen and lower amounts in muscle. At the subcellular level, the bulk of the protein appears to be present in the cytosol and in large cytosolic complexes. However, a significant portion of the protein is detected on vesicle-like cytoplasmic structures as well as on the cis-Golgi. In the Golgi region, crn7 staining appears broader than that of the cis-Golgi markers Erd2p and beta-COP, still, the trans-Golgi network appears predominantly crn7-negative. Importantly, the membrane-associated form of crn7 protein is phosphorylated on tyrosine residues, whereas the cytosolic form is not. Crn7 is the first coronin protein proven to localize to the Golgi membrane. We conclude that it plays a role in the organization of intracellular membrane compartments and vesicular trafficking rather than in remodeling the cytoskeleton.

3T3 Cells↗
PubMed1990

New evidence for endoplasmic reticulum and Golgi apparatus in rabbit reticulocytes.

The incorporation of 14C-galactose and 14C-fucose in rabbit reticulocytes obtained by bleeding anaemia and separated according to their stage of maturation was studied. Only the youngest reticulocytes were shown to incorporate the two sugars with galactosylation being three times higher. The glycosylation behaved different from the synthesis of membrane proteins with respect to the dependence on temperature and time. Among the newly synthesized membrane proteins the signal sequence receptor (SSR), a compound of the system addressing protein translocation, was detected immunologically.

Animals↗
PubMed1996

Subcellular distribution of "intersecting' beta-N-acetylglucosaminyltransferase in Dictyostelium discoideum. A likely marker for the Golgi apparatus.

Glycoprotein processing in Dictyostelium discoideum is characterized by enzyme catalyzed steps not reported in other organisms. One of these is the formation of a beta 1 --> 4 linkage between GlcNAc and the mannose linked to the core mannose in the alpha 1 --> 6 position of N-glycosides. A simple and sensitive assay for this GlcNAc transferase activity, using a tri-mannose acceptor and a low concentration of UDP-GlcNAc, was developed. Homogenates of the organism were subjected to sub-cellular fractionation by centrifugation in discontinuous sucrose gradients. The specific activity was enriched 4-5-fold in a crude membrane fraction. The transferase was purified 10-12-fold in a membrane fraction that bands on top of 1.1 M sucrose. This fraction was also enriched in nucleotidyldiphosphatase. The enriched fraction was deficient in glucose-6-phosphatase, an endoplasmic reticulum marker. Approx. 80% of the transferase activity was latent, and unavailable to protease. Purified membranes were either subjected to phase separation in Triton X-114, or sodium carbonate extraction or sonication. In each case, the transferase behaved as an intrinsic membrane protein. Several secreted and lysosomal proteins are modified by the enzyme. These data support the idea that the GlcNAc transferase is present as an integral Golgi membrane protein and that at least the catalytic center of the transferase is on the lumenal side of the vesicles.

Acid Anhydride Hydrolases↗
PubMed2002

The Saccharomyces cerevisiae alg12delta mutant reveals a role for the middle-arm alpha1,2Man- and upper-arm alpha1,2Manalpha1,6Man- residues of Glc3Man9GlcNAc2-PP-Dol in regulating glycoprotein glycan processing in the endoplasmic reticulum and Golgi apparatus.

N-glycosylation in nearly all eukaryotes proceeds in the endoplasmic reticulum (ER) by transfer of the precursor Glc(3)Man(9)GlcNAc(2) from dolichyl pyrophosphate (PP-Dol) to consensus Asn residues in nascent proteins. The Saccharomyces cerevisiae alg (asparagine-linked glycosylation) mutants fail to synthesize oligosaccharide lipid properly, and the alg12 mutant accumulates a Man(7)GlcNAc(2)-PP-Dol intermediate. We show that the Man(7)GlcNAc(2) released from alg12Delta-secreted invertase is Manalpha1,2Manalpha1,2Manalpha1,3(Manalpha1,2Manalpha1,3Manalpha1,6)-Manbeta1,4-GlcNAcbeta1-4GlcNAcalpha/beta, confirming that the Man(7)GlcNAc(2) is the product of the middle-arm terminal alpha1,2-mannoslytransferase encoded by the ALG9 gene. Although the ER glucose addition and trimming events are similar in alg12Delta and wild-type cells, the central-arm alpha1,2-linked Man residue normally removed in the ER by Mns1p persists in the alg12Delta background. This confirms in vivo earlier in vitro experiments showing that the upper-arm Manalpha1,2Manalpha1,6-disaccharide moiety, missing in alg12Delta Man(7)GlcNAc(2), is recognized and required by Mns1p for optimum mannosidase activity. The presence of this Man influences downstream glycan processing by reducing the efficiency of Ochlp, the cis-Golgi alpha1,6-mannosyltransferase responsible for initiating outer-chain mannan synthesis, leading to hypoglycosylation of external invertase and vacuolar protease A.

Carbohydrate Sequence↗
PubMed1977

Cytochemical contributions to differentiating GERL from the Golgi apparatus.

Recent studies from our laboratory are described which deal with endocrine cells (insulinoma, beta-cells of the pancreas, thyroid epithelial cells), pancreatic exocrine cells, and hepatocytes. These emphasize the importance of the hydrolase-rich specialized region of endoplasmic reticulum, known as GERL, in secretory cells. Also reviewed in this paper are the varied molecular transformations which apparently occur in GERL in different cell types, as reported from other laboratories as well as our own. Evidence of the continuity of GERL with rough endoplasmic reticulum is presented. Two hydrolytic enzyme activities in GERL, in addition to acid phosphatase activity, are recorded. Finally, the use of cytochemical staining procedures in the study of microperoxisomes is briefly described.

Acid Phosphatase↗
PubMed1977

Cytoplasmic effects of X-irradiation on cultured cells 2. Alterations in lysosomes, plasma membrane, Golgi apparatus, and related structures.

Topoinhibited human glia cells in vitro were exposed to X-radiation generated by an 8-MeV linear accelerator at a dose of 20,000 rad. Transmission electron microscopy of irradiated cells at intervals varying between 30 min. and 5 days following irradiation revealed alterations mainly in the plasma membrane and the lysosomal vacuome. Increased ruffling of plasma membranes, augmented endocytosis and extensive intracellular autophagy developed within 24 hours after irradiation. The implications of the plasma membrane alterations are discussed, and a tentative model covering possible mechanism involved in the development of autophagic vacuoles is presented. The possibility is entertained that alternative mechanism may be operative during the formation of the autophagic vacuole in irradiated glia cells. The origin of the isolation membrane appears to be (a) preexisting lysosomes; and (b) flattened vacuolar cytoplasmic elements.

Cell Membrane↗
PubMed1990

The export pathway of the pseudorabies virus gB homolog gII involves oligomer formation in the endoplasmic reticulum and protease processing in the Golgi apparatus.

The pseudorabies virus gII gene shares significant homology with the gB gene of herpes simplex virus type 1. Unlike gB, however, gII is processed by specific protease cleavage events after the synthesis of its precursor. The processed forms are maintained in an oligomeric complex that includes disulfide linkages. In this report, we demonstrate the kinetics of modification, complex formation, and subsequent protease processing. In particular, we suggest that gII oligomer formation in the endoplasmic reticulum is an integral part of the export pathway and that protease cleavage occurs only after oligomers have formed. Furthermore, through the use of glycoprotein gene fusions between the gIII glycoprotein and the gII glycoprotein genes of pseudorabies virus, we have mapped a functional cleavage domain of gII to an 11-amino-acid segment.

Amino Acid Sequence↗
PubMed1995

Mitotic disassembly of the mammalian Golgi apparatus.

A cell-free system that mimics mitotic fragmentation of Golgi stacks has provided a working model for the disassembly process. Two distinct pathways, one COP-dependent and one COP-independent, act on Golgi stacks to give rise to two types of end products: transport vesicles and larger, more heterogeneous vesicles and tubules. We suggest that both mitotic end products result from enhanced fission of Golgi membranes under conditions where membrane fusion is generally inhibited.

Journal Article↗
PubMed2002

The Golgi apparatus: balancing new with old.

Most models put forward to explain cellular processes do not stand the test of time. The 'lucky' few that are able to survive extensive experimental tests and peer critique may eventually become dogmas or paradigms. When this happens, the amount of experimental data required to overturn the paradigm is extensive. To some, such inertia may seem prohibitive to scientific progress but rather, in our opinion, this helps to maintain a degree of coherence. It is needed so that experiments and interpretations may be conducted within relatively safe boundaries. In the field of protein transport in the secretory pathway, we have enjoyed a relatively stable and productive period for quite some time (more than 30 years!). It is only very recently that the field has entered into a phase where all bets seem to be off. As in any paradigm shift, the accumulation of experimental observations inconsistent with the old dogma eventually reached a critical point. As we 'reluctantly' dispense with the long-standing paradigm of forward vesicular transport, we face a time that is bound to be trying as well as exciting.

Animals↗
PubMed1979

On the morphology of the Golgi apparatus and its relation to glucose-6-phosphate dehydrogenase in neurosecretory nuclei of rats under normal and thirsting conditions (application of karyometry).

Histochemical studies on the distribution of TPPase and G6PD in SON and PVN of rats have been conducted by using the new TPPase method, karyometry, and statistics under normal and thirsting conditions. All differences in nuclear volume among the 4 groups classified by the TPPase and G6PD reactions were significant after thirsting for 2 and 4 days, respectively. The intimate parallel relationship was found statistically between the GA shape and G6PD activity in neurons of both nuclei. These results may substantiate the usefulness of classification of neurons of both nuclei. These results may substantiate the usefulness of classification of neurons by the TPPase method on the LM level. They also suggest strongly that the GA shape may directly indicate its synthesizing activity, and that the complicate GA shape may correspond to the phase of vigorous synthesizing activity in the neuron. Both nuclei included some neurons whose GA appeared intact under activation. This suggests that secretory cycles of individual cells may proceed asynchromously under thirsting as well as normal conditions. The unique peak revealed by the curves of mean nuclear volumes of GA Type IV and G6PD. strongly positive groups only in PVN must be representative of phenomenon of exhaustion of PVN.

Animals↗
PubMed2006

[Effect of p-nitrophenyl-xyloside on the biosynthesis of proteoglycan in rat ovarian granulosa cells--analyses of glycosaminoglycan synthesis in the Golgi apparatus].

Biosynthesis of proteoglycans and glycosaminoglycans in the presence of p-nitrophenyl-xyloside was studied using a primary rat ovarian granulosa cell culture system. Addition of p-nitrophenyl-xyloside into cell culture medium caused about a 700% increase of [³⁵S]sulfate incorporation (ED50 at 0.03 mM) into macromolecules, which included free chondroitin sulfate chains initiated on xyloside and native proteoglycans. Free chondroitin sulfate chains initiated on xyloside were almost exclusively secreted into the medium. The molecular size of chondroitin sulfate chains decreased from 40,000 to 21,000 as the total [³⁵S]sulfate incorporation was enhanced, suggesting that enhanced synthesis of chondroitin sulfate perturbed the normal mechanism of glycosaminoglycan chain termination. Biosynthesis of heparan sulfate proteoglycans was reduced by approximately 50%, likely due to competition at the level of UDP-sugar precursors. [³⁵S]Sulfate incorporation was shut down by the addition of cycloheximide with an initial half time of approximately 2 hr in the presence of xyloside, while that in the absence of xyloside was about 20 min. The difference likely reflects the turnover rate of glycosaminoglycan synthesizing capacity as a whole. The turnover rate of glycosaminoglycan synthesizing capacity observed in ovarian granulosa cells was much shorter than that observed in chondrocytes, reflecting the relative dominance of proteoglycan biosynthetic activity in the total metabolic activity of the cells.

Animals↗