Subcellular Distribution of 65,000 Calmodulin-Binding Protein (p65) and Synaptophysin (p38) in Adrenal Medulla

Both neuronal and endocrine cells contain secretory vesicles that store and release neurotransmitters and peptides. Neuronal cells release their secretory material from both small synaptic vesicles and large dense-core vesicles (LDCVs), whereas endocrine cells release secretory products from LDCVs. Neuronal small synaptic vesicles are known to express three integral membrane proteins: 65,000 calmodulin-binding protein (65-CMBP) (p65), synaptophysin (p38), and SV2. A controversial question surrounding these three proteins is whether they are present in LDCV membranes of endocrine and neuronal cells. Sucrose density centrifugation of adrenal medulla was performed to study and compare the subcellular distribution of two of these small synaptic vesicle proteins (65-CMBP and synaptophysin). Subsequent immunoblotting and 125I-Protein A binding experiments performed on the fractions obtained from sucrose gradients showed that 65-CMBP was present in fractions corresponding to granule membranes and intact chromaffin granules. Similar immunoblotting and 125I-Protein A binding experiments with synaptophysin antibodies showed that this protein was also present in intact granules and granule membrane fractions. However, an additional membrane component, equilibrating near the upper portion of the sucrose gradient, also showed strong immunoreactivity with anti-synaptophysin and high 125I-Protein A binding activity. In addition, immunoblotting experiments on purified plasma and granule membranes demonstrated that 65-CMBP was a component of both membranes, whereas synaptophysin was only present in granule membranes. Thus, there appears to be a different subcellular localization between 65-CMBP and synaptophysin in the chromaffin cell.     

In Adrenal Medulla Synaptophysin (Protein p38) Is Present in Chromaffin Granules and in a Special Vesicle Population

We have analyzed the properties and subcellular localization of synaptophysin (protein p38) in bovine adrenal medulla. In one-dimensional immunoblotting the adrenal antigen appears identical to synaptophysin of rat synaptic vesicles. In two-dimensional immunoblotting it migrates as a heterogeneous band varying in pI from 4.5 to 5.8. Subcellular fractionation by various sucrose gradients revealed that synaptophysin was present in two different cell particles. More than half of the antigens present in adrenal medulla were confined to special membranes that sedimented both with the "large granules" and with microsomal elements. These membranes could be removed from the large granule sediment by washing. In gradients it equilibrated in regions of low sucrose density. These membranes did not contain any markers for chromaffin granules. Less than half of the amount of synaptophysin present in adrenal medulla copurified with chromaffin granules. Despite several variations in the fractionation scheme synaptophysin could not be removed from chromaffin granules. After washing of granule membranes with alkaline solution synaptophysin still cosedimented in gradients with typical granule markers. The concentration of synaptophysin in membranes of chromaffin granules is low (less than 10%) when compared with synaptic vesicles. It is concluded that in adrenal medulla synaptophysin is present in special membranes, probably in high concentration, and in membranes of chromaffin granules, either in a low concentration in all or in a higher concentration in some of them.     

Endocrine secretory granules and neuronal synaptic vesicles have three integral membrane proteins in common

In response to an external stimulus, neuronal cells release neurotransmitters from small synaptic vesicles and endocrine cells release secretory proteins from large dense core granules. Despite these differences, endocrine cells express three proteins known to be components of synaptic vesicle membranes. To determine if all three proteins, p38, p65, and SV2, are present in endocrine dense core granule membranes, monoclonal antibodies bound to beads were used to immunoisolate organelles containing the synaptic vesicle antigens. [3H]norepinephrine was used to label both chromaffin granules purified from the bovine adrenal medulla and rat pheochromocytoma (PC12) cells. Up to 80% of the vesicular [3H]norepinephrine was immunoisolated from both labeled purified bovine chromaffin granules and PC12 postnuclear supernatants. In PC12 cells transfected with DNA encoding human growth hormone, the hormone was packaged and released with norepinephrine. 90% of the sedimentable hormone was also immunoisolated by antibodies to all three proteins. Stimulated secretion of PC12 cells via depolarization with 50 mM KCl decreased the amount of [3H]norepinephrine or human growth hormone immunoisolated. Electron microscopy of the immunoisolated fractions revealed large (greater than 100 nm diameter) dense core vesicles adherent to the beads. Thus, large dense core vesicles containing secretory proteins possess all three of the known synaptic vesicle membrane proteins.     

Demonstration of immunochemical identity between the synaptic vesicle proteins synaptin and synaptophysin/p38

The synaptic vesicle proteins synaptin and synaptophysin/p38 were shown to be immunochemically identical. Western immunoblot analysis of Triton X-100 extracts from rat brain showed that polyclonal polyspecific anti-synaptin antibodies and monoclonal antibody SY38 against synaptophysin both reacted with a band of 38 kDa. In two-dimensional immunoblots of chromaffin granule membranes from bovine adrenal medulla anti-synaptin and anti-synaptophysin antibodies also recognized the same component. Finally, in a Western immunoblotting experiment SY38 reacted with an immuno-isolated synaptin antigen.     

Membrane Composition of Adrenergic Large and Small Dense Core Vesicles and of Synaptic Vesicles: Consequences for Their Biogenesis

The membrane proteins of adrenergic large dense core vesicles, in particular those of chromaffin granules, have been characterized in detail. With the exception of the nucleotide carrier all major peptides have been cloned. There has been a controversy whether these vesicles contain antigens like synaptophysin, synaptotagmin and VAMP or synaptobrevin found in high concentration in synaptic vesicles. One can now conclude that large dense core vesicles also contain these peptides although in lower concentrations. The biosynthesis of large dense core vesicles is analogous to that of other peptide secreting vesicles of the regulated pathway. One cannot yet definitely define the biosynthesis of small dense core vesicles which apparently have a very similar membrane composition to that of large dense core vesicles. They may form directly from large dense core vesicles when their membranes have been retrieved after exocytosis. These membranes may become sorted in an endosomal compartment where peptides may be deleted or added. Such an addition could be derived from synaptophysin-rich vesicles present in adrenergic axons. However small dense core vesicle peptides may also be transported axonally independent of large dense core vesicles. For proving one of these possibilities some crucial experiments have been suggested.     

Fractionation of synaptophysin-containing vesicles from rat brain and cultured PC12 pheochromocytoma cells

Synaptophysin is a transmembrane glycoprotein of neuroendocrine vesicles. Its content and distribution in subcellular fractions from cultured PC12 cells, rat brain and bovine adrenal medulla were determined by a sensitive dot immunoassay. Synaptophysin-containing fractions appeared as monodispersed populations similar to synaptic vesicles in density and size distribution. Membranes from synaptic vesicles contained approximately 100-times more synaptophysin than chromaffin granules. In conclusion, synaptophysin is located almost exclusively in vesicles of brain and PC12 cells which are distinct from dense core granules.     

Adrenal chromaffin granules and secretory granules from thyroid parafollicular cells have several common antigens

The presence of various antigens in two types of isolated endocrine vesicles (chromaffin granules and secretory vesicles of thyroid parafollicular cells) was investigated by immunoblotting. The two types of vesicles have three common secretory proteins: chromogranin A, chromogranin B and secretogranin II. Furthermore, six common membrane antigens were found: cytochrome b-561, carboxypeptidase H, glycoprotein II, glycoprotein III, synaptin/synaptophysin and SV 2. These results demonstrate that vesicles obtained from neural crest-derived endocrine cells not only share several common secretory peptides and proteins, but also have common properties as far as their membrane antigens are concerned.     

Immunocytochemical Localization of Synaptic Proteins at Vesicular Organelles in PC12 Cells

The distribution of the three synaptic vesicle proteins SV2, synaptophysin and synaptotagmin, and of SNAP-25, a component of the docking and fusion complex, was investigated in PC12 cells by immunocytochemistry. Colloidal gold particle-bound secondary antibodies and a preembedding protocol were applied. Granules were labeled for SV2 and synaptotagmin but not for synaptophysin. Electron-lucent vesicles were labeled most intensively for synaptophysin but also for SV2 and to a lesser extent for synaptotagmin. The t-SNARE SNAP-25 was found at the plasma membrane but also at the surface of granules. Labeling of Golgi vesicles was observed for all antigens investigated. Also components of the endosomal pathway such as multivesicular bodies and multilamellar bodies were occasionally marked. The results suggest that the three membrane-integral synaptic vesicle proteins can have a differential distribution between electron-lucent vesicles (of which PC12 cells may possess more than one type) and granules. The membrane compartment of granules appears not to be an immediate precursor of that of electron-lucent vesicles.     

Domain structure of synaptotagmin (p65)

Synaptotagmin (p65) is an abundant and evolutionarily conserved protein of synaptic vesicles that contains two copies of an internal repeat homologous to the regulatory region of protein kinase C. In the current study, we have investigated the biochemical properties of synaptotagmin, demonstrating that it contains five protein domains: an intravesicular amino-terminal domain that is glycosylated but lacks a cleavable signal sequence; a single transmembrane region; a sequence separating the transmembrane region from the two repeats homologous to protein kinase C; the two protein kinase C-homologous repeats; and a conserved carboxyl-terminal sequence following the two repeats homologous to protein kinase C. Sucrose density gradient centrifugations and gel electrophoresis indicate that synaptotagmin monomers associate into dimers and are part of a larger molecular weight complex. A sequence predicted to form an amphipathic alpha-helix that may cause the stable dimerization of synaptotagmin is found in its third domain between the transmembrane region and the protein kinase C-homologous repeats. Synaptotagmin contains a single hypersensitive proteolytic site that is located immediately amino-terminal to the amphipathic alpha-helix, suggesting that synaptotagmin contains a particularly exposed region as the peptide backbone emerges from the dimer. Finally, subcellular fractionation and antibody bead purification demonstrate that synaptotagmin co-purifies with synaptophysin and other synaptic vesicle markers in brain. However, in the adrenal medulla, synaptotagmin was found in both synaptophysin-containing microvesicles and in chromaffin granules that are devoid of synaptophysin, suggesting a shared role for synaptotagmin in the exocytosis of small synaptic vesicles and large dense core catecholaminergic vesicles.     

Most Synaptic Vesicles Isolated from Rat Brain Carry Three Membrane Proteins, SV2, Synaptophysin, and p65

We have prepared highly purified synaptic vesicles from rat brain by subjecting vesicles purified by our previous method to a further fractionation step, i.e., equilibrium centrifugation on a Ficoll gradient. Monoclonal antibodies to three membrane proteins enriched in synaptic vesicles--SV2, synaptophysin, and p65--each were able to immunoprecipitate specifically approximately 90% of the total membrane protein from Ficoll-purified synaptic vesicle preparations. Anti-SV2 precipitated 96% of protein, anti-synaptophysin 92%, and anti-p65 83%. These results demonstrate two points: (1) Ficoll-purified synaptic vesicles appear to be greater than 90% pure, i.e., less than 10% of membranes in the preparation do not carry synaptic vesicle-associated proteins. These very pure synaptic vesicles may be useful for direct biochemical analyses of mammalian synaptic vesicle composition and function. (2) SV2, synaptophysin, and p65 coexist on most rat brain synaptic vesicles. This result suggests that the functions of these proteins are common to most brain synaptic vesicles. However, if SV2, synaptophysin, or p65 is involved in synaptic vesicle dynamics, e.g., in vesicle trafficking or exocytosis, separate cellular systems are very likely required to modulate the activity of such proteins in a temporally or spatially specific manner.     

Characterization of a membrane protein from cholinergic synaptic vesicles isolated from the electric organ of Torpedo marmorata

Rabbits were immunized with cholinergic synaptic vesicles isolated from the electric organ of Torpedo marmorata. The resultant antiserum had one major antibody activity against an antigen called the Torpedo vesicle antigen. This antigen could not be demonstrated in muscle, liver or blood and is therefore, suggested to be nervous-tissue specific. The vesicle antigen was quantified in various parts of the nervous system and in subcellular fractions of the electric organ of Torpedo marmorata and was found to be highly enriched in synaptic vesicle membranes. The antigen bound to concanavalin A, thereby demonstrating the presence of a carbohydrate moiety. By means of charge-shift electrophoresis, amphiphilicity was demonstrated, indicating that the Torpedo vesicle antigen is an intrinsic membrane protein. The antigen was immunochemically unrelated to other brain specific proteins such as 14-3-2, S-100, the glial fibrillary acidic protein and synaptin. Furthermore, it was unrelated to two other membrane proteins, the nicotinic acetylcholine receptor and acetylcholinesterase, present in Torpedo electric organ. The antiserum against Torpedo synaptic vesicles did not react with preparations of rat brain synaptic vesicles or ox adrenal medullary chromaffin granules.     

Exocytosis by synaptic terminals innervating the adrenal gland of the goldfish reveals multiple domains within the plasmalemma.

The adrenal chromaffin gland of the goldfish has typical synaptic terminals embedded in its surface which are homologues of the cholinergic fibres innervating the mammalian adrenal medulla. The terminals contain both lucent synaptic vesicles and larger secretory granules with dense cores, known to be storage sites for transmitters and peptides, respectively. Three domains are present within the terminal plasmalemma. Exocytosis of vesicles is thought to be associated with a 'synaptic domain' marked by synaptic thickenings around which the vesicles cluster. Exocytosis of granules, stimulated by high K+ and visualized with the aid of tannic acid, is almost exclusively associated with areas of the membrane adjacent to chromaffin cells, and in particular with unspecialized regions which constitute the 'parasynaptic domain', creating a pattern of targeted secretory discharge. Sites of release within the 'non-synaptic domain', which is sheathed in glial cell lamellae, are extremely rare, despite the expansive character of this domain and the close association of granules with the plasmalemma within it. The pattern of secretory release described may be correlated with the position of the terminals at the surface of the innervated organ.     

Catecholamines Are Present in a Synaptic-Like Microvesicle-Enriched Fraction from Bovine Adrenal Medulla

Abstract: "Synaptic-like microvesicles" are present in all neuroendocrine cells and cell lines. Despite their resemblance to small synaptic vesicles of the CNS. a thorough biochemical characterization is lacking. Moreover, the subcellular distribution of synaptophysin, the most abundant integral membrane protein of small synaptic vesicles, in adrenal medulla is still controversial. Using gradient centrifugation. we were able to compare the distribution of several markers for small synaptic vesicles and chromaffin granules. Synaptophysin was found at a high density (1.16 g/ml), purifying away from dopamine β-hydroxylase and cytochrome b₅₆₁. Both noradrenaline and adrenaline showed a parallel distribution with synaptophysin, suggesting their presence in synaptic-like microvesicles. Experiments in the presence of tetrabenazine did not influence the catecholamine content. Additionally, tetrabenazine binding showed a consistent shoulder in the region of synaptophysin. [³H]-Noradrenaline uptake was blocked by tetrabenazine, but not by desipramine. Also chromogranin A parallels the distribution of synaptophysin: however, a localization in the Golgi cannot be ruled out. Synaptophysin was shown to undergo very fast phosphorylation, together with another triplet protein of ∼ 18 kDa. In contrast, the latter showed a rather bimodal distribution coinciding with synaptophysin and dopamine β-hydroxylase. Immunoelectron microscopy of synaptic-like microvesicle fractions showed an intense labeling for synaptophysin on 60-90-nm organelles. Whereas abundant gold labeling for cytochrome b₅₆₁ was found over the entire surface of chromaffin granules, synaptophysin labeling was encountered mostly on vesicles adsorbed to granules. We conclude that catecholamines might be stored in synaptic-like microvesicles of the chromaffin cell.     

Sorting during transport to the surface of PC12 cells: divergence of synaptic vesicle and secretory granule proteins

PC12 cells, a cell line derived from a rat pheochromocytoma, have both regulated and constitutive secretory pathways. Regulated secretion occurs via large dense core granules, which are related to chromaffin granules and are abundant in these cells. In addition, PC12 cells also contain small electron-lucent vesicles, whose numbers increase in response to nerve growth factor and which may be related to cholinergic synaptic vesicles. These could characterize a second regulated secretory pathway. We have investigated the trafficking of protein markers for both these organelles. We have purified and characterized the large dense core granules from these cells using sequential velocity and equilibrium gradients. We demonstrate the copurification of the major PC12 soluble regulated secretory protein (secretogranin II) with this organelle. As a marker for the synaptic vesicle-like organelles in this system, we have used the integral membrane glycoprotein p38 or synaptophysin. We show that the p38-enriched fraction of PC12 cells comigrates with rat brain synaptic vesicles on an equilibrium gradient. We also demonstrate that p38 purifies away from the dense core granules; less than 5% of this protein is found in our dense granule fraction. Finally we show that p38 does not pass through the dense granule fraction in pulse-chase experiments. These results rule out the possibility of p38 reaching the small clear vesicles via mature dense granules and imply that these cells may have two independently derived regulated pathways.     

A Similar Calmodulin-Binding Protein Expressed in Chromaffin, Synaptic, and Neurohypophyseal Secretory Vesicles

The presence of calmodulin-binding proteins in three neurosecretory vesicles (bovine adrenal chromaffin granules, bovine posterior pituitary secretory granules, and rat brain synaptic vesicles) was investigated. When detergent-solubilized membrane proteins from each type of secretory organelle were applied to calmodulin-affinity columns in the presence of calcium, several calmodulin-binding proteins were retained and these were eluted by EGTA from the columns. In all three membranes, a 65-kilodalton (63 kilodaltons in rat brain synaptic vesicles) and a 53-kilodalton protein were found consistently in the EGTA eluate. 125I-Calmodulin overlay tests on nitrocellulose sheets containing transferred chromaffin and posterior pituitary secretory granule membrane proteins showed a similarity in the protein bands labeled with radioactive calmodulin. In the presence of 10(-4) M calcium, eight major protein bands (240, 180, 145, 125, 65, 60, 53, and 49 kilodaltons) were labeled with 125I-calmodulin. The presence of 10 microM trifluoperazine (a calmodulin antagonist) significantly reduced this labeling, while no labeling was seen in the presence of 1 mM EGTA. Two monoclonal antibodies (mAb 30, mAb 48), previously shown to react with a cholinergic synaptic vesicle membrane protein of approximate molecular mass of 65 kilodaltons, were tested on total membrane proteins from the three different secretory vesicles and on calmodulin-binding proteins isolated from these membranes using calmodulin-affinity chromatography. Both monoclonal antibodies reacted with a 65-kilodalton protein present in membranes from chromaffin and posterior pituitary secretory granules and with a 63-kilodalton protein present in rat brain synaptic vesicle membranes. When the immunoblotting was repeated on secretory vesicle membrane calmodulin-binding proteins isolated by calmodulin-affinity chromatography, an identical staining pattern was obtained. These results clearly indicate that an immunologically identical calmodulin-binding protein is expressed in at least three different neurosecretory vesicle types, thus suggesting a common role for this protein in secretory vesicle function.     

Heterogeneous distribution of synaptophysin and protein 65 in synaptic vesicles isolated from rat cerebral cortex

Rat brain cerebral cortex derived synaptic vesicles sedimenting on a 0.4 M sucrose solution were further fractionated according to size by column chromatography on Sephacryl-1000 and analyzed for their binding activities of antibodies directed against the vesicle-associated proteins synaptophysin, synapsin I, protein 65 and clathrin. Whereas synapsin I and particularly protein 65 and clathrin are associated with a large range of vesicle sizes, synaptophysin elutes with small vesicles only. Using monoclonal antibodies against either synaptophysin or protein 65 and polyacrylamide beads for solid matrix immunoprecipitation, significant differences could be revealed in the protein composition of the resulting vesicle populations. Whereas synapsin I is associated with both synaptophysin and protein 65 immunoprecipitated vesicle populations, synaptophysin appears to be only a minor constituent of vesicles precipitated with anti-protein 65. Vesicles precipitated with anti-synaptophysin antibodies are enriched in acetylcholine. Our results suggest that the vesicle membrane protein synaptophysin and protein 65 may not have a ubiquitous distribution among synaptic vesicles. Protein 65 containing large vesicle populations contain little synaptophysin and synaptophysin is mainly associated with synaptic vesicles of small diameter.     

Quantification of p38/synaptophysin in highly purified adrenal medullary chromaffin vesicles

Chromaffin vesicles were first purified by differential and density gradient centrifugation in isotonic (Percoll) gradients. In subsequent sucrose gradients p38/synaptophysin exhibited the same distribution as established marker substances of chromaffin vesicles. Quantification of immunoblots revealed that 750 ng p38/synaptophysin per mg of protein were present in the chromaffin vesicles recovered from the sucrose gradient. Thus the amount of p38/synaptophysin per mg protein of chromaffin vesicles is about 100 times lower than that observed in clear (synaptic) vesicles. However, because of the large difference in surface area and protein content, the amount of p38/synaptophysin per single vesicle is the same in both types of organelles.     

The synaptic vesicle proteins SV2, synaptotagmin and synaptophysin are sorted to separate cellular compartments in CHO fibroblasts

We expressed the synaptic vesicle proteins SV2, synaptotagmin, and synaptophysin in CHO fibroblasts to investigate the targeting information contained by each protein. All three proteins entered different cellular compartments. Synaptotagmin was found on the plasma membrane. Both SV2 and synaptophysin were sorted to small intracellular vesicles, but synaptophysin colocalized with early endosomal markers, while SV2 did not. SV2-containing vesicles did not have the same sedimentation characteristics as authentic synaptic vesicles, even though transfected SV2 was sorted from endosomal markers. We also created cell lines expressing both SV2 and synaptotagmin, both synaptotagmin and synaptophysin, and lines expressing all three synaptic vesicle proteins. In all cases, the proteins maintained their distinct compartmentalizations, were not found in the same organelle, and did not created synaptic vesicle-like structures. These results have important implications for models of synaptic vesicle biogenesis.     

Preferential localization of a vesicular monoamine transporter to dense core vesicles in PC12 cells

Neurons and endocrine cells have two types of secretory vesicle that undergo regulated exocytosis. Large dense core vesicles (LDCVs) store neural peptides whereas small clear synaptic vesicles store classical neurotransmitters such as acetylcholine, gamma-aminobutyric acid (GABA), glycine, and glutamate. However, monoamines differ from other classical transmitters and have been reported to appear in both LDCVs and smaller vesicles. To localize the transporter that packages monoamines into secretory vesicles, we have raised antibodies to a COOH- terminal sequence from the vesicular amine transporter expressed in the adrenal gland (VMAT1). Like synaptic vesicle proteins, the transporter occurs in endosomes of transfected CHO cells, accounting for the observed vesicular transport activity. In rat pheochromocytoma PC12 cells, the transporter occurs principally in LDCVs by both immunofluorescence and density gradient centrifugation. Synaptic-like microvesicles in PC12 cells contain relatively little VMAT1. The results appear to account for the storage of monoamines by LDCVs in the adrenal medulla and indicate that VMAT1 provides a novel membrane protein marker unique to LDCVs.     

Opossum adrenal medulla: II. Differentiation of the chromaffin cell

The ultrastructure of the opossum adrenal medulla was examined in its postnatal development. Maturation of chromaffin cells and genesis of chromaffin vesicles were of particular interest. The primitive sympathetic cell was seen to contain few organelles with no apparent polarity. Initial pheochromoblasts contained more organelles with some polarity. Endoplasmic reticulum and the Golgi complex increased as the pheochromoblasts matured, which suggested increased synthetic activity. Structures resembling Golgi/endoplasmic reticulum/lysosome (GERL) systems were seen in the pheochromoblasts. It is suggested that some of the components of the chromaffin vesicle may be processed by the GERL while others come directly through the Golgi complex. It is stressed that the developing pheochromoblast in the opossum presents an interesting model in which to study the genesis of the chromaffin vesicle.