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...

We used thin-section fracture-label to determine the distribution of wheat-germ agglutinin binding sites in intracellular membranes of secretory and nonsecretory rat tissues as well as in human leukocytes . In all cases, analysis of the distribution of wheat germ agglutinin led to the definition of two endomembrane compartments : one, characterized by absence of the label, includes the membranes of mitochondria and peroxisomes as well as those of the endoplasmic reticulum and nuclear envelope ; the other, strongly labeled, comprises the membrane of lysosomes, phagocytic vacuoles, and secretory granules, as well as the plasma membrane. The Golgi apparatus was weakly labeled in all studied tissues .