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.     

Detection with Monoclonal Antibodies of a 15-kDa Proteolipid in Both Presynaptic Plasma Membranes and Synaptic Vesicles in Torpedo Electric Organ

A protein, the mediatophore, has been purified from Torpedo electric organ presynaptic plasma membranes. This protein mediates the release of acetylcholine through artificial membranes when activated by calcium and is made up of 15-kDa proteolipid subunits. After immunization with purified delipidated mediatophore, monoclonal antibodies binding to the 15-kDa proteolipid band on Western blots of purified mediatophore were selected. A 15-kDa proteolipid antigen was also detected in cholinergic synaptic vesicles. Using an immunological assay, it was estimated that presynaptic plasma membranes and synaptic vesicles contain similar proportions of 15-kDa proteolipid antigen. Detection by immunofluorescence in the electric organ showed that only nerve endings were labeled. In electric lobes, the staining was associated with intracellular membranes of the electroneuron cell bodies and in axons. Nerve endings at Torpedo neuromuscular junctions were also labeled with anti-15-kDa proteolipid monoclonal antibodies.     

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.     

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.     

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.     

Enhanced Acetylcholine Release from Cells that Have More 15-kDa Proteolipid in Their Membrane, a Constituent V-ATPase, and Mediatophore

Abstract: Mediatophore is a protein that translocates acetylcholine (ACh) on calcium action. It is a homopolymer of a 15-kDa proteolipid that is also a constituent of the membrane sector of vacuolar H⁺-adenosine trisphosphatase (V-ATPase; vacuolar proton pump). Experiments on neuroblastoma cell lines (N18TG-2) that are deficient for ACh release and on cells that are competent for release, such as the glioma C6BU-1 or the N18TG-2/C6BU-1 fusion product NG108-15, show that there is a correlation between ACh release and the 15-kDa proteolipid content of the cell membrane. In another cell line, L-M(TK⁻), it has been possible to up-regulate ACh release and the membrane proteolipid content after treating the cells with dibutyryl-cyclic AMP or dexamethasone. As mediatophore translocates ACh and as V-ATPase may help vesicular ACh storage, it was interesting to determine the respective role of the two proteins in the observed correlation between release and proteolipid content. After blocking vesicular loading with vesamicol, we did not affect release from these cells, suggesting that the observed correlation may be attributed to mediatophore. The acquisition of an ACh release mechanism would then depend on the process that guides the proteolipid to the plasma membrane of the cell.     

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     

Kinetic Properties of Purified Pseudomonas aeruginosa Phosphorylcholine Phosphatase Indicated That This Enzyme May Be Utilized by the Bacteria to Colonize in Different Environments

A protein oligomer with an approximate molecular weight of its 37-kDa monomer form was purified from the cell envelope fraction of Vibrio damsela cells. This oligomer exhibited strong porin activity when reconstituted into proteoliposomes with phosphatidyl choline. The functional properties for the 37-kDa protein suggest that it is a nonspecific or general porin, with an apparent pore size of 1.6 nm. This porin allows penetration of a variety of hydrophilic solutes according to their molecular mass. After electroelution, the oligomer was partially dissociated into monomers, whereas treatment with EDTA did not affect its dissociation. The monomers of the 37-kDa protein were not active in the reconstitution assay. The effect of culture media on the composition of the outer membrane protein of V. damsela was examined. Only one outer membrane protein with an apparent molecular weight of 37 kDa (37-kDa protein) was formed in cells grown in 3% NaCl–BHI broth and in 3% NaCl–nutrient broth with the addition of 2% glucose. Three outer membrane proteins, with apparent molecular weights of 37 kDa, 40 kDa, and 46 kDa, were produced in cells grown in 3% NaCl–nutrient broth. An additional outer membrane protein with an apparent molecular weight of 44 kDa (44-kDa protein) was found in cells grown in 3% NaCl–nutrient broth with the addition of 2% maltose. This protein was found to exhibit specificity to maltose derivatives. The results obtained in this study confirm the porin-like character of discussed proteins and give a basis for advanced study of those proteins. Received: 16 June 1998 / Accepted: 18 July 1998     

The lipid requirements of mediatophore for acetylcholine release activity. Large-scale purification of this protein in a reactive form

The mediatophore, a protein which translocates acetylcholine, has been recently purified from the plasma membrane of the Torpedo electric organ nerve terminals. The functional integrity of the mediatophore requires the presence of lipids. Removal of associated lipids led to an irreversible loss of activity when obtained by ether precipitation of the protein. A two-phase extraction procedure was developed to gently remove the lipids. Conditions were found to reactivate the protein by emulsifying it with electric organ lipids. A large-scale preparation procedure of the delipidified mediatophore in a reactive form is described.     

Monoclonal antibodies that bind the renal Na+/glucose symport system. 1. Identification

Phlorizin is a specific, high-affinity ligand that binds the active site of the Na+/glucose symporter by a Na+-dependent mechanism but is not itself transported across the membrane. We have isolated a panel of monoclonal antibodies that influence high-affinity, Na+-dependent phlorizin binding to pig renal brush border membranes. Antibodies were derived after immunization of mice either with highly purified renal brush border membranes or with apical membranes purified from LLC-PK1, a cell line of pig renal proximal tubule origin. Antibody 11A3D6, an IgG2b, reproducibly stimulated Na+-dependent phlorizin binding whereas antibody 18H10B12, an IgM, strongly inhibited specific binding. These effects were maximal after 30-min incubation and exhibited saturation at increased antibody concentrations. Antibodies did not affect Na+-dependent sugar uptake in vesicles but significantly prevented transport inhibition by bound phlorizin. Antibodies recognized a 75-kDa antigen identified by Western blot analysis of brush border membranes, and a 75-kDa membrane protein could be immunoprecipitated by 18H10B12. These properties, taken together with results in the following paper [Wu, J.-S.R., & Lever, J.E. (1987) Biochemistry (following paper in this issue)], provide compelling evidence that the 75-kDa antigen recognized by these antibodies is a component of the renal Na+/glucose symporter.     

An ultrastructural comparison of neuromuscular junctions in normal and developmentally arrested Rana pipiens larvae: limited maturation in the absence of metamorphosis

Neuromuscular junctions in the rectus abdominis muscles of normal and developmentally arrested Rana pipiens larvae were studied with freeze fracture and conventional electron microscopy to determine whether structural aspects of junctional maturation depend on metamorphosis. Comparison was made between junctions in premetamorphic larvae 1-3 months old and junctions in larvae that had remained in premetamorphosis for more than a year (more than four times as long as normal). In most respects, junctions from the two groups of larvae were similar. Unlike adult junctions, nerve-muscle contacts in both larval groups were pleomorphic and often involved more than one neuronal process; Schwann cell processes very rarely extended between nerve and muscle. Active zone structure ranged from total disorganization to an adult pattern of highly ordered double rows of particles aligned over junctional folds. Only quantitative analysis revealed differences between junctions in old and young larvae. The older larvae had fewer nerve-muscle contact sites involving multiple neuronal elements and a higher ratio of active zone length to presynaptic membrane area, although the mean active zone length was the same in the two groups. The results indicate that the maturation of junctional shape, the branching pattern of the axons, and the relationship of presynaptic axons to Schwann cells must be directly or indirectly dependent on the hormonal or behavioral changes associated with metamorphosis.     

Release of acetylcholine by Xenopus oocytes injected with mRNAs from cholinergic neurons.

Xenopus laevis oocytes were injected with poly(A)+ mRNAs extracted from the electric lobes of Torpedo marmorata. The electric lobes contain the perikarya of approximately 120,000 cholinergic neurons that innervate the electric organs and are homologous to motor neurons. The injected oocytes accumulated acetylcholine and were able to synthesize [14C]acetylcholine from 1-[14C]acetate. With KCl depolarization and upon treatment with a Ca2+ ionophore, they released their endogenous as well as the radiolabelled neurotransmitter in a Ca(2+)-dependent manner. No synthesis or release were obtained from control oocytes. With respect to their dependency upon Ca2+ concentration, the oocytes injected with Torpedo electric lobe mRNAs released acetylcholine in a manner which closely resembled that found in the native synapses. In contrast to the controls, primed oocytes were also able to release [14C]acetylcholine that was injected a few hours prior to the release trial. Immunoblot analysis demonstrated that the 15 kd proteolipid antigen of the purified mediatophore, a 200 kd presynaptic protein able to translocate acetylcholine, was expressed in the ACh-releasing oocytes but not in the controls. The present observation may provide a useful approach for investigating the proteins involved in the release of acetylcholine and of other neurotransmitter substances.     

Solubilization and Partial Purification of a Presynaptic Membrane Protein Ensuring Calcium-Dependent Acetylcholine Release from Proteoliposomes

In previous work, it was shown that cytoplasmic acetylcholine decreased on stimulation of Torpedo electric organ or synaptosomes in a strictly calcium-dependent manner. This led to the hypothesis that the presynaptic membrane contained an element translocating acetylcholine when activated by calcium. To test this hypothesis, the presynaptic membrane constituents were incorporated into the membranes of liposomes filled with acetylcholine. The proteoliposomes thus obtained released the transmitter in response to a calcium influx. The kinetics and calcium dependency of acetylcholine release were comparable for proteoliposomes and synaptosomes. The presynaptic membrane element ensuring calcium-dependent acetylcholine release is most probably a protein, since it was susceptible to Pronase, but only when the protease had access to the intracellular face of the presynaptic membrane. Postsynaptic membrane fractions contained very low amounts of this protein. It was extracted from the presynaptic membrane under alkaline conditions in the form of a protein-lipid complex of large size and low density which was partially purified. The specificity of the calcium-dependent release for acetylcholine was tested with proteoliposomes filled with equal amounts of acetylcholine and choline or acetylcholine and ATP. In both cases, acetylcholine was released preferentially. After cholate solubilization and gel filtration, the protein ensuring the calcium-dependent acetylcholine release was recovered at a high apparent molecular weight (between 600,000 and 200,000 daltons), its apparent sedimentation coefficient being 17S after cholate elimination. This protein is probably an essential coin of the transmitter release mechanism. We propose to name it mediatophore.     

Rab2 regulates presynaptic precursor vesicle biogenesis at the trans-Golgi

Reliable delivery of presynaptic material, including active zone and synaptic vesicle proteins from neuronal somata to synaptic terminals, is prerequisite for successful synaptogenesis and neurotransmission. However, molecular mechanisms controlling the somatic assembly of presynaptic precursors remain insufficiently understood. We show here that in mutants of the small GTPase Rab2, both active zone and synaptic vesicle proteins accumulated in the neuronal cell body at the trans-Golgi and were, consequently, depleted at synaptic terminals, provoking neurotransmission deficits. Ectopic presynaptic material accumulations consisted of heterogeneous vesicles and short tubules of 40 × 60 nm, segregating in subfractions either positive for active zone or synaptic vesicle proteins and LAMP1, a lysosomal membrane protein. Genetically, Rab2 acts upstream of Arl8, a lysosomal adaptor controlling axonal export of precursors. Collectively, we identified a Golgi-associated assembly sequence of presynaptic precursor biogenesis dependent on a Rab2-regulated protein export and sorting step at the trans-Golgi.     

Calcium-Induced Desensitization of Acetylcholine Release from Synaptosomes or Proteoliposomes Equipped with Mediatophore, a Presynaptic Membrane Protein

A "fatigue" of acetylcholine (ACh) release is described in cholinergic synaptosomes stimulated with the calcium ionophore A23187 or gramicidin. A small conditioning calcium entry, which did not trigger a large ACh release, led to a decrease of transmitter release elicited by a second large calcium influx. This fatigue was half-maximal at approximately 30 microM external calcium and developed in a few minutes. In contrast, activation of release by calcium was very rapid and was half-maximal at approximately 0.5 mM external calcium. Activation and desensitization of release could be attributed to the recently identified presynaptic membrane protein, the "mediatophore." Proteoliposomes equipped with purified mediatophore showed a calcium-dependent activation and "fatigue" of ACh release similar to that of synaptosomes. It was found that the ionophore A23187 rapidly equilibrated internal and external calcium concentrations in proteoliposomes. Thus, the external calcium concentration gave the internal concentration required for activation or desensitization of proteoliposomal ACh release. The mediatophore showed remarkable calcium binding properties (20 sites/molecule) with a KD of 25 microM. The physiological implications of desensitization on the organization of release sites are discussed.     

Evaluation of a Recombinant 27-kDa Outer Membrane Protein of Coxiella burnetii as an Immunodiagnostic Reagent

The 27-kDa outer membrane protein from eight strains of Coxiella burnetii was expressed in the pET-21c protein expression system. Two fusion proteins with molecular masses of 30 and 32 kDa were evident in all eight of the recombinants by SDS-PAGE and immunoblotting. A protein having an approximate size of 30 kDa was purified from the Escherichia coli lysates by one-step affinity purification. The utility of the purified recombinant protein in ELISA was also evaluated by testing its reactivity with human sera and comparing this reactivity with that of Nine Mile phase II antigen. All of the 40 IF-positive serum samples were ELISA-positive for both the Nine Mile phase II and recombinant antigens, and negative serum controls were negative for both antigens. These results suggest that ELISA with the 27-kDa recombinant antigen is a sensitive and specific method for detecting anti-C. burnetii antibodies in human sera.     

Acetylcholine Translocating Protein: Mediatophore at Rat Neuromuscular Synapses

The neuromuscular synapses of the rat sternomastoid muscles contain a membrane protein, mediatophore, that endows artificial membranes with a calcium-dependent acetylcholine release mechanism. Mediatophore and choline acetylase had similar distributions along the muscle. Sciatic nerve membranes contain mediatophore, and a purified preparation was obtained from the nerve.     

The cytotoxicity of Bacillus thuringiensis subsp. coreanensis A1519 strain against the human leukemic T cell

A novel cytotoxic protein was isolated from the crystal produced by Bacillus thuringiensis subsp. coreanensis A1519 strain. Upon treatment of the crystal proteins by proteinase K, the significant cytotoxicity toward the leukemic T cell, MOLT-4, was exhibited. The microscopic observation indicated that the cell death was accompanied by no extensive rupture of the cell membrane. It was, therefore, suggested that the cell death of MOLT-4 was induced through a mechanism other than the colloid-osmotic swelling and cell lysis as caused by hitherto known B. thuringiensis crystal proteins. The 29-kDa polypeptide proved to be an active component of the proteinase K-digested A1519 crystal proteins. EC(50) of the purified 29-kDa polypeptide was 0.078 microg/ml. The N-terminal amino acid sequence of the 29-kDa polypeptide shared no significant homology with all the known proteins, suggesting that this polypeptide belong to a new family of B. thuringiensis crystal proteins. In the ligand blotting analysis, specific binding proteins for the 29-kDa polypeptide were detected from the cell membrane of MOLT-4.     

Identification of a 220-kDa membrane tumor-associated antigen by human anti-UK114 monoclonal antibodies selected from the immunoglobulin repertoire of a cancer patient

Human monoclonal antibodies (HuMAb) specific for a 14-kDa perchloric acid-soluble protein (defined as UK114) were produced by somatic fusion of the human-mouse myeloma K6H6/B5 with Epstein-Barr virus-transformed peripheral B lymphocytes from a cancer patient previously treated with UK101 preparations, containing the UK114 protein. Three IgM-secreting clones were selected on the criteria of specificity for the purified UK114 protein immobilized onto plastic and adapted to grow in a serum-free medium. The reactivity of these antibodies showed a broad distribution pattern restricted to fresh tumor tissues and tumor cell lines, mainly of the adenocarcinoma type. None of the normal cells, nonmalignant cell lines, and normal tissues surrounding the neoplastic lesions were reactive. The immunochemical analysis of the target antigens showed that the HuMAb recognize a molecule of 220 kDa selectively expressed by the surface of tumor cells, as well as a cytoplasmic 14-kDa protein. The 220-kDa antigen was different from other tumor-associated antigens with similar molecular mass and, so far, unique. In the presence of human complement, two of three HuMAb are cytotoxic for tumor cells expressing the 220-kDa surface antigen. The tumor specificity and the lytic ability attributed to these HuMAb are promising features for the exploration of future clinical applications.