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PubMed1998

Targeting of active sialyltransferase to the plant Golgi apparatus.

Glycosyltransferases in the Golgi apparatus synthesize cell wall polysaccharides and elaborate the complex glycans of glycoproteins. To investigate the targeting of this type of enzyme to plant Golgi compartments, we generated transgenic Arabidopsis plants expressing alpha-2,6-sialyltransferase, a glycosyltransferase of the mammalian trans-Golgi cisternae and the trans-Golgi network. Biochemical analysis as well as immunolight and immunoelectron microscopy of these plants indicate that the protein is targeted specifically to the Golgi apparatus. Moreover, the protein is predominantly localized to the cisternae and membranes of the trans side of the organelle. When supplied with the appropriate substrates, the enzyme has significant alpha-2,6-sialyltransferase activity. These results indicate a conservation of glycosyltransferase targeting mechanisms between plant and mammalian cells and also demonstrate that glycosyltransferases can be subcompartmentalized to specific cisternae of the plant Golgi apparatus.

Animals↗
PubMed2005

Dynamics of COPII vesicles and the Golgi apparatus in cultured Nicotiana tabacum BY-2 cells provides evidence for transient association of Golgi stacks with endoplasmic reticulum exit sites.

Despite the ubiquitous presence of the COPI, COPII, and clathrin vesicle budding machineries in all eukaryotes, the organization of the secretory pathway in plants differs significantly from that in yeast and mammalian cells. Mobile Golgi stacks and the lack of both transitional endoplasmic reticulum (ER) and a distinct ER-to-Golgi intermediate compartment are the most prominent distinguishing morphological features of the early secretory pathway in plants. Although the formation of COPI vesicles at periphery of Golgi cisternae has been demonstrated in plants, exit from the ER has been difficult to visualize, and the spatial relationship of this event is now a matter of controversy. Using tobacco (Nicotiana tabacum) BY-2 cells, which represent a highly active secretory system, we have used two approaches to investigate the location and dynamics of COPII binding to the ER and the relationship of these ER exit sites (ERES) to the Golgi apparatus. On the one hand, we have identified endogenous COPII using affinity purified antisera generated against selected COPII-coat proteins (Sar1, Sec13, and Sec23); on the other hand, we have prepared a BY-2 cell line expressing Sec13:green fluorescent protein (GFP) to perform live cell imaging with red fluorescent protein-labeled ER or Golgi stacks. COPII binding to the ER in BY-2 cells is visualized as fluorescent punctate structures uniformly distributed over the surface of the ER, both after antibody staining as well as by Sec13:GFP expression. These structures are smaller and greatly outnumber the Golgi stacks. They are stationary, but have an extremely short half-life (<10 s). Without correlative imaging data on the export of membrane or lumenal ER cargo it was not possible to equate unequivocally these COPII binding loci with ERES. When a GDP-fixed Sar1 mutant is expressed, ER export is blocked and the visualization of COPII binding is perturbed. On the other hand, when secretion is inhibited by brefeldin A, COPII binding sites on the ER remain visible even after the Golgi apparatus has been lost. Live cell imaging in a confocal laser scanning microscope equipped with spinning disk optics allowed us to investigate the relationship between mobile Golgi stacks and COPII binding sites. As they move, Golgi stacks temporarily associated with COPII binding sites at their rims. Golgi stacks were visualized with their peripheries partially or fully occupied with COPII. In the latter case, Golgi stacks had the appearance of a COPII halo. Slow moving Golgi stacks tended to have more peripheral COPII than faster moving ones. However, some stationary Golgi stacks entirely lacking COPII were also observed. Our results indicate that, in a cell type with highly mobile Golgi stacks like tobacco BY-2, the Golgi apparatus is not continually linked to a single ERES. By contrast, Golgi stacks associate intermittently and sometimes concurrently with several ERES as they move.

Binding Sites↗
PubMed1996

Ceramides are transported through the Golgi apparatus in human keratinocytes in vitro.

The intercellular lipid sheets of the stratum corneum constitute the epidermal permeability barrier that permits terrestrial life. Although lamellar granules are known to deliver the precursors of the stratum corneum lipids into the intercellular spaces, their site of origin remains unknown. Lamellar granules have characteristics of both secretory granules and lysosomes, which are known to originate from the Golgi apparatus in other cell types. Glucosylceramides, a major component of lamellar granule contents and the precursors of stratum corneum ceramides, have been found to be synthesized primarily in the early compartments of the Golgi apparatus in other cell types. We have investigated the transport and metabolism of a fluorescently labeled ceramide in human keratinocyte cultures using laser-scanning confocal microscopy and lipid analysis. We found that ceramide is metabolized to glucosylceramide and sphingomyelin as it passes through the Golgi apparatus and the metabolites are then delivered to the plasma membrane. Cold temperature, Brefeldin A, and monensin, all known to inhibit transport from the Golgi to the plasma membrane, prevented ceramide metabolites from appearing at the plasma membrane. Because glucosylceramides are one of the most important lipid constituents of lamellar granules, these results support the hypothesis that the Golgi is the origin of lamellar granules.

Biological Transport↗
PubMed1997

Rat liver Golgi apparatus contains a protein kinase similar to the casein kinase of lactating mammary gland.

By using a beta-casein-derived specific peptide substrate for mammary gland Golgi-enriched-fraction casein kinase, phosphorylating activity has been detected in the Golgi apparatus of rat liver, spleen and to a lesser extent, kidney and brain, while the other post-nuclear cytoplasmic fractions are totally devoid of such a casein kinase activity. In contrast ubiquitous protein kinases CK1 and CK2 (casein kinases 1 and 2), tested with their specific peptide substrates, display different subcellular distribution and are almost undetectable in the Golgi fraction. The absence of CK2 in the Golgi fraction has been also confirmed using specific antibodies. The relatedness between the liver Golgi apparatus casein kinase (G-CK) and the bona fide mammary gland Golgi-enriched-fraction casein kinase (GEF-CK) is supported by a variety of observations, notably: (a) identical peptide substrate specificity, consistent with an S-X-E-X consensus sequence; (b) preference for Mn2+, and, to a lesser extent, Co2+, over Mg2+, as activating cation; (c) superimposable elution profiles from DEAE-Sepharose, heparin-Sepharose, and Superdex 200, this latter consistent with a molecular mass around 500 kDa; (d) insensitivity to staurosporine and heparin (a potent inhibitor of CK2) and inability to use GTP as phosphate donor (by contrast to CK2). These data provide the evidence for the existence of a third class of ubiquitous casein kinases here termed G-CK, distinct from CK1 and CK2, specifically located to the Golgi apparatus and related to the bona fide casein kinase(s) responsible for the phosphorylation of casein secreted from lactating mammary gland. The possible involvement of G-CK in the phosphorylation of secretory pathways proteins at S-X-E motifs is discussed.

Amino Acid Sequence↗
PubMed2006

Expanding the role of the dynein regulatory complex to non-axonemal functions: association of GAS11 with the Golgi apparatus.

The mammalian GAS11 gene is a candidate tumor suppressor of unknown function that was previously identified as one of several genes upregulated upon growth arrest. Interestingly, although GAS11 homologs in Trypanosoma brucei (trypanin) and Chlamydomonas reinhardtii (PF2) are integral components of the flagellar axoneme and are necessary for regulating flagellar beat, the GAS11 gene was discovered based on its expression in cells that do not assemble a motile cilium. This suggests that GAS11 function might not be restricted to the cilium. To investigate this possibility, we generated GAS11-specific antibodies and demonstrate here that GAS11 is expressed in a variety of mammalian cells that lack a motile cilium. In COS7 cells, GAS11 is associated with the detergent-insoluble cytoskeleton and exhibits a juxtanuclear localization that overlaps with the pericentrosomal Golgi apparatus. This localization is dependent upon intact microtubules and is cell-cycle regulated, such that GAS11 is dispersed throughout the cytoplasm as cells progress through mitosis. GAS11 remains associated with Golgi fragments following depolymerization of cytoplasmic microtubules but is dispersed upon disruption of the Golgi with brefeldin A. These data suggest that GAS11 is associated with the Golgi apparatus. In support of this, recombinant GAS11 binds Golgi membranes in vitro. In growth-arrested mIMCD3 cells, GAS11 co-localizes with gamma-tubulin at the base of the primary cilium. The pericentrosomal Golgi apparatus and base of the cilium both represent convergence points for microtubule minus ends and correspond to sites where dynein regulation is required. The algal GAS11 homolog functions as part of a dynein regulatory complex (DRC) in the axoneme (Rupp and Porter. J Cell Biol 2003;162:47-57) and our findings suggest that components of this axonemal dynein regulatory system have been adapted in mammalian cells to participate in non-axonemal functions.

Animals↗
PubMed1987

Tridimensional structure of the Golgi apparatus of nonciliated epithelial cells of the ductuli efferentes in rat: an electron microscope stereoscopic study.

The 3-dimensional structure of the Golgi apparatus has been analyzed in thin and thick sections of nonciliated epithelial cells of ductuli efferentes of rat by use of low- and high-voltage electron microscopes and a stereoscopic approach. In thick sections of tissue impregnated with osmium, the Golgi apparatus appeared at low magnification as a continuous network forming a corona at the apical pole of the nucleus. At higher magnification and in thin sections of tissue postfixed with reduced osmium and stained with lead citrate or treated to demonstrate phosphatase activity, the following structural features were observed. In the longitudinal axis of the Golgi network there were alternating compact and noncompact zones. The compact zones were composed of 6-8 flattened, poorly fenestrated saccules in close apposition to each other and forming stacks. The noncompact zones were composed of a number of highly fenestrated and slightly distended saccules, which were continuous with and bridged the saccules of the compact zones. In the cis-trans axis of the Golgi apparatus the following compartments were observed: (a) On the cis face there was a continuous osmiophilic tubular network referred to as the cis element; (b) a cis compartment composed of 3 or 4 NADPase-positive saccules perforated with pores in register forming wells that contained small vesicles; (c) a trans compartment composed of 1 or 2 TPPAse-positive elements underlying the NADPase ones, followed by 1 or 2 CMPase-positive elements that showed a flattened saccular part continuous with a network of anastomotic tubules. These tubular networks curved away from the overlying elements, giving these elements a 'peeling-off" configuration. These elements referred to as sacculotubular elements were discontinuous along the Golgi network. This compartment also included shriveled trans-tubular networks detached from the overlying sacculotubular elements and seemingly undergoing fragmentation into vesicles and tubules. The structural features of the elements of the trans compartment were indicative of continuous renewal.

Animals↗
PubMed2002

Partitioning of the matrix fraction of the Golgi apparatus during mitosis in animal cells.

The Golgi apparatus is partitioned during mitosis in animal cells by a process of fragmentation, dispersal, and reassembly in each daughter cell. We fractionated the Golgi apparatus in vivo using the drug brefeldin A or a dominant-negative mutant of the Sar1p protein. After these treatments, Golgi enzymes moved back to the endoplasmic reticulum, leaving behind a matrix of Golgi structural proteins. Under these conditions, cells still entered and exited mitosis normally, and their Golgi matrix partitioned in a manner very similar to that of the complete organelle. Thus, the matrix may be the partitioning unit of the Golgi apparatus and may carry the Golgi enzyme-containing membranes into the daughter cells.

Anaphase↗
PubMed1985

A vital stain for the Golgi apparatus.

The Golgi complex, a membranous organelle with important functions in membrane traffic and macromolecular synthesis, has been stained in living cells with a fluorescent sphingolipid. Cells were first incubated with liposomes containing N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]-6-aminocaproyl sphingosine (C6-NBD-ceramide), or with a bovine serum albumin complex of the fluorescent lipid, and then examined by fluorescence microscopy. An intensely fluorescent perinuclear structure was identified as the Golgi apparatus by its colocalization with known Golgi markers in fixed cells. C6-NBD-ceramide was used to observe the morphology of the Golgi apparatus in living cells in the presence or absence of monensin or Colcemid, and during mitosis. In all cases, C6-NBD-ceramide revealed a Golgi apparatus in the living cell that was identical to that obtained with conventional procedures that require fixation.

4-Chloro-7-nitrobenzofurazan↗
PubMed2002

Molecular and phenotypic analysis of CaVRG4, encoding an essential Golgi apparatus GDP-mannose transporter.

Cell surface mannan is implicated in almost every aspect of pathogenicity of Candida albicans. In Saccharomyces cerevisiae, the Vrg4 protein acts as a master regulator of mannan synthesis through its role in substrate provision. The substrate for mannosylation of proteins and lipids in the Golgi apparatus is GDP-mannose, whose lumenal transport is catalyzed by Vrg4p. This nucleotide sugar is synthesized in the cytoplasm by pathways that are highly conserved in all eukaryotes, but its lumenal transport (and hence Golgi apparatus-specific mannosylation) is a fungus-specific process. To begin to study the role of Golgi mannosylation in C. albicans, we isolated the CaVRG4 gene and analyzed the effects of loss of its function. CaVRG4 encodes a functional homologue of the S. cerevisiae GDP-mannose transporter. CaVrg4p localized to punctate spots within the cytoplasm of C. albicans in a pattern reminiscent of localization of Vrg4p in the Golgi apparatus in S. cerevisiae. Like partial loss of ScVRG4 function, partial loss of CaVRG4 function resulted in mannosylation defects, which in turn led to a number of cell wall-associated phenotypes. While heterozygotes displayed no growth phenotypes, a hemizygous strain, containing a single copy of CaVRG4 under control of the methionine-repressible MET3 promoter, did not grow in the presence of methionine and cysteine, demonstrating that CaVRG4 is essential for viability. Mutant Candida vrg4 strains were defective in hyphal formation but exhibited a constitutive polarized mode of pseudohyphal growth. Because the VRG4 gene is essential for yeast viability but does not have a mammalian homologue, it is a particularly attractive target for development of antifungal therapies.

Amino Acid Sequence↗
PubMed1994

The herpes simplex virus UL20 protein compensates for the differential disruption of exocytosis of virions and viral membrane glycoproteins associated with fragmentation of the Golgi apparatus.

The Golgi apparatus is fragmented and dispersed in Vero cells but not in human 143TK- cells infected with wild-type herpes simplex virus 1. Moreover, a recombinant virus lacking the gene encoding the membrane protein UL20 (UL20- virus) accumulates in the space between the inner and outer nuclear membranes of Vero cells but is exported and spreads from cell to cell in 143TK- cell cultures. Here we report that in Vero cells infected with UL20- virus, the virion envelope glycoproteins were of the immature type, whereas the viral glycoproteins associated with cell membranes were fully processed up to the addition of sialic acid, a trans-Golgi function. Moreover, the amounts of viral glycoproteins accumulating in the plasma membranes were considerably smaller than those detected on the surface of Vero cells infected with wild-type virus. In contrast, the amounts of viral glycoproteins present on the plasma membranes of 143TK- cells infected with wild-type or UL20- virus were nearly identical. We conclude that (i) in Vero cells infected with UL20- virus the block in the export of virions is at the entry into the exocytic pathway, and a second block in the exocytosis of viral glycoproteins associated with cytoplasmic membranes is due to an impairment of transport beyond Golgi fragments containing trans-Golgi enzymes and not to a failure of the Golgi oligosaccharide-processing functions; (ii) these defects are manifested in cells in which the Golgi apparatus is fragmented; and (iii) the UL20 protein compensates for these defects by enabling transport to and from the fragmented Golgi apparatus.

Animals↗
PubMed2002

Molecular motor KIF1C is not essential for mouse survival and motor-dependent retrograde Golgi apparatus-to-endoplasmic reticulum transport.

KIF1C is a new member of the kinesin superfamily of proteins (KIFs), which act as microtubule-based molecular motors involved in intracellular transport. We cloned full-length mouse kif1C cDNA, which turned out to have a high homology to a mitochondrial motor KIF1Balpha and to be expressed ubiquitously. To investigate the in vivo significance of KIF1C, we generated kif1C(-/-) mice by knocking in the beta-galactosidase gene into the motor domain of kif1C gene. On staining of LacZ, we detected its expression in the heart, liver, hippocampus, and cerebellum. Unexpectedly, kif1C(-/-) mice were viable and showed no obvious abnormalities. Because immunocytochemistry showed partial colocalization of KIF1C with the Golgi marker protein, we compared the organelle distribution in primary lung fibroblasts from kif1C(+/+) and kif1C(-/-) mice. We found that there was no significant difference in the distribution of the Golgi apparatus or in the transport from the Golgi apparatus to the endoplasmic reticulum (ER) facilitated by brefeldin A between the two cells. This retrograde membrane transport was further confirmed to be normal by time-lapse analysis. Consequently, KIF1C is dispensable for the motor-dependent retrograde transport from the Golgi apparatus to the ER.

Amino Acid Sequence↗
PubMed2004

Ras activation in Jurkat T cells following low-grade stimulation of the T-cell receptor is specific to N-Ras and occurs only on the Golgi apparatus.

Ras activation is critical for T-cell development and function, but the specific roles of the different Ras isoforms in T-lymphocyte function are poorly understood. We recently reported T-cell receptor (TCR) activation of ectopically expressed H-Ras on the the Golgi apparatus of T cells. Here we studied the isoform and subcellular compartment specificity of Ras signaling in Jurkat T cells. H-Ras was expressed at much lower levels than the other Ras isoforms in Jurkat and several other T-cell lines. Glutathione S-transferase-Ras-binding domain (RBD) pulldown assays revealed that, although high-grade TCR stimulation and phorbol ester activated both N-Ras and K-Ras, low-grade stimulation of the TCR resulted in specific activation of N-Ras. Surprisingly, whereas ectopically expressed H-Ras cocapped with the TCRs in lipid microdomains of the Jurkat plasma membrane, N-Ras did not. Live-cell imaging of Jurkat cells expressing green fluorescent protein-RBD, a fluorescent reporter of GTP-bound Ras, revealed that N-Ras activation occurs exclusively on the Golgi apparatus in a phospholipase Cgamma- and RasGRP1-dependent fashion. The specificity of N-Ras signaling downstream of low-grade TCR stimulation was dependent on the monoacylation of the hypervariable membrane targeting sequence. Our data show that, in contrast to fibroblasts stimulated with growth factors in which all three Ras isoforms become activated and signaling occurs at both the plasma membrane and Golgi apparatus, Golgi-associated N-Ras is the critical Ras isoform and intracellular pool for low-grade TCR signaling in Jurkat T cells.

Amino Acid Sequence↗
PubMed1977

Glycosylation of apoproteins of rat very low density lipoproteins during transit through the hepatic Golgi apparatus.

The glycosylation of apo very low density lipoproteins (apo-VLDL) in vivo was studied by following the incorporation of [14C]glucosamine into several groups of apoproteins of VLDL isolated from hepatic Golgi fractions and from serum of sucrose-fed, colchicine-treated rats. Simultaneous incorporation of [3H]leucine was used to quantitate the apoproteins following separation by polyacrylamide gel electrophoresis. Experimental conditions were selected so that the 14C:3H ratio in the apoproteins permitted estimations of the extent of glycosylation by glucosamine and its metabolites. A rapidly decreasing 14C:3H ratio was noted in serum apo-VLDL for the first 30 min after administration of the isotopically labelled precursors, followed by stabilization of the ratio. These data are consistent with the glycosylation of a preformed pool of apo-VLDL, probably apo-B. Glucosamine was progressively incorporated into apo-VLDL during transition from the forming face of the Golgi apparatus to the secretory vesicles, as indicated by an increasing 14C:3H ratio. On the other hand, the ratio of the rapidly migrating apoproteins of VLDL, corresponding to the apo-C-II and apo-C-III, showed the opposite trend, as did total apo high density lipoprotein (apo-HDL) and the rapidly migrating bands of apo-HDL. Division of the rapidly migrating apoproteins of VLDL into upper bands (probably apo-C-II and apo-C-III-0) and lower bands (probably apo-C-III-3) resulted in a 14C:3H ratio near zero in the upper band apoproteins, consistent with the absence of carbohydrates. The lower band showed a rising 14C:3H ratio during transition through the Golgi apparatus, suggesting increased glycosylation, The decreasing 14C:3H ratio in the rapidly migrating proteins is therefore due to the acquisition of apo-C-II and apo-C-III-0 by VLDL during passage from the forming face to the secretory vesicles of the Golgi apparatus.

Animals↗
PubMed1983

Alkaline phosphatase biosynthesis in the endoplasmic reticulum and its transport through the Golgi apparatus to the plasma membrane: cytochemical evidence.

Enzyme induction of HeLa cell placental alkaline phosphatase with various agents such as prednisolone, sodium butyrate, hyperosmolality (NaCl), or combination of these inducers resulted in the appearance of enzyme activity in the rough endoplasmic reticulum, nuclear envelope, Golgi apparatus, and plasma membrane. In the Golgi apparatus, intense reaction product deposits tended to be concentrated on its trans side, with small vesicles and granules also being positively stained. Inhibition of protein synthesis with cycloheximide was followed by the disappearance of enzyme activity from these cytoplasmic organelles but not from the plasma membrane. Treatment with monensin, a secretory protein transport inhibitor, uniformly increased activity in the rough endoplasmic reticulum while causing marked dilatation of the intensely positive Golgi cisternae. These results suggest that intracellular alkaline phosphatase is newly synthesized in the endoplasmic reticulum and then passes en route through the Golgi apparatus to the plasma membrane. Accordingly, the present system could represent the biosynthesis, transport, and incorporation of the model cell surface enzyme protein to add to the vesicular stomatitus virus glyco-1 (VSV-G) protein and acetylcholine receptor model systems for studying the dynamics of cell surface protein genesis, transport, and membrane integration.

Alkaline Phosphatase↗
PubMed1984

The Golgi apparatus and GERL during postnatal differentiation of rat parotid acinar cells: an electron microscopic cytochemical study.

Morphological and cytochemical changes in the Golgi apparatus and GERL of differentiating parotid acinar cells were examined in Sprague-Dawley rats from 5 days to young adult. At day 5, the Golgi apparatus consisted of 3-6 narrow saccules, with short segments of GERL lying adjacent to the trans Golgi saccule. As the glands matured, the Golgi apparatus increased in size and the saccules became broadened and fenestrated reaching a maximum from days 15-20. The saccules subsequently narrowed slightly and by day 25 resembled those seen in young adults. Numerous cisternae of GERL could be seen at the trans face during this period. While the glands were maturing, marked changes occurred in the distribution of thiamine pyrophosphatase (TPPase) activity in the Golgi saccules. In the immature cells, TPPase activity was restricted to 1 or 2 trans Golgi saccules. However, by day 10 TPPase could also be localized in immature secretory granules and in GERL-like cisternae. Unreactive segments of GERL were also present. This pattern of localization persisted until day 20, after which the TPPase activity in the GERL-like cisternae diminished gradually until by day 40 TPPase again was localized in 1-2 trans Golgi saccules and an occasional immature secretory granule. Acid phosphatase (AcPase) activity was localized primarily in lysosomes in the very young animals and increased in GERL with age up to day 15. From days 15 to 20 there was a decrease in the amount of activity seen in GERL, but from day 20 on, the AcPase activity increased until it reached that seen in young adult animals. These results indicate that the presence of TPPase activity in GERL-like cisternae and immature secretory granules may be dependent upon the developmental as well as the physiologic state of the acinar cells and lend further support to the suggestion that GERL is derived from the trans Golgi saccules.

Acid Phosphatase↗
PubMed1989

Identification of a novel protein (G210) specific to the Golgi apparatus.

A monoclonal antibody, 3C9, has enabled the detection of a novel Golgi-specific protein in bovine tissues. Immunohistochemical studies at the light microscopic level have detected the 3C9 antigen only in certain cells: exocrine pancreas, gut epithelium, and thymus epithelium. Examination of gut and pancreas by immunoelectron microscopy showed a localization exclusive to the Golgi apparatus. The relative molecular weight of the antigen detected by immunoblotting is 210,000 daltons. The antigen is not extracted from microsomal membranes of bovine gut epithelium by sodium carbonate solutions. Furthermore, the 3C9 antigen enters into the detergent phase when Triton X-114 partitioning methods are used. These data strongly suggest that this novel antigen is an intrinsic membrane protein, resident in the Golgi apparatus of certain cells. Moreover, they enhance the hypothesis that the distribution of enzymes and polypeptides in the Golgi apparatus is cell specific.

Animals↗
PubMed1992

Lectin cytochemistry in the gastrointestinal tract with special reference to glycosylation in the Golgi apparatus of Brunner's gland cells.

Two hydrophilic, low temperature-embedding resins, Lowicryl K4M and LR White, were compared in lectin cytochemistry. Post-embedding staining of colloidal gold-labeled Griffonia symplicifolia agglutinin II (GSA-II) resulted in staining of the Golgi apparatus and mucous granules of mucous neck cells in the gastric fundic gland, pylorocytes, and Brunner's gland cells embedded in either resin, although it was much easier to make ultra-thin sections with LR White-embedded material than with the other. Post-fixation with uranyl acetate followed by LR White embedding improved general ultrastructure so that lectin binding sites were identified precisely. All examined lectins, soybean agglutinin (SBA), Maclura pomifera agglutinin (MPA), GSA-II, and Ulex europaeus agglutinin I (UEA-I), stained mucous granules and the Golgi apparatus, in which the staining pattern was characteristic of each lectin: cis cisternae were labeled with SBA and MPA, intermediate cisternae with GSA-II, and trans cisternae and mucous granules with SBA, GSA-II, UEA-I, and lightly with MPA. No labeling was observed in the rough endoplasmic reticulum with any lectin. These findings suggest that the Golgi apparatus is the site of O-linked glycosylation and can be divided into at least three distinct compartments with regard to the glycosylation.

Animals↗
PubMed1993

Monoclonal antibodies to the Golgi apparatus of serous exocrine cells.

We demonstrated that a common antigen (Golgi-associated antigen, GAA 108) is present in the Golgi apparatus of serous exocrine cells, using an immunohistochemical method with monoclonal antibodies (MAb) 108 (IgG1) and 18 (IgM), raised to the microsomal fractions of rat parotid gland. The MAb reacted with polypeptides of molecular weights in the 58-170 KD range in parotid gland on Western blot analysis. The Golgi apparatus of the following cells was immunostained with these MAb: acinar cells of parotid gland, pancreas, and exorbital lacrimal gland, serous cells of sublingual gland, chief cells of stomach, and epithelial cells of rat prostate. However, positive reaction occurred throughout the entire cytoplasm of submandibular gland acinar cells. Immunoelectron microscopy (IM) revealed antigen (GAA 108) localization in the medial and trans-Golgi cisternae and trans-Golgi network (TGN), including condensing vacuoles, in parotid, exorbital lacrimal, and pancreatic acinar cells, and serous acinar cells of sublingual gland. Lysosomes and apical cell membranes also stained positively in some cells. In the submandibular gland reactions were observed in the medial and trans-Golgi cisternae, condensing vacuoles, secretory granule contents, cell membrane, and in some duct lumens. These results suggest that although GAA 108 is found in the Golgi apparatus of most serous exocrine cells, it is secreted by a regulated pathway in the acinar cells of submandibular gland.

Animals↗