Abstract. The exocytotic exposure and retrieval of an antigen of chromaffin granule membranes were studied with chromaffin cells isolated from bovine adrenal medulla. Cells were incubated with an antiserum against glycoprotein III followed by fluorescein-or gold-labeled anti-IgG. Immunofluorescence on the cell surface was present in a patchy distribution irrespective of whether bivalent antibodies or Fab fragments were used. During subsequent incubation these fluorescent membrane patches were internalized within 45 min. At the ultrastructural level immunogold-labeled patches were present on the surface of stimulated cells. During incubation (5 rain to 6 h) these immunolabeled membrane patches became coated, giving rise to coated vesicles and finally to smooth vesicles. These latter vesicles were found spread throughout the cytoplasm including the Golgi region, but Golgi stacks did not become labeled. Part of the immunolabel was transferred to multivesicular bodies, which probably represent a lysosomal pathway. 30 min after incubation immunolabel was also found in electron-dense vesicles apparently representing newly formed chromaffin granules. After 6 h of incubation immunolabel was found in vesicles indistinguishable from mature chromaffin granules. These results provide direct evidence that after exocytosis membranes of chromaffin granules are selectively retrieved from the plasma membrane and are partly recycled to newly formed chromaffin granules, providing a shuttle service from the Golgi region to the plasma membrane. SECRETION from adrenal medulla occurs by exocylosis (2, 21,26). This process, which adds membranes of chromaffin granules to the plasma membrane, is followed by endocytosis, as shown in early studies (l l, 15). In 1972 we demonstrated (34) that the synthesis rate of the membrane proteins of chromaffin granules was significantly lower than that of the secretory proteins (chromogranins) of the contents (for similar results in exocrine pancreas see reference 4). Based on this study we proposed that the membranes of chromaffin granules are reused for several secretory cycles (see also the discussion in reference 31). This concept gained considerable support when it was shown that in stimulated endocrine cells exogenous markers were taken up by endocytosis and were subsequently found in newly formed secretory vesicles (see references 5, 8, 9, and 24). These experiments demonstrated recycling of membranes but did not yet establish the specific nature of the recycled membranes. To achieve this, specific antigens of the secretory vesicle membrane have to be followed throughout the exo/endocytosis cycle. Two antigens of the chromaffin granules, i.e., dopamine, 13-hydroxylase and glycoprotein III, proved suitable for such an approach. In a first series of immunofluorescent studies it was established that these granule antigens become exposed on the cell surface of isolated chromaffin cells during exocytotic activity (3,13,18, 19,27). By quantitative evaluation we were able to show that after stimulati...

Abstract. A cDNA encoding the full-length 75-kD human nerve growth factor receptor was transfected into MDCK cells and its product was found to be expressed predominantly (80%) on the apical membrane, as a result of vectorial targeting from an intracellular site . Apical hNGFR bound NGF with low affinity and internalized it inefficiently (6% of surface bound NGF per hour) . Several mutant hNGFRs were analyzed, after transfection in MDCK cells, for polarized surface expression, ligand binding, and endocytosis . Deletion of juxtamembrane attachment sites for a cluster of O-linked sugars did not alter apical localization . A mutant receptor lacking the entire cytoplasmic tail (except for the five proximal amino acids) was also expressed on the apical membrane, suggesting that information for apical sorting was contained in the ectoplasmic or transmembrane domains. However, a 58 T HE plasma membrane of epithelial cells displays distinct apical and basolateral domains with different sets of proteins and lipids (9,50,58) . To establish and maintain these polarized surface domains, epithelial cells use diverse strategies including sorting and packaging of proteins and lipids into different post-Golgi transport vesicles, targeting of these vesicles to the correct domain, and stabilization and/or accurate recycling of the proteins specific for a given domain . Using different epithelial models such as liver, intestine, and kidney cells, progress was recently made in defining the pathways followed by apical and basolateral proteins from the Golgi complex to their respective domains (2, 25, 37) . Two major sorting sites were established by these studies, the trans-Golgi network (TGN) ' (17, 27, 28, 31, 36, 41) and the basolateral membrane (2,25,35,37) . To date, all basolateral proteins studied appear to be targeted directly from an intracellular sorting site (presumably 1. Abbreviations used in this paper: GPI, glycosyl-phosphatidylinositol ; hNGFR, human nerve growth factor ; PIgR, poly-IG receptor ; PLAP, placental alkaline phosphatase ; S-NHS-biotin, sulfo-N-hydroxyl-succinimidobiotin ; TGN, trans-Golgi network; WT, wild type .C The Rockefeller University Press, 0021-9525/91/11/607/12 $2 .00

The exocytotic exposure of antigens of chromaffin granule membranes was studied with chromaffin cells isolated from bovine adrenal medulla. Antigens on the cell surface were visualized by indirect membrane immunofluorescence employing antisera against glycoprotein III and dopamine ß-hydroxylase . With unstimulated cells, only weak immunofluorescence on the cell surface was observed, whereas stimulated cells (with carbachol or Bat +) exhibited much stronger reactions . In all cases the staining appeared as dots and patches . To quantitatively prove these observations, we analyzed the immunostained cells using a fluorescenceactivated cell sorter . After stimulation, the average fluorescence intensity of the cell population was enhanced . This increase correlated with the degree of catecholamine secretion . The fluorescence intensity of stimulated cells varied over a broad range indicating that individual cells reacted variably to the secretagogues . When stimulated cells were incubated at 37°C for up to 45 min after stimulation, a decrease of membrane immunofluorescence approaching that of unstimulated control cells was observed . Apparently, the membranes of chromaffin granules, which had been incorporated into the plasma membrane, were retrieved by a specific and relatively fast process . This retrieval of the antigen from the cell surface was blocked by sodium azide, but not influenced by colchicine, cytochalasin B, and trifluoperazine . The quantitative methods established in this paper should prove useful for further study of the kinetics of the exo-endocytotic cycle in secretory tissues .Secretion of catecholamines from adrenal medulla occurs by exocytosis (23,27) . During this process the membranes of the secretory organelles, i .e ., the chromaffin granules, are incorporated into the plasma membrane . In order to prevent enlargement of the cell surface, membrane retrieval has to accompany exocytotic activity (17). In adrenal medulla, evidence for endocytotic processes subsequent to secretion has been obtained by studying the uptake of exogenous markers (10,16,26). These investigations have not established whether the membranes of chromaffin granules are retrieved specifically and, if so, how fast this specific endocytosis proceeds. To answer these questions, specific components of the membranes of chromaffin granules have to be followed through the exo-endocytotic cycle . The biochemical composition and topology of these membranes has already been characterized

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