Distribution of terminal glycosyltransferases in hepatic Golgi fractions.

The combined application of thin-section and critical-point-drying "fracture-label" is used to determine the pattern of distribution and partition of wheat-germ agglutinin and concanavalin A binding sites on the membrane faces of freeze-fractured exocrine and endocrine rat pancreatic cells. Whereas the exoplasmic face of plasma membranes is preferentially labeled by both lectins, the endoplasmic reticulum and nuclear envelope are strongly and uniformly labeled by concanavalin A but not by wheat-germ agglutinin . The results support current views in the glycosylation of membrane proteins and do not support the backflow of sialidated glycoproteins to the endoplasmic reticulum.Recently, we showed that cytochemical and immunochemical techniques can be combined with freeze-fracture to gain generalized access for direct labeling of the fracture faces of biological membranes as well as of exposed groups in crossfractured cytoplasm (14-16) . The results of these "fracturelabel" techniques can be assessed either by observation of thin sections of freeze-fractured specimens ("thin-section fracturelabel") (15, 16) or of platinum/carbon replicas of critical point dried, freeze-fractured preparations ("critical-point-drying fracture-label") (14).Initial application of thin-section fracture-label showed that numerous concanavalin A binding sites could be labeled on the membrane faces of freeze-fractured plasma membranes, endoplasmic reticulum, and nuclear envelope membranes of leukocytes, HeLa cells, and hepatocytes (16). We report here the results of thin-section and critical-point-drying fracturelabel of isolated rat exocrine and endocrine pancreatic cells. We used colloidal gold and ferritin conjugates to determine and compare the patterns of distribution of wheat-germ agglutinin (WGA) and concanavalin A (Con A) binding sites on the fracture face of their plasma and intracellular membranes. Our results show that whereas Con A binding sites are present on nuclear envelope, endoplasmic reticulum, secretory vesicle, and plasma membranes, wheat-germ agglutinin binding sites are absent from the nuclear envelope and the endoplasmic reticu-THE JOURNAL OF CELL BIOLOGY " VOLUME 91 NOVEMBER 1981 361-372 lum. These results are consistent with current views on the pathways of glycosylation of membrane proteins (5,12, 18,19,22,23,24) and do not support the reflux of fully glycosylated products to the endoplasmic reticulum (3,7,25,29). They demonstrate the capacity of fracture-label to investigate the topochemistry of plasma and intracellular membranes in situ . MATERIALS AND METHODS CellsPancreas tissue was excised from 3-to 8-d-old rats (Sprague-Dawley) and digested with collagenase type IV (5 g/ml in Hanks' solution, from Worthington Biochemical Corp ., Freehold, N . J .) for 6 min at 37°C, and isolated cells or small clusters of acinar and islet cells were obtained. The cells and cell clusters were harvested by centrifugation (800 rpm, 6 min), washed twice in Hanks' solution and fixed in 1% glutaraldehyde for 2 h at 4°C . A...

supporting

confidence: 82%

Section: Cytochemical Labelingmentioning

confidence: 99%

Freeze-fracture cytochemistry: localization of wheat-germ agglutinin and concanavalin A binding sites on freeze-fractured pancreatic cells.

Silva¹,

Torrisi²,

Kachar³

1981

“…Once terminal sugar addition is completed, intracellular transport of these molecules to the cell surface is unaffected by the drug (43) . Our finding that monensin depresses incorporation of galactose but not fucose was unexpected since there is little evidence that terminal sugars are each added in a separate region of the GA (2,43) . That the drug may be directly affecting galactosyltransferase is not likely since many of the individual fasttransported proteins that are depressed by monensin do not contain either galactose or fucose (36) .…”

Section: Discussionmentioning

confidence: 68%

Evidence that all newly synthesized proteins destined for fast axonal transport pass through the Golgi apparatus.

Hammerschlag¹,

Stone²,

Bolen³

et al. 1982

124

Effects of the sodium ionophore, monensin, were examined on the passage from neuronal cell body to axon of materials undergoing fast intracellular transport. In vitro exposure of bullfrog dorsal root ganglia to concentrations of drug <1 .0 UM led to a dose-dependent depression in the amount of fast-transported [3H]leucine-or [3H]glycerol-labeled material appearing in the nerve trunk. Incorporation of either precursor was unaffected . Exposure of a desheathed nerve trunk to similar concentrations of monensin, while ganglia were incubated in drug-free medium, had no effect on transport. With [3H]fucose as precursor, fast transport of labeled glycoproteins was depressed to the same extent as with [3H]leucine; synthesis, again, was unaffected . By contrast, with [3 H]galactose as precursor, an apparent reduction in transport of labeled glycoproteins was accounted for by a marked depression in incorporation . The inference from these findings, that monensin acts to block fast transport at the level of the Golgi apparatus, was supported by ultrastructural examination of the drug-treated neurons. An extensive and selective disruption of Golgi saccules was observed, accompanied by an accumulation of clumped smooth membranous cisternae.Quantitative analyses of 48 individual fast-transported protein species, after separation by two-dimensional gel electrophoresis, revealed that monensin depresses all proteins to a similar extent . These results indicate that passage through the Golgi apparatus is an obligatory step in the intracellular routing of materials destined for fast axonal transport.

“…The trimmed GlcNAcMansGIcNAc2 structure can then be further processed by the addition of fucose to the innermost GlcNAc residue and by additions of various amounts of GlcNAc, galactose, sialic acid, and fucose to the outer residues. These latter reactions all take place in or near the Golgi apparatus (16,21,115). Inasmuch as there four a(l,2)-linked mannoses and two other c~-mannoses are removed during processing to complex carbohydrates, there are quite conceivably six different mannosidases.…”

Section: Discussionmentioning

confidence: 99%

Correlation of glycosylation forms with position in amino acid sequence

Pollack¹,

Atkinson²

1983

105

We surveyed published reports on about 50 glycoproteins whose amino acid sequence, glycosylation sites, and type of glycosylation at a particular site have been established. We note that high-mannose substances were rarely found at the N-terminal side of a previously glycosylated complex site. There was a very definite distribution of complex sites about the N-terminal region. Furthermore, secreted glycoproteins usually contained only complex oligosaccharides whereas membrane proteins contained both types. We suggest that the position of the glycosylation site with respect to the N-terminus affects the extent of oligosaccharide processing and subsequent presentation of complex or high-mannose structures in the mature glycoprotein. This review relates glycosylation type to its position in the known sequence of given proteins and discusses these observations in light of known glycosylation processing reactions.