Overlapping distribution of two glycosyltransferases in the Golgi apparatus of HeLa cells.

Nilsson, Tommy; Pypaert, Marc; Hoe, Mee H.; Slusarewicz, Paul; Berger, Eric G.; Warren, Graham

doi:10.1083/jcb.120.1.5

Cited by 213 publications

(163 citation statements)

References 47 publications

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“…If the connections between nonequivalent cisternae described here facilitate the rapid passage of protein cargo through the Golgi by means of the lumen that forms, it will be important to characterize the molecular machinery that restricts and͞or regulates mixing between the membranes of cisternae if they are indeed heterotypic. Further, Nilsson et al (27) showed that the distributions of processing enzymes overlap between medial-and trans-cisternae, and they proposed that Golgi cisternae may be differentiated, not by distinct sets of processing enzymes, but rather by the ratios in which they are mixed. The connections shown here may play a role in regulating the distribution of Golgi enzymes.…”

Section: Resultsmentioning

confidence: 99%

Direct continuities between cisternae at different levels of the Golgi complex in glucose-stimulated mouse islet beta cells

Marsh

Volkmann

McIntosh

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

169

137

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Direct continuity between the membranes of cisternae in the Golgi complex in mammalian cells rarely has been observed; when seen, its documentation has been equivocal. Here we have used dualaxis electron microscope tomography to examine the architecture of the Golgi in three dimensions at Ϸ6-nm resolution in rapidly frozen, freeze-substituted murine cells that make and secrete insulin in response to glucose challenge. Our data show three types of direct connections between Golgi cisternae that are normally distinct from one another. These connections all ''bypass'' interceding cisternae. We propose that when pancreatic beta cells are stimulated to synthesize and secrete insulin rapidly in vivo, such connections provide a continuous lumen that facilitates the rapid transit of large amounts of newly made protein for secretion. The heterotypic fusion of cisternae, even transiently, raises important questions about the molecular mechanisms that (i) facilitate the fusion͞fission of cisternal membranes and control the directionality and specificity of such events, and (ii) retain Golgi processing enzymes at specific places within individual cisternae when two cisternae at different levels in the Golgi have fused, maintaining the sequential processing hierarchy that is a hallmark of Golgi organization.T he mechanisms by which proteins and lipids move through the Golgi complex remain controversial. There are three prevailing theories for how membrane and luminal cargo are transported: (i) by carrier vesicles that move cargo between physically distinct Golgi cisternae in both the anterograde and retrograde directions (1, 2), (ii) by the forward movement or ''progression'' of the cisternae themselves, accompanied by vesicle movement in the retrograde direction (3-5), or (iii) by both mechanisms acting in concert (6-8). Direct membrane connections between cisternae at different levels of the Golgi also have been proposed (8-11). Lippincott-Schwartz and colleagues (11) have suggested that such connections might constitute a major mechanism for transport through the Golgi stack, based on data that show first order kinetics for the rate of transport of exogenous viral glycoprotein of vesicular stomatitis virus through the Golgi following its overexpression in cultured cells. Convincing evidence for direct continuity between nonequivalent cisternae has been limited or lacking, although connectivity between cisternae at equivalent levels of the Golgi ribbon is well documented (12)(13)(14).In this study, rapid freezing followed by freeze-substitution fixation of endocrine tissue isolated from mouse pancreas, together with techniques for analyzing electron microscope (EM) data in 3D at Ϸ6-nm resolution (15), have allowed us to capture and visualize such events in ''professional'' secretory cells stimulated to make and secrete large amounts of protein.Our observations of membrane trafficking in primary cells maintained in organ culture and stimulated to secrete at high levels suggest that the overexpression of proteins from transg...

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Section: Resultsmentioning

confidence: 99%

Direct continuities between cisternae at different levels of the Golgi complex in glucose-stimulated mouse islet beta cells

Marsh

Volkmann

McIntosh

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

169

137

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“…The fact that afew xylosylated and fucosylated glycoproteins are immunodetected early in the cis Golgi cisternae indicates that the corresponding glycosyltransferases are also present in this compartment. A diffuse distribution of glycosyltransferases over the Golgi cisternae has already been described for a2+6 sialyltransferases and N-acetylglucosaminyltransferase I in mammalian cells (Lee et al, 1989;Nilsson et a/., 1993). To characterize further the glycan-processing pathway in plant cells, our next goal is to purify glycosyltransferases and raise antisera against them.…”

Section: Discussionmentioning

confidence: 99%

Distribution of xylosylation and fucosylation in the plant Golgi apparatus

et al. 1994

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SummaryAntibodies have been immunopurified which are specific for carbohydrate epitopes containing the pl+2 xylose or a1 4 3 fucose residues found on complex Nlinked glycans in plants. The antibody specificity was determined by taking advantage of an Arabidopsis thaliana N-glycosylation mutant which lacks N-acetylglucosaminyltransferase I and is unable to synthesize complex glycans. These antibodies were used to immunolocalize xylose-and fucose-containing glycoproteins in suspension-cultured sycamore cells (Acer pseudoplatanus). By mapping the enzymatic reaction products within the Golgi apparatus, the fucosyl-and xylosyltransferase subcellular localization was made possible using immunocytochemistry on thin sections of high-pressure frozen and freezesubstituted sycamore cells. This procedure allows a much better preservation of organelles, and particularly of the Golgi stack morphology, than that obtained with conventionally fixed samples. Glycoproteins containing p1+2 xylose and al+3 fucose residues were immunodetected in the cell wall, the vacuole, and the Golgi cisternae. The extent of immunolabeling over the different cisternae of 50 Golgi stacks was quantified after treatment with antixylose or anti-fucose antibodies. Labeling for xylosecontaining glycoproteins was predominent in the medial cisternae, while fucose-containing glycoproteins were mainly detected in the trans compartment. Therefore, in plants, complex N-linked glycan xylosylation probably occurs mostly at the medial Golgi level and a1 4 3 fucose is mainly incorporated in the trans cisternae. Finally, fucose-and xylosecontaining glycoproteins were also immunolocalized, albeit to a lesser extent, in earlier Golgi compartments. This indicates that the glycosylation events are a continuous process with some maxima in given compartments, rather than a succession of discrete and compartment-dependent steps.

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“…The monoclonal mouse ascite 9E10 (gift from T. Nillson, Heidelberg, Germany) was used to detected the myc epitope (Nilsson et al, 1993).…”

Section: Drosophila Golgi Markers and Antibodiesmentioning

confidence: 99%

Biogenesis of Golgi Stacks in Imaginal Discs ofDrosophila melanogaster

Kondylis

Goulding

Dunne

et al. 2001

MBoC

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We provide a detailed description of Golgi stack biogenesis that takes place in vivo during one of the morphogenetic events in the lifespan of Drosophila melanogaster. In early third-instar larvae, small clusters consisting mostly of vesicles and tubules were present in epithelial imaginal disk cells. As larvae progressed through mid-and late-third instar, these larval clusters became larger but also increasingly formed cisternae, some of which were stacked. In white pupae, the typical Golgi stack was observed. We show that larval clusters are Golgi stack precursors by 1) localizing various Golgi-specific markers to the larval clusters by electron and immunofluorescence confocal microscopy, 2) driving this conversion in wild-type larvae incubated at 37°C for 2 h, and 3) showing that this conversion does not take place in an NSF1 mutant (comt 17). The biological significance of this conversion became clear when we found that the steroid hormone 20-hydroxyecdysone (ecdysone) is critically involved in this conversion. In its absence, Golgi stack biogenesis did not occur and the larval clusters remained unaltered. We showed that dGM130 and sec23p expression increases approximately three-and fivefold, respectively, when discs are exposed to ecdysone in vivo and in vitro. Taken together, these results suggest that we have developed an in vivo system to study the ecdysone-triggered Golgi stack biogenesis.

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Overlapping distribution of two glycosyltransferases in the Golgi apparatus of HeLa cells.

Cited by 213 publications

References 47 publications

Direct continuities between cisternae at different levels of the Golgi complex in glucose-stimulated mouse islet beta cells

Direct continuities between cisternae at different levels of the Golgi complex in glucose-stimulated mouse islet beta cells

Distribution of xylosylation and fucosylation in the plant Golgi apparatus

Biogenesis of Golgi Stacks in Imaginal Discs ofDrosophila melanogaster

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