Brefeldin A inhibits the formation of constitutive secretory vesicles and immature secretory granules from the trans-Golgi network.

The effects of brefeldin A (BFA) on membrane traffic between the trans-Golgi network (TGN) and the plasma membrane were investigated in intact PC12 cells and in a cell-free system derived from PC12 cells. In intact cells, BFA caused a virtually complete block of constitutive secretion, as indicated by the lack of release from, and accumulation in, the cells of a [35S]sulfate-labeled heparan sulfate proteoglycan (hsPG). Pulse-chase experiments with [35S]sulfate followed by subcellular fractionation showed that this block was due to the inhibition of formation of constitutive secretory vesicles (CSVs) from the TGN. BFA did not block the depolarization-induced release of [35S]sulfate-labeled chromogranin B (CgB) and secretogranin II (SgII) from secretory granules formed prior to the addition of the drug, showing that BFA does not block secretory granule fusion with the plasma membrane. The presence of BFA did, however, prevent the appearance of [35S]sulfate-labeled CgB and SgII in secretory granules, indicating that the drug inhibits the formation of secretory granules from the TGN. Evidence for a direct block of vesicle formation by BFA was obtained using a cell-free system derived from [35S]sulfate-labeled PC12 cells. In this system, low concentrations of BFA (5 micrograms/ml) inhibited the formation of the hsPG-containing CSVs and that of the SgII-containing secretory granules from the TGN to the same extent (50-60%) as, and in a non-additive manner with, the nonhydrolyzable GTP analogue GTP gamma S. Consistent with the inhibitory effects of BFA on vesicle formation from the TGN, BFA treatment of intact PC12 cells led to the hypersialylation of CgB, which presumably was due to the increased residence time of the protein in the TGN. In conclusion, our data are consistent with, and allow the generalization of, the concept that the BFA-induced block of anterograde membrane traffic results from the inhibition of vesicle formation from a donor compartment.     

Synaptic-like Microvesicles of Neuroendocrine Cells Originate from a Novel Compartment That Is Continuous with the Plasma Membrane and Devoid of Transferrin Receptor

We have characterized the compartment from which synaptic-like microvesicles (SLMVs), the neuroendocrine counterpart of neuronal synaptic vesicles, originate. For this purpose we have exploited the previous observation that newly synthesized synaptophysin, a membrane marker of synaptic vesicles and SLMVs, is delivered to the latter organelles via the plasma membrane and an internal compartment. Specifically, synaptophysin was labeled by cell surface biotinylation of unstimulated PC12 cells at 18°C, a condition which blocked the appearance of biotinylated synaptophysin in SLMVs and in which there appeared to be no significant exocytosis of SLMVs. The majority of synaptophysin labeled at 18°C with the membraneimpermeant, cleavable sulfo-NHS-SS–biotin was still accessible to extracellularly added MesNa, a 150-D membrane-impermeant thiol-reducing agent, but not to the 68,000-D protein avidin. The SLMVs generated upon reversal of the temperature to 37°C originated exclusively from the membranes containing the MesNaaccessible rather than the MesNa-protected population of synaptophysin molecules. Biogenesis of SLMVs from MesNa-accessible membranes was also observed after a short (2 min) biotinylation of synaptophysin at 37°C followed by chase. In contrast to synaptophysin, transferrin receptor biotinylated at 18° or 37°C became rapidly inaccessible to MesNa. Immunofluorescence and immunogold electron microscopy of PC12 cells revealed, in addition to the previously described perinuclear endosome in which synaptophysin and transferrin receptor are colocalized, a sub-plasmalemmal tubulocisternal membrane system distinct from caveolin-positive caveolae that contained synaptophysin but little, if any, transferrin receptor. The latter synaptophysin was selectively visualized upon digitonin permeabilization and quantitatively extracted, despite paraformaldehyde fixation, by Triton X-100. Synaptophysin biotinylated at 18°C was present in these subplasmalemmal membranes. We conclude that SLMVs originate from a novel compartment that is connected to the plasma membrane via a narrow membrane continuity and lacks transferrin receptor.     

Reduction of the disulfide bond of chromogranin B (secretogranin I) in the trans-Golgi network causes its missorting to the constitutive secretory pathways.

The role of the single, highly conserved disulfide bond in chromogranin B (secretogranin I) on the sorting of this regulated secretory protein to secretory granules was investigated in the neuroendocrine cell line PC12. Treatment of PC12 cells with dithiothreitol (DTT), a membrane permeable thiol reducing agent known to prevent disulfide bond formation in intact cells, resulted in the secretion of newly synthesized chromogranin B, but only slightly decreased the intracellular storage of newly synthesized secretogranin II, a regulated secretory protein devoid of cysteines. The secretion of newly synthesized chromogranin B in the presence of DTT occurred with similar kinetics to those of a heparan sulfate proteoglycan, a known marker of the constitutive secretory pathway in PC12 cells. Analysis of the various secretory vesicles derived from the trans-Golgi network (TGN) indicated that DTT treatment diverted newly synthesized chromogranin B to constitutive secretory vesicles, whereas the packaging of secretogranin II into immature secretory granules was unaffected by the reducing agent. The chromogranin B molecules diverted to constitutive secretory vesicles, in contrast to those stored in secretory granules, were found to contain free sulfhydryl residues. The effect of DTT on chromogranin B occurred in the TGN rather than in the endoplasmic reticulum. We conclude that the sorting of CgB in the TGN to secretory granules is dependent upon the integrity of its single disulfide bond.     

Characterization of the immature secretory granule, an intermediate in granule biogenesis

The events in the biogenesis of secretory granules after the budding of a dense-cored vesicle from the trans-Golgi network (TGN) were investigated in the neuroendocrine cell line PC12, using sulfate-labeled secretogranin II as a marker. The TGN-derived dense-cored vesicles, which we refer to as immature secretory granules, were found to be obligatory organellar intermediates in the biogenesis of the mature secretory granules which accumulate in the cell. Immature secretory granules were converted to mature secretory granules with a half-time of approximately 45 min. This conversion entailed an increase in their size, implying that the maturation of secretory granules includes a fusion event involving immature secretory granules. Pulse-chase labelling of PC12 cells followed by stimulation with high K+, which causes the release of secretogranin II, showed that not only mature, but also immature secretory granules were capable of undergoing regulated exocytosis. The kinetics of secretion of secretogranin II, as well as those of a constitutively secreted heparan sulfate proteoglycan, were reduced by treatment of PC12 cells with nocodazole, suggesting that both secretory granules and constitutive secretory vesicles are transported to the plasma membrane along microtubules. Our results imply that certain membrane proteins, e.g., those involved in the fusion of post-TGN vesicles with the plasma membrane, are sorted upon exit from the TGN, whereas other membrane proteins, e.g., those involved in the interaction of post-TGN vesicles with the cytoskeleton, may not be sorted.     

ADP ribosylation factor and a 14-kD polypeptide are associated with heparan sulfate-carrying post-trans-Golgi network secretory vesicles in rat hepatocytes

Constitutive secretory vesicles carrying heparan sulfate proteoglycan (HSPG) were identified in isolated rat hepatocytes by pulse-chase experiments with [35S]sulfate and purified by velocity-controlled sucrose gradient centrifugation followed by equilibrium density centrifugation in Nycodenz. Using this procedure, the vesicles were separated from plasma membranes, Golgi, trans-Golgi network (TGN), ER, endosomes, lysosomes, transcytotic vesicles, and mitochondria. The diameter of these vesicles was approximately 100-200 nm as determined by electron microscopy. A typical coat structure as described for intra- Golgi transport vesicles or clathrin-coated vesicles could not be seen, and the vesicles were not associated with the coat protein beta-COP. Furthermore, the vesicles appear to represent a low density compartment (1.05-1.06 g/ml). Other constitutively secreted proteins (rat serum albumin, apolipoprotein E, and fibrinogen) could not be detected in purified HSPG-carrying vesicles, but banded in the denser fractions of the Nycodenz gradient. Moreover, during pulse-chase labeling with [35S]methionine, labeled albumin did not appear in the post-TGN vesicle fraction carrying HSPGs. These findings indicate sorting of HSPGs and albumin into different types of constitutive secretory vesicles in hepatocytes. Two proteins were found to be tightly associated with the membranes of the HSPG carrying vesicles: a member of the ADP ribosylation factor family of small guanine nucleotide-binding proteins and an unknown 14-kD peripheral membrane protein (VAPP14). Concerning the secretory pathway, we conclude from these results that ADP ribosylation factor proteins are not only involved in vesicular transport from the ER via the Golgi to the TGN, but also in vesicular transport from the TGN to the plasma membrane.     

Cholesterol binds to synaptophysin and is required for biogenesis of synaptic vesicles

Here, to study lipid-protein interactions that contribute to the biogenesis of regulated secretory vesicles, we have developed new approaches by which to label proteins in vivo, using photoactivatable cholesterol and glycerophospholipids. We identify synaptophysin as a major specifically cholesterol-binding protein in PC12 cells and brain synaptic vesicles. Limited cholesterol depletion, which has little effect on total endocytic activity, blocks the biogenesis of synaptic-like microvesicles (SLMVs) from the plasma membrane. We propose that specific interactions between cholesterol and SLMV membrane proteins, such as synaptophysin, contribute to both the segregation of SLMV membrane constituents from plasma-membrane constituents, and the induction of synaptic-vesicle curvature.     

Developing Dictyostelium cells contain the lysosomal enzyme alpha- mannosidase in a secretory granule

The prespore vesicle (PSV) is an organelle which secretes spore coat proteins and gal/galNAc polysaccharides from prespore cells of Dictyostelium. By combining the techniques of protein A-gold immunocytochemistry and ricin-gold affinity cytochemistry we have demonstrated colocalization of the lysosomal enzyme alpha-mannosidase with gal/galNAc polysaccharides in prespore vesicles and the spore coat. To determine the origin of prespore vesicles a series of pulse- chase experiments were performed. Cells were labeled with [35S]methionine or [35S]sulfate at different times during development and allowed to differentiate in the presence of unlabeled methionine or sulfate for various periods of time. The cells were homogenized and intracellular organelles were separated using Percoll density gradient centrifugation. The distribution of [35S]methionine-labeled alpha- mannosidase and [35S]sulfate-labeled glycoproteins in the Percoll gradients was determined. It was found that prespore vesicles contained protein which was previously found in lysosomes. Newly labeled protein also entered these vesicles. The data suggest that developing Dictyostelium cells either restructure preexisting lysosomes into prespore vesicles or transport protein between these two organelles. We propose that secretory granules and lysosomes may have a common biosynthetic origin and may be evolutionarily related.     

Synaptic vesicles form by budding from tubular extensions of sorting endosomes in PC12 cells

The putative role of sorting early endosomes (EEs) in synaptic-like microvesicle (SLMV) formation in the neuroendocrine PC12 cell line was investigated by quantitative immunoelectron microscopy. By BSA-gold internalization kinetics, four distinct endosomal subcompartments were distinguished: primary endocytic vesicles, EEs, late endosomes, and lysosomes. As in other cells, EEs consisted of vacuolar and tubulovesicular subdomains. The SLMV marker proteins synaptophysin and vesicle-associated membrane protein 2 (VAMP-2) localized to both the EE vacuoles and associated tubulovesicles. Quantitative analysis showed that the transferrin receptor and SLMV proteins colocalized to a significantly higher degree in primary endocytic vesicles then in EE-associated tubulovesicles. By incubating PC12 cells expressing T antigen-tagged VAMP (VAMP-TAg) with antibodies against the luminal TAg, the recycling pathway of SLMV proteins was directly visualized. At 15 degrees C, internalized VAMP-TAg accumulated in the vacuolar domain of EEs. Upon rewarming to 37 degrees C, the labeling shifted to the tubular part of EEs and to newly formed SLMVs. Our data delineate a pathway in which SLMV proteins together with transferrin receptor are delivered to EEs, where they are sorted into SLMVs and recycling vesicles, respectively.     

Prohormone processing in the trans-Golgi network: endoproteolytic cleavage of prosomatostatin and formation of nascent secretory vesicles in permeabilized cells

Many peptide hormones are synthesized as larger precursors which undergo endoproteolytic cleavage at paired basic residues to generate a bioactive molecule. Morphological evidence from several laboratories has implicated either the TGN or immature secretory granules as the site of prohormone cleavage. To identify the site where prohormone cleavage is initiated, we have used retrovirally infected rat anterior pituitary GH3 cells which express high levels of prosomatostatin (proSRIF) (Stoller, T. J., and D. Shields. J. Cell Biol. 1988. 107:2087- 2095). By incubating these cells at 20 degrees C, a temperature that prevents exit from the Golgi apparatus, proSRIF accumulated quantitatively in the TGN and no proteolytic processing was evident; processing resumed upon shifting the cells back to 37 degrees C. After the 20 degrees C block, the cells were mechanically permeabilized and pro-SRIF processing determined. Cleavage of proSRIF to the mature hormone was approximately 35-50% efficient, required incubation at 37 degrees C and ATP hydrolysis, but was independent of GTP or cytosol. The in vitro ATP-dependent proSRIF processing was inhibited by inclusion of chloroquine, a weak base, CCCP, a protonophore, or by preincubating the permeabilized cells with low concentrations of N- ethylmaleimide, an inhibitor of vacuolar-type ATP-dependent proton pumps. These data suggest that: (a) proSRIF cleavage is initiated in the TGN, and (b) this reaction requires an acidic pH which is facilitated by a Golgi-associated vacuolar-type ATPase. A characteristic feature of polypeptide hormone-producing cells is their ability to store the mature hormone in dense core secretory granules. To investigate the mechanism of protein sorting to secretory granules, the budding of nascent secretory vesicles from the TGN was determined. No vesicle formation occurred at 20 degrees C; in contrast, at 37 degrees C, the budding of secretory vesicles was approximately 40% efficient and was dependent on ATP, GTP, and cytosolic factors. Vesicle formation was inhibited by GTP gamma S suggesting a role for GTP- binding proteins in this process. Vesicle budding was dependent on cytosolic factors that were tightly membrane associated and could be removed only by treating the permeabilized cells with high salt. After high salt treatment, vesicle formation was dependent on added cytosol or the dialyzed salt extract. The formation of nascent secretory vesicles contrasts with prosomatostatin processing which required only ATP for efficient cleavage. Our results demonstrate that prohormone cleavage which is initiated in the TGN, precedes vesicle formation and that processing can be uncoupled from the generation of nascent secretory vesicles.     

Cellugyrin and synaptogyrin facilitate targeting of synaptophysin to a ubiquitous synaptic vesicle-sized compartment in PC12 cells

Cellugyrin represents a ubiquitously expressed four-transmembrane domain protein that is closely related to synaptic vesicle protein synaptogyrin and, more remotely, to synaptophysin. We report here that, in PC12 cells, cellugyrin is localized in synaptic-like microvesicles (SLMVs), along with synaptogyrin and synaptophysin. Upon overexpression of synaptophysin in PC12 cells, it is localized in rapidly sedimenting membranes and practically is not delivered to the SLMVs. On the contrary, the efficiency of the SLMV targeting of exogenously expressed cellugyrin and synaptogyrin is high. Moreover, expression of cellugyrin (or synaptogyrin) in PC12 cells dramatically and specifically increases SLMV targeting of endogenous synaptophysin. Finally, we utilized the SLMV purification scheme on a series of non-neuroendocrine cell types including the mouse fibroblast cell line 3T3-L1, the Chinese hamster ovary cell line CHO-K1, and the monkey kidney epithelial cell line COS7 and found that a cellugyrin-positive microvesicular compartment was present in all cell types tested. We suggest that synaptic vesicles have evolved from cellugyrin-positive ubiquitous microvesicles and that neuroendocrine SLMVs represent a step along that pathway of evolution.     

Recycling of the dense-core vesicle membrane protein phogrin in Min6 beta-cells

Phogrin (IA2-beta) is an integral membrane protein of dense-core vesicles in neuroendocrine cells. We have examined the recycling of endogenous phogrin following exocytosis in insulin secreting Min6 beta-cells by monitoring stimulus dependent-uptake of antibodies directed against the lumenal domain of the protein. While low levels of internalized phogrin accumulated in LAMP1-positive lysosomes, more than 35% of internalized phogrin recycled back to an insulin-positive compartment and could return to the cell surface during a second exocytic stimulation. The recycling phogrin transited a syntaxin 6-positive compartment but did not appear to go through the TGN38-positive trans Golgi network. The results suggest a model in which secretory membrane components can recycle from the endosomal system to immature secretory granules without interaction with the major portion of the TGN.     

Sorting of synaptophysin into special vesicles in nonneuroendocrine epithelial cells

Synaptophysin is a major transmembrane glycoprotein of a type of small vesicle with an electron-translucent content (SET vesicles), including the approximately 50-nm presynaptic vesicles in neuronal cells, and of similar, somewhat larger (< or = approximately 90 nm) vesicles (SLMV) in neuroendocrine (NE) cells. When certain epithelial non-NE cells, such as human hepatocellular carcinoma PLC cells, were cDNA transfected to synthesize synaptophysin, the new molecules appeared in specific SET vesicles. As this was in contrast to other reports that only NE cells were able to sort synaptophysin away from other plasma membrane proteins into presynaptic- or SLMV-type vesicles, we have further characterized the vesicles containing synaptophysin in transfected PLC cells. Using fractionation and immunoisolation techniques, we have separated different kinds of vesicles, and we have identified a distinct type of synaptophysin-rich, small (30-90-nm) vesicle that contains little, if any, protein of the constitutive secretory pathway marker hepatitis B surface antigen, of the fluid phase endocytosis marker HRP, and of the plasma membrane recycling endosomal marker transferrin receptor. In addition, we have found variously sized vesicles that contained both synaptophysin and transferrin receptor. A corresponding result was also obtained by direct visualization, using double-label immunofluorescence microscopy for the endocytotic markers and synaptophysin in confocal laser scan microscopy and in double- immunogold label electron microscopy. We conclude that diverse non-NE cells of epithelial nature are able to enrich the "foreign" molecule synaptophysin in a category of SET vesicles that are morphologically indistinguishable from SLMV of NE cells, including one type of vesicle in which synaptophysin is sorted away from endosomal marker proteins. Possible mechanisms of this sorting are discussed.     

Rab8 regulates basolateral secretory, but not recycling, traffic at the recycling endosome

Rab8 is a monomeric GTPase that regulates the delivery of newly synthesized proteins to the basolateral surface in polarized epithelial cells. Recent publications have demonstrated that basolateral proteins interacting with the mu1-B clathrin adapter subunit pass through the recycling endosome (RE) en route from the TGN to the plasma membrane. Because Rab8 interacts with these basolateral proteins, these findings raise the question of whether Rab8 acts before, at, or after the RE. We find that Rab8 overexpression during the formation of polarity in MDCK cells, disrupts polarization of the cell, explaining how Rab8 mutants can disrupt basolateral endocytic and secretory traffic. However, once cells are polarized, Rab8 mutants cause mis-sorting of newly synthesized basolateral proteins such as VSV-G to the apical surface, but do not cause mis-sorting of membrane proteins already at the cell surface or in the endocytic recycling pathway. Enzymatic ablation of the RE also prevents traffic from the TGN from reaching the RE and similarly results in mis-sorting of newly synthesized VSV-G. We conclude that Rab8 regulates biosynthetic traffic through REs to the plasma membrane, but not trafficking of endocytic cargo through the RE. The data are consistent with a model in which Rab8 functions in regulating the delivery of TGN-derived cargo to REs.     

Trans-Golgi network and subapical compartment of HepG2 cells display different properties in sorting and exiting of sphingolipids

In HepG2 cells, the subapical compartment (SAC) is involved in the biogenesis of membrane polarity. By contrast, direct apical transport originating from the trans-Golgi network (TGN), which may contribute to polarity establishment, has been poorly defined in these cells. Thus, although newly synthesized sphingolipids can be directly transported from the TGN to the apical membrane, numerous apical resident proteins are traveling via the transcytotic route. Here, we developed an in vitro transport assay and compared the molecular sorting of 6-[N-(7-nitrobenz-2-oxa-1,3 diazol-4-yl)amino] hexanoyl-sphingomyelin (C(6)NBD-SM) and C(6)NBD-glucosylceramide (C(6)NBD-GlcCer) in TGN and SAC. SM is released from both TGN and SAC in the lumenal leaflet of transport vesicles. This holds also for GlcCer released from the SAC but not for a substantial fraction that departed from the Golgi. Distinct transport vesicles, enriched in either SM or GlcCer are released from SAC, consistent with their rigid sorting in this compartment. Different vesicle populations could not be recovered from TGN, although in situ experiments reveal that GlcCer is preferentially transported to the apical membrane, reflecting different transport mechanisms. The results indicate that in HepG2 cells sphingolipids are mainly sorted in the SAC membrane and that the release of SM from SAC and TGN is differentially regulated.     

Interaction of furin in immature secretory granules from neuroendocrine cells with the AP-1 adaptor complex is modulated by casein kinase II phosphorylation.

The composition of secretory granules in neuroendocrine and endocrine cells is determined by two sorting events; the first in the trans-Golgi complex (TGN), the second in the immature secretory granule (ISG). Sorting from the ISG, which may be mediated by the AP-1 type adaptor complex and clathrin-coated vesicles, occurs during ISG maturation. Here we show that furin, a ubiquitously expressed, TGN/endosomal membrane endoprotease, is present in the regulated pathway of neuroendocrine cells where it is found in ISGs. By contrast, TGN38, a membrane protein that is also routed through the TGN/endosomal system does not enter ISGs. Furin, however, is excluded from mature secretory granules, suggesting that the endoprotease is retrieved from the clathrin-coated ISGs. Consistent with this, we show that the furin cytoplasmic domain interacts with AP-1, a component of the TGN/ISG-localized clathrin sorting machinery. Interaction between AP-1 and furin is dependent on phosphorylation of the enzyme's cytoplasmic domain by casein kinase II. Finally, in support of a requirement for the phosphorylation-dependent association of furin with AP-1, expression of furin mutants that mimic either the phosphorylated or unphosphorylated forms of the endoprotease in AtT-20 cells demonstrates that the integrity of the CKII sites is necessary for removal of furin from the regulated pathway.     

Membrane retrieval in the guinea pig neurohypophysis: biochemical characterization of a retrieval structure

[3H]Choline and [35S]methionine injected into the guinea pig hypothalamus in vivo were incorporated into the lipids and proteins, respectively, of secretory vesicles transported to the neural lobe. Prolonged in vivo stimulation of hormone secretion by dehydration decreased the [3H]choline content of secretory vesicles, with a concomitant increase in the [3H]choline content of a membrane fraction isolated on sucrose gradients. After stimulation of neural lobes in vitro in the presence of horseradish peroxidase, this extracellular fluid marker was found in the same membrane fraction. SDS electrophoresis of membrane proteins radiolabelled by [35S]methionine in vivo demonstrated that this fraction contained at least one major protein also present in the secretory vesicle membrane. These results suggest that we have isolated a membrane fraction containing the structure(s) involve in membrane retrieval in the neurohypophysis.     

Trimeric G-proteins of the trans-Golgi network are involved in the formation of constitutive secretory vesicles and immature secretory granules.

Non-hydrolysable analogues of GTP, such as GTP gamma S and GMP-PNP, have previously been shown to inhibit the formation of constitutive secretory vesicles (CSVs) and immature secretory granules (ISGs) from the trans-Golgi network (TGN). Using a cell-free system, we show here that the formation of these vesicles is also inhibited by [A1F4]-, a compound known to act on trimeric G-proteins. Addition of highly purified G-protein beta gamma subunits stimulated, in a differential manner, the cell-free formation of both CSVs and ISGs. ADP-ribosylation experiments revealed the presence of a pertussis toxin-sensitive G-protein alpha subunit in the TGN. We conclude that trimeric G-proteins regulate the formation of secretory vesicles from the TGN.     

Association of a phosphatidylinositol-specific 3-kinase with a human trans-Golgi network resident protein

The eukaryotic trans-Golgi network (TGN) is a key site for the formation of transport vesicles destined for different intracellular compartments [1]. A key marker for the mammalian TGN is TGN38/46 [2]. This integral membrane glycoprotein cycles between the TGN and the cell surface and is implicated in recruitment of cytosolic factors and regulation of at least one type of vesicle formation at the mammalian TGN [2] and [3]. In this study, we have identified a phosphatidylinositol (PtdIns)-specific 3-kinase activity associated with the human orthologue (TGN46), which is sensitive to lipid kinase inhibitors. Treatment of HeLa cells with low levels of these inhibitors reveals subtle morphological changes in TGN46-positive compartments. Our findings suggest a role for PtdIns 3-kinases and presumably for the product, PtdIns 3-phosphate (PtdIns3P), in the formation of secretory transport vesicles by mechanisms conserved in yeast and mammals.     

Serotonin organelles of rabbit platelets contain synaptophysin

Synaptophysin, an integral membrane protein of synaptic vesicles in nerve terminals and a class of small translucent vesicles in neuroendocrine cells, was detected in intact rabbit platelets by immunoblotting, immunofluorescence staining and immuno-electron microscopy. In a highly purified preparation of serotonin organelles isolated from rabbit platelets, synaptophysin was enriched approximately 10-15-fold over platelet homogenate. About 80% of total platelet synaptophysin was present in this purified fraction. The apparent molecular mass (approximately 38 kDa) and the extent of glycosylation of platelet-derived synaptophysin was more similar to the neuronal than to the neuroendocrine form of the protein. Immunofluorescence microscopy revealed that synaptophysin was compartmentalized in intact rabbit platelets and immuno-electron microscopy of subcellular fractions showed that it was localized exclusively to the membrane surface of serotonin organelles. No synaptophysin-like immunoreactivity was detected in platelets from other species such as human, guinea pig and rat. Another integral membrane protein of synaptic vesicles, p65, and a family of synaptic vesicle-associated phosphoproteins, the synapsins, were not detected in platelets of any species tested. These results provide evidence that serotonin organelles from rabbit platelets share a subset of protein components with synaptic vesicles from neurons. Synaptophysin in serotonin organelles from rabbit platelets, as suggested for small synaptic vesicles in neurons, might play a role in the formation of protein channels for the exocytotic release of serotonin.     

Sulfation and constitutive secretion of dopamine beta-hydroxylase from rat pheochromocytoma (PC12) cells

The biosynthesis and secretion of dopamine beta-hydroxylase were investigated by radiolabeling rat pheochromocytoma (PC12) cells in culture. Intracellular dopamine beta-hydroxylase from a crude chromaffin vesicle fraction and secreted dopamine beta-hydroxylase from culture medium were immunoprecipitated using antiserum made against purified bovine soluble dopamine beta-hydroxylase. Analysis of the immunoprecipitated enzyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that: 1) the membrane-bound form of the hydroxylase from crude secretory vesicle membrane extracts contained two nonidentical subunits in approximately stoichiometric amounts (Mr = 77,000 and 73,000); 2) the soluble hydroxylase from the lysate of these secretory vesicles was composed predominantly of a single subunit (Mr = 73,000); and 3) the hydroxylase secreted into the medium under resting conditions was also composed of a single subunit (approximate Mr = 73,000). All subunits of the multiple forms of hydroxylase were glycoproteins. Under resting conditions, the rate of secretion of hydroxylase was approximately 6% of total cellular enzyme/15 min. The secreted form of the hydroxylase incorporated [35S]sulfate, whereas no significant [35S]sulfate was incorporated into the cellular forms of enzyme. We propose that in addition to the dopamine beta-hydroxylase which is found in catecholamine storage vesicles and released during stimulus-coupled exocytosis, PC12 cells also have a constitutive secretory pathway for dopamine beta-hydroxylase and that the enzyme released by this second pathway is sulfated.