The same 15 kDa proteolipid subunit is a constituent of two different proteins in torpedo, the acetylcholine releasing protein mediatophore and the vacuolar HATPase

Using the monoclonal antibody 15KI, we have studied, at the cellular and subcellular levels, the distribution of a 15 kDa proteolipid, identified as the subunit of mediatophore, a presynaptic membrane protein able to release acetylcholine when activated by calcium. Aside from the electric lobe, the antigen distribution in the brain of Torpedo paralleled that of the synaptic vesicle antigen SV2 and did not appear to be related to that of acetylcholine and choline acetyltransferase. The 15 kDa proteolipid antigen was therefore present in all nerve endings and not restricted to cholinergic ones. At the ultrastructural level, on cholinergic nerve endings, the antigen was detected associated to synaptic vesicles and, to a lesser extent, to the presynaptic plasma membrane. Indeed, considering the high sequence homology between the mediatophore subunit (Birman et al., 1990) and the proteolipid subunit of the vacuolar type H⁺ATPase, a major enzyme constituent of synaptic vesicles, this distribution was not surprising.

To determine whether antibody 15KI recognizes the vacuolar type H⁺ATPase, we chose a non neuronal cell type which possesses a high content of this enzyme, the kidney proton secreting epithelial cells. Indeed, antibody 15KI intensely labelled the apical plasma membrane of mitochondria rich epithelial cells in kidney tubules. A high density of the antigen was also found associated to intracellular membrane structures such as lysosomal multivesicular bodies, both in kidney epithelial cells and in electromotoneurons. The 15 kDa proteolipid antigen was associated with other vacuolar H⁺ATPase subunits in kidney membranes which was not the case in presynaptic plasma membranes. This illustrates that the 15 kDa proteolipid antigen is a constituent of two different protein complexes, which exhibit very different functional properties.     

ASSOCIATION OF SYNTAXIN WITH SNAP 25 AND VAMP (SYNAPTOBREVIN) IN TORPEDO SYNAPTOSOMES

Two proteins of the presynaptic plasma membrane, syntaxin and SNAP 25, and VAMP/synaptobrevin, a synaptic vesicle membrane protein, form stable protein complexes which are involved in the docking and fusion of synaptic vesicles at the mammalian brain presynaptic membrane. Similar protein complexes were revealed in an homogeneous population of cholinergic synaptosomes purified from Torpedo electric organ by combining velocity sedimentation and immunoprecipitation experiments. After CHAPS solubilization, virtually all the nerve terminal syntaxin was found in the form of large 16 S complexes, in association with 65% of SNAP 25 and 15% of VAMP. Upon Triton X100 solubilization, syntaxin was still recovered in association with SNAP 25 and VAMP but in smaller 8 S complexes. A small (2–5%) percentage of the nerve terminal 15 kDa proteolipid subunit of the v-H⁺ ATPase and of mediatophore was copurified with syntaxin, using two different antisyntaxin monoclonal antibodies. The use of an homogeneous population of peripheral cholinergic nerve terminals allowed us to extend results on the composition of the brain presynaptic protein complexes to the Torpedo electric organ synapse, a model of the rapid neuromuscular synapses. Copyright © 1996 Elsevier Science Ltd     

Immunological detection of mediatophore in motor end-plates and electric organ subcellular fractions of torpedo marmorata

A rabbit antiserum to mediatophore, a nerve terminal membrane protein involved in calcium dependent ACh release, was raised after immunization with the purified protein. An immunological assay for mediatophore was then developed and the subcellular distribution of this protein in Torpedo electric organ fractions was studied. A good agreement was obtained between the distribution in the different fractions of the antigen and of mediatophore related acetylcholine releasing activity as determined by reconstitution in proteoliposomes. Mediatophore was highly concentrated in presynaptic plasma membranes of electric organ, while very low contents were observed in electric nerves and electric lobes. Although some mediatophore was found in synaptic vesicle fractions, this most probably resulted from presynaptic membrane contamination as evaluated with other presynaptic membrane markers. Nerve terminals of motor end-plates were strongly stained with anti-mediatophore antibodies.     

Immunolabelling of the presynaptic membrane of Torpedo electric organ nerve terminals with an antiserum towards the acetylcholine releasing protein mediatophore

Mediatophore is a nerve terminal membrane protein purified from Torpedo electric organ on its ability to translocate acetylcholine upon calcium action. An antiserum able to immunoprecipitate mediatophore activity was used to study the subcellular distribution of this protein. The presynaptic membrane exhibited a strong and discontinuous immunogold labelling, especially at the active zone where ACh is thought to be released. Two antigens were recognized on immunoblots of synaptosomal membranes: the 15-kDa subunit of mediatophore and a 14-kDa membrane protein that has a wide non-neuronal distribution. Antibodies purified from the serum on native mediatophore and monospecific towards the 15-kDa antigen still gave a high presynaptic membrane localized labelling. In addition, a few 14-kDa protein sites were present at the active zone. The Schwann cell finger interposed between the presynaptic membrane and the postsynaptic arch also exhibited the 14-kDa antigen raising the question of a possible interaction of mediatophore with the 14-kDa protein originating from the Schwann cell.     

Effect of N,N-Dicyclohexylcarbodiimide on Acetylcholine Release from Torpedo Synaptosomes and Proteoliposomes Reconstituted with the Proteolipid Mediatophore

The mediatophore is a presynaptic membrane protein that has been shown to translocate acetylcholine (ACh) under calcium stimulation when reconstituted into artificial membranes. The mediatophore subunit, a 15-kDa proteolipid, presents a very high sequence homology with the N,N'-dicyclohexylcarbodiimide (DCCD)-binding proteolipid subunit of the vacuolar-type H(+)-ATPase. This prompted us to study the effect of DCCD, a potent blocker of proton translocation, on calcium-dependent ACh release. The present work shows that DCCD has no effect on ACh translocation either from Torpedo synaptosomes or from proteoliposomes reconstituted with purified mediatophore. However, using [14C]DCCD, we were able to demonstrate that the drug does bind to the 15-kDa proteolipid subunit of the mediatophore. These results suggest that although the 15-kDa proteolipid subunits of the mediatophore and the vacuolar H(+)-ATPase may be identical, different domains of these proteins are involved in proton translocation and calcium-dependent ACh release and that the two proteins have a different membrane organization.     

Evidence for an Association of the 15-kDa Proteolipid of Mediatophore with a 14-kDa Polypeptide

The present report shows that mediatophore, a nerve terminal membrane protein that translocates acetylcholine on calcium action, forms a complex with a 14-kDa polypeptide. The complex was identified based on the following results. (a) A polyclonal antimediatophore antiserum that immunoprecipitates activity precipitates both the 15- and 14-kDa polypeptides. (b) After HPLC purification of mediatophore, both antigens were found in the same peak. (c) After 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate solubilization of presynaptic membranes or of the purified mediatophore, an immunoaffinity column made with the anti-14-kDa antigen monoclonal antibody retained both the 14-kDa and the 15-kDa polypeptide. Similarly, immunoprecipitation experiments using protein A-coated beads sedimented an immunocomplex in which both antigens were found. (d) The 14-kDa antigen could be localized in the synaptosomal membrane where mediatophore and its 15-kDa component are found.     

Presynaptic plasma membranes and synaptic vesicles of cholinergic nerve endings demonstrated by means of specific antisera

Summary Antisera were raised to cholinergic presynaptic plasma membranes and synaptic vesicles isolated from the electric organ of Torpedo marmorata and tested by immunochemical and immunohistochemical methods. The antisera responded to many antigens not specific to nerve endings, but it was possible to eliminate these antibodies by means of simple absorption procedures with fractions containing the unwanted antigens. After absorption, staining of thin sections of electric organ by immunofluorescence was limited to the region of nerve endings in the tissue.The remaining antibodies responded in the case of the plasma membrane antisera predominantly to a 33,000 molecular-weight polypeptide and a chloroform/methanol-soluble antigen. In cross reactivity studies it was found that this antiserum not only stains cholinergic nerve endings in Torpedo but also those in mammalian tissue. The antigen responsible for the cross reactivity is restricted to the chloroform/methanol-soluble material.The vesicle antiserum labels cholinergic nerve endings in mammalian tissue as well; the relevant antigen in this case is different from the one described above and is likely to be a glycosaminoglycan. The antisera provide valuable markers for cholinergic nerve terminals. In addition, the vesicle antiserum may now be used to study axonal transport and the life cycle of this organelle in the cholinergic neurone.Abbreviations SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - EGTA ethylenebis (oxoethylenenitrilo) tetra-acetic acid - MW apparent molecular weight Enzymes. Na⁺, K⁺-activated ATPase (EC 3.6.1.3); acetylcholine esterase (EC 3.1.1.7); choline acetyl-transferase (EC 2.3.1.6)     

Is the acetylcholine releasing protein mediatophore present in rat brain?

Mediatophore is a protein purified from the nerve terminal membranes of Torpedo electric organ. It confers to artificial membranes a calcium-dependent mechanism that translocates acetylcholine. When similar reconstitution experiments are applied to rat brain synaptosomal membranes they reveal the presence of mediatophore activity with properties close to those described for the Torpedo protein (extractability, sensitivity to calcium, and effect of the drug cetiedil). The activity was more abundant in synaptosomal membranes than in mitochondrial or myelinic membranes and in cholinergic areas as compared to cerebellum.     

Large-scale purification of presynaptic plasma membranes from Torpedo marmorata electric organ

The presynaptic plasma membrane (PSPM) of cholinergic nerve terminals was purified from Torpedo electric organ using a large-scale procedure. Up to 500 g of frozen electric organ were fractioned in a single run, leading to the isolation of greater than 100 mg of PSPM proteins. The purity of the fraction is similar to that of the synaptosomal plasma membrane obtained after subfractionation of Torpedo synaptosomes as judged by its membrane-bound acetylcholinesterase activity, the number of Glycera convoluta neurotoxin binding sites, and the binding of two monoclonal antibodies directed against PSPM. The specificity of these antibodies for the PSPM is demonstrated by immunofluorescence microscopy.     

Comparison of the postsynaptic 43-kDa protein from muscle cells that differ in acetylcholine receptor clustering activity

A peripheral membrane protein of Mr = 43,000 (43-kDa protein) is closely associated with the acetylcholine receptor (AChR) in Torpedo electrocyte postsynaptic membranes and may play a role in anchoring receptors at synaptic sites. A component immunologically related to the 43-kDa protein also occurs specifically at mammalian muscle synapses and in association with receptor clusters on cultured muscle cells. We have studied this mammalian protein in two mouse muscle cell lines, C2 and BC3H1, that differ in AChR clustering activity. The 43-kDa-related protein was purified from muscle cell detergent extracts by immunoaffinity chromatography using monoclonal antibodies (mAbs) prepared against the Torpedo 43-kDa protein and identified by immunoblotting. In both C2 and BC3H1 cells, a protein of molecular mass of approximately 43,000 was recognized by two mAbs with different epitope specificity. To measure the 43-kDa protein in mammalian muscle cells, we designed a quantitative immunological assay utilizing these two mAbs. As in Torpedo electric organ, the concentration of the 43-kDa protein and receptor was approximately equimolar in C2 cells and in BC3H1 cells. Furthermore, during differentiation of both muscle cell lines, the appearance of the 43-kDa protein correlated closely with that of the receptor, raising the intriguing possibility that the expression of these two proteins is controlled by similar regulatory mechanisms. These results indicate that the inability of BC3H1 cells to form AChR clusters apparently does not result from a deficiency in the 43-kDa protein.     

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.     

Evoked Acetylcholine Release Expressed in Neuroblastoma Cells by Transfection of Mediatophore cDNA

Abstract: Transmitter release was elicited in two ways from cultured cells filled with acetylcholine: (a) in a biochemical assay by successive addition of a calcium ionophore and calcium and (b) electrophysiologically, by electrical stimulation of individual cells and real-time recording with an embryonic Xenopus myocyte. Glioma C6-Bu-1 cells were found to be competent for Ca²⁺-dependent and quantal release. In contrast, no release could be elicited from mouse neuroblastoma N18TG-2 cells. However, acetylcholine release could be restored when N18TG-2 cells were transfected with a plasmid coding for mediatophore. Mediatophore is a protein of nerve terminal membranes purified from the Torpedo electric organ on the basis of its acetylcholine-releasing capacity. The transfected N18TG-2 cells expressed Torpedo mediatophore in their plasma membrane. In response to an electrical stimulus, they generated in the myocyte evoked currents that were curare sensitive and calcium dependent and displayed discrete amplitude levels, like in naturally occurring synapses.     

Putative Synaptic Vesicle Nucleotide Transporter Identified as Glyceraldehyde-3-Phosphate Dehydrogenase

Abstract: Synaptic vesicles isolated from electric ray electric organ have been shown previously to contain a 34-kDa protein that binds azido-ATP, azido-AMP, and N -ethylmaleimide. The protein was found to share similarities with the mitochondrial ADP/ATP carrier and assumed to represent the synaptic vesicle nucleotide transporter. Synaptic vesicles were purified by sucrose density gradient centrifugation and subsequent chromatography on Sephacryl S-1000 from both Torpedo electric organ and bovine brain cerebral cortex. They contained ATP-binding proteins of 35 kDa and 34 kDa, respectively. ATP binding was inhibited by AMP. Both proteins were highly enriched after column chromatography of vesicle proteins of AMP-Sepharose. Antibodies were obtained against both proteins. Antibodies against the bovine brain synaptic vesicle protein of 34 kDa bound specifically to the 35-kDa protein of Torpedo vesicles. An N-terminal sequence obtained against the 34-kDa protein of bovine brain synaptic vesicles identified it as glyceraldehyde-3-phosphate dehydrogenase. The previously observed molecular characteristics of the putative vesicular nucleotide transporter in Torpedo fit those of glyceraldehyde-3-phosphate dehydrogenase. We, therefore, suggest that the protein previously identified as putative nucleotide transporter is, in fact, glyceraldehyde-3-phosphate dehydrogenase.     

Binding of a Glycera convoluta neurotoxin to cholinergic nerve terminal plasma membranes

The crude extract of venom glands of the polychaete annelid Glycera convoluta triggers a large Ca2+-dependent acetylcholine release from both frog motor nerve terminals and Torpedo electric organ synaptosomes. This extract was partially purified by Concanavalin A affinity chromatography. The biological activity was correlated in both preparations to a 300,000-dalton band, as shown by gel electrophoresis. This confirmed previous determinations obtained with chromatographic methods. This glycoprotein binds to presynaptic but not postsynaptic plasma membranes isolated from Torpedo electric organ. Pretreatment of intact synaptosomes by pronase abolished both the binding and the venom- induced acetylcholine release without impairing the high K+-induced acetylcholine release. Pretreatment of nerve terminal membranes by Concanavalin A similarly prevented the binding and the biological response. Binding to Torpedo membranes was still observed in the presence of EGTA. An antiserum directed to venom glycoproteins inhibited the neurotoxin so we could directly follow its binding to the presynaptic membrane. Glycera convoluta neurotoxin has to bind to a ectocellularly oriented protein of the presynaptic terminal to induce transmitter release.     

G-proteins in Torpedo marmorata electric organ. Differential distribution in pre- and post-synaptic membranes and synaptic vesicles

The nature of the G-proteins present in the pre- and post-synaptic plasma membranes and in the synaptic vesicles of cholinergic nerve terminals purified from the Torpedo electric organ was investigated. In pre- and post-synaptic plasma membranes, Bordetella pertussis toxin, known to catalyze the ADP-ribosylation of the alpha-subunit of several G-proteins, labels two substrates at 41 and 39 kDa. The 39 kDa subunit detected by ADP-ribosylation in the synaptic plasma membrane fractions was immunologically similar to the Go alpha-subunit purified from calf brain. In contrast to bovine chromaffin cell granules, no G-protein could be detected in Torpedo synaptic vesicles either by ADP-ribosylation or by immunoblotting.     

Calcium-induced acetylcholine release and intramembrane particle occurrence in proteoliposomes equipped with mediatophore.

Proteoliposomes obtained from the mediatophore, a purified Torpedo electric organ nerve terminals protein, and endogenous lipids were used for a study of calcium-induced release of acetylcholine and freeze-fracture electron microscopy. Large intramembrane particles were induced by the influx of calcium into proteoliposomes, as previously observed for synaptosomes or stimulated electric organ nerve terminals. The involvement of mediatophore in a calcium dependent acetylcholine translocation seems therefore to be related to the occurrence of a category of intramembrane particles in the course of the release process.     

300-kD subsynaptic protein copurifies with acetylcholine receptor-rich membranes and is concentrated at neuromuscular synapses

Acetylcholine receptor-rich membranes from the electric organ of Torpedo californica are enriched in the four different subunits of the acetylcholine receptor and in two peripheral membrane proteins at 43 and 300 kD. We produced monoclonal antibodies against the 300-kD protein and have used these antibodies to determine the location of the protein, both in the electric organ and in skeletal muscle. Antibodies to the 300-kD protein were characterized by Western blots, binding assays to isolated membranes, and immunofluorescence on tissue. In Torpedo electric organ, antibodies to the 300-kD protein stain only the innervated face of the electrocytes. The 300-kD protein is on the intracellular surface of the postsynaptic membrane, since antibodies to the 300-kD protein bind more efficiently to saponin-permeabilized, right side out membranes than to intact membranes. Some antibodies against the Torpedo 300-kD protein cross-react with amphibian and mammalian neuromuscular synapses, and the cross-reacting protein is also highly concentrated on the intracellular surface of the post-synaptic membrane.     

Electron microscopic localization of calelectrin, a Mr 36 000 calcium-regulated protein, at the cholinergic electromotor synapse of Torpedo

Calelectrin is a calcium-binding protein of Mr 36 000 which has previously been shown to be associated with membranes of the cholinergic synapse in a calcium-dependent manner. We report here that calelectrin was solubilized from the electric organ of Torpedo marmorata in the absence of calcium together with proteins of Mr 54 000 and Mr 15 000. In cholinergic nerve endings isolated from the electric organ only calelectrin was solubilized in a calcium-dependent manner. A specific antiserum to calelectrin was used to localize the antigen by immunofluorescence microscopy on sections of electric organ and showed that calelectrin is distributed throughout the postsynaptic cell. Calelectrin was also detected in axons and in the cell bodies of the cholinergic neurones where it was concentrated in discrete patches throughout the cells. Electric organ tissue was processed to localize calelectrin with the electron microscope using an immunoperoxidase method. The most intense staining was observed on the cytoplasmic face of the acetylcholine receptor-containing postsynaptic membrane and also associated with the intracellular filaments of the electrocyte. The intensity of staining associated with these structures could be greatly reduced by preincubating the tissue with calcium chelators. In nerve terminals calelectrin was associated with synaptic vesicles in a polarized fashion. Calelectrin was also found on the cytoplasmic face of the synaptosomal plasma membrane and associated with neurofilaments. No extracellular staining was ever observed. Our results strongly support our original hypothesis that calelectrin is a calcium-regulated component of intracellular structure associated both with membranes and filaments.