Proteomic analysis of the presynaptic active zone.

Synaptic vesicles are key organelles in chemical signaling, allowing neurons to communicate with each other and with neighboring cells. Vesicle integral or membrane-associated proteins mediate the various tasks the organelle fulfills during its life cycle. These include organelle transport, interaction with the nerve terminal cytoskeleton, uptake and storage of low molecular weight constituents, and the regulated interaction with the presynaptic plasma membrane, the active zone, during exo- and endocytosis. Converging work from several laboratories within the last 30 years resulted in the molecular and functional characterization of the protein inventory of the synaptic vesicle compartment. Nowadays advances in membrane protein separation and mass spectrometry have dramatically promoted this field resulting in a detailed description of the synaptic vesicle proteome and making synaptic vesicles the best characterized organelles. Recently, the proteome of the active zone was identified using the docked synaptic vesicles as target for immunoisolation. Combining gel-based protein separation techniques, mass spectrometry, and immunodetection, a considerable variety of proteins has been detected in the active zone. This includes synaptic vesicle proteins, components of the presynaptic fusion and retrieval machinery, proteins involved in intracellular signal transduction, a large variety of adhesion molecules and proteins potentially involved in regulating the functional and structural dynamics of the presynapse. Here, we discuss recent information concerning the proteome of the presynaptic active zone, focusing on proteins that are potentially involved in the short- and long-term structural modulation of the mature presynaptic compartment. In addition, we discuss the functional relevance of amyloid precursor protein in these membrane fractions and the putative interplay with direct or indirect interaction partners in the active zone.

The hemicholinium-3 sensitive high affinity choline transporter is internalized by clathrin-mediated endocytosis and is present in endosomes and synaptic vesicles.

Synthesis of acetylcholine depends on the plasma membrane uptake of choline by a high affinity choline transporter (CHT1). Choline uptake is regulated by nerve impulses and trafficking of an intracellular pool of CHT1 to the plasma membrane may be important for this regulation. We have generated a hemagglutinin (HA) epitope tagged CHT1 to investigate the organelles involved with intracellular trafficking of this protein. Expression of CHT1-HA in HEK 293 cells establishes Na+-dependent, hemicholinium-3 sensitive high-affinity choline transport activity. Confocal microscopy reveals that CHT1-HA is found predominantly in intracellular organelles in three different cell lines. Importantly, CHT1-HA seems to be continuously cycling between the plasma membrane and endocytic organelles via a constitutive clathrin-mediated endocytic pathway. In a neuronal cell line, CHT1-HA colocalizes with the early endocytic marker green fluorescent protein (GFP)-Rab 5 and with two markers of synaptic-like vesicles, VAMP-myc and GFP-VAChT, suggesting that in cultured cells CHT1 is present mainly in organelles of endocytic origin. Subcellular fractionation and immunoisolation of organelles from rat brain indicate that CHT1 is present in synaptic vesicles. We propose that intracellular CHT1 can be recruited during stimulation to increase choline uptake in nerve terminals.

Major vault protein is a substrate of endogenous protein kinases in CHO and PC12 cells.

Major vault protein (MVP) is the predominant member of a large cytosolic ribonucleoprotein particle, termed vault. We have previously shown that MVP derived from electric ray electric organ becomes phosphorylated by protein kinase C in vitro and by tyrosine kinase in vivo. Here we show that MVP from two mammalian cell lines (CHO and PC12 cell) becomes highly phosphorylated by endogenous protein kinases in cell-free systems. The susceptibility to protein kinases differs substantially from those observed in MVP derived from electric organ. Phosphorylation of MVP depends on the presence of Mg2+ and can be inhibited by the chelating agent EDTA. Inhibitors of casein kinase II attenuate the phosphorylation of MVP. In contrast to CHO cells, addition of recombinant casein kinase II enhances the phosphorylation of MVP in PC12 cells. Endogenous kinase activity is of particulate nature and copurifies with vault particles. Immuno-affinity purified vaults containing recombinant tagged MVP expressed in CHO cells reveal no autophosphorylation, suggesting that protein kinase activity is not an intrinsic property of vaults. Our results suggest that cell-specific phosphorylation of MVP may play a critical role in vault function.

Venom gland of the digger wasp Liris niger: morphology, ultrastructure, age-related changes and biochemical aspects.

Females of the parasitoid digger wasp species Liris niger hunt crickets as food for their future brood. The wasps paralyse the prey by injecting their venom directly into the CNS. The venom is produced in a gland consisting of two ramified glandular tubules terminating in a common reservoir. The reservoir contents enter the sting bulb via a ductus venatus. Secretory units of dermal gland type III line the two free gland tubules, the afferent ducts to the reservoir and the cap region within the reservoir. Secretion products of tubules reach the reservoir through the cuticle-lined central funnel. Secretory cells in the distal and middle parts of the tubules contain extensive rough endoplasmic reticulum and numerous electron-dense vesicles, whereas secretory cells of the afferent ducts and the cap region of the reservoir lack electron-dense vesicles and the endoplasmic reticulum is poorly developed. The secretory apparatus undergoes age-related changes. The secretory units in the venom gland tubules and inside the reservoir complete differentiation 1 day after imaginal ecdysis. After 30 days, massive autolytic processes occur in the secretory cells and in the epithelial cells of the reservoir. Analysis of the polypeptide composition demonstrates that the venom reservoir contains numerous proteins ranging from 3.4 to 200 kDa. A dominant component is a glycoprotein of about 90 kDa. In contrast the polypeptide composition of Dufour's gland is completely different and contains no glycoproteins. Comparison of the venom reservoir contents with the polypeptide pattern of venom droplets reveals that all of the major proteinaceous constituents become secreted. Thus the secreted venom contains exclusively proteins present in the soluble contents of the venom gland.

A comparison of synaptic protein localization in hippocampal mossy fiber terminals and neurosecretory endings of the neurohypophysis using the cryo-immunogold technique.

In central synapses synaptic vesicle docking and exocytosis occurs at morphologically specialized sites (active zones) and requires the interaction of specific proteins in the formation of a SNARE complex. In contrast, neurosecretory terminals lack active zones. Using the cryo-immunogold technique we analyzed the localization of synaptic vesicle proteins and of proteins of the docking complex at active zones. This was compared to the localization of the identical proteins in neurosecretory terminals. In addition we compared the vesicular and granular localization of the proteins investigated. Synaptic vesicles in rat hippocampal mossy fiber synapses and microvesicles in the neurosecretory terminals of the neurohypophysis contained in common the proteins VAMP II (a v-SNARE), SV2, rab3A, and N-type Ca(2+) channels. Only minor immunolabeling for these proteins was observed at neurosecretory granules. These results support the notion of a close functional identity of microvesicles from neurosecretory endings of the neurohypophysis and of synaptic vesicles. The vesicular pool of N-type Ca(2+) channels may serve their stimulation-induced translocation into the plasma membrane. We find increased labeling for VAMP II, SNAP-25, N-type Ca(2+) channels and of rab3A at the active zones of mossy fiber synapses. Labeling at release sites is by far highest for Bassoon, a high molecular weight protein of the active zone. The labeling pattern implies an association of Bassoon with presynaptic dense projections. Bassoon is absent from neurosecretory terminals and VAMP II, SNAP-25, rab3A, and N-type Ca(2+) channels reveal a scattered distribution over the plasma membrane. The competence of the presynaptic active zone for selective vesicle docking may not primarily result from its contents in SNARE proteins but rather from the preformation of presynaptic dense projections as structural guides for vesicle exocytosis.

Age-dependent pre- and postsynaptic distribution of AMPA receptors at synapses in CA3 stratum radiatum of hippocampal slice cultures compared with intact brain.

Organotypic slice cultures of rat hippocampus are widely used as experimental preparations for the study of synaptic plasticity, but their degree of correspondence with intact brain is not fully known. Here, using postembedding immunogold labelling, we describe the ultrastructural distribution of AMPA-type glutamate receptors (GluR1-4) in CA3 stratum radiatum of organotypic hippocampal slice cultures at 10 days to 11 weeks in vitro and compare the labelling with intact brain of corresponding age. In both types of preparation, the 11-week-old samples contained the highest proportion of AMPA receptor-like immunoreactive synapses. The incidence of labelled synapses, however, was higher in vivo (49%) than in vitro (24%). The intensity of labelling (number of gold particles per labelled synapse) also increased with age and was also higher in vivo than in vitro. In both organotypic cultures and intact brain, labelling was frequently found at presynaptic sites, often attached to vesicular structures. The specificity of these findings was supported both by light microscopic immunolabelling of GluR2/3 subunits and by electron microscopic double labelling of different epitopes of the GluR2 subunit. The vesicular localization of AMPA receptors was supported by Western blot analysis of subcellular fractions. Morphological evidence of presynaptic excitatory innervation of glutamatergic neurons supports a functional role for presynaptically located AMPA receptors. Our results therefore suggest that AMPA receptors occur in both pre- and postsynaptic profiles and that the distribution of AMPA receptors in cultured brain slices is fundamentally similar to intact brain, but that synaptic maturation may be retarded in vitro.

Membrane association of presynaptic cytomatrix protein bassoon.

Components of the specialized cytomatrix at active zones of presynaptic nerve terminals are thought to be involved in organizing synaptic events such as immobilisation or translocation of synaptic vesicles and assemblingactive zone components. The 420-kDa non-transmembraneprotein Bassoon is a specific componentof the presynaptic cytomatrix that shares features with both cytoskeleton-associated and peripheral-membrane proteins. Using immunogold electron microscopy we show here that synapse associated Bassoon is distributed in a subregion of active zones. Using a biochemical assay we show that a fraction of Bassoon is membrane associated. Electron microscopy performed on the same biochemical fraction further revealed that Bassoon is associated with vesicular structures. Together these data suggest that at least a fraction of Bassoon is associated with a membraneous compartment in neurons.

Endocytic vacuoles formed following a short pulse of K+ -stimulation contain a plethora of presynaptic membrane proteins.

It is now well established that the membrane of synaptic vesicles is recycled following exocytosis. However, little is known concerning the identity of the primary or secondary endocytic structures and their molecular composition. Using cultured rat cerebellar granule cells we combined uptake of horseradish peroxidase as a fluid phase marker and immunogold labeling for a variety of presynaptic proteins to assess the molecular identity of the stimulation-induced endocytic compartments. Short periods (5 or 30 s) of stimulation with 50 mM KCl were followed by periods of recovery for up to 30 min. Stimulation resulted in the formation of horseradish-peroxidase-filled vacuoles in the axonal varicosities as the apparent primary endocytic compartment. Horseradish peroxidase-filled synaptic vesicles were formed when stimulated cells were allowed to recover in horseradish peroxidase-free culture medium. Horseradish peroxidase-filled vacuoles as wells as vesicles contained the synaptic vesicle membrane proteins VAMP II, synaptotagmin, SV2, and synaptophysin, the vesicle-associated proteins rab 3A and synapsin I, and in addition SNAP-25. No incorporation of vesicle proteins into the plasma membrane was observed. Horseradish peroxidase-filled vesicles and vacuoles generated on incubation of unstimulated granule cells with horseradish peroxidase for prolonged periods of time were equally immunolabeled. Renewed stimulation of prestimulated granule cells with either 100 mM KCl or 30 microM Ca2+ ionophore A23187 resulted in a reduction of horseradish peroxidase-filled vacuoles suggesting that the vacuolar membrane compartment was exocytosis-competent. Our results suggest that varicosities of cultured cerebellar granule cells possess a fast stimulation-induced pathway for recycling the entire synaptic vesicle membrane compartment. The primary endocytic compartment represents not a synaptic vesicle but a somewhat larger vesicle protein-containing vacuolar entity from which smaller vesicles of identical protein composition may be regenerated. Endocytic vacuoles and synaptic vesicles share membrane and membrane-associated proteins and presumably also major functional properties.

Axonal transport of ribonucleoprotein particles (vaults).

RNA was previously shown to be transported into both dendritic and axonal compartments of nerve cells, presumably involving a ribonucleoprotein particle. In order to reveal potential mechanisms of transport we investigated the axonal transport of the major vault protein of the electric ray Torpedo marmorata. This protein is the major protein component of a ribonucleoprotein particle (vault) carrying a non-translatable RNA and has a wide distribution in the animal kingdom. It is highly enriched in the cholinergic electromotor neurons and similar in size to synaptic vesicles. The axonal transport of vaults was investigated by immunofluorescence, using the anti-vault protein antibody as marker, and cytofluorimetric scanning, and was compared to that of the synaptic vesicle membrane protein SV2 and of the beta-subunit of the F1-ATPase as a marker for mitochondria. Following a crush significant axonal accumulation of SV2 proximal to the crush could first be observed after 1 h, that of mitochondria after 3 h and that of vaults after 6 h, although weekly fluorescent traces of accumulations of vault protein were observed in the confocal microscope as early as 3 h. Within the time-period investigated (up to 72 h) the accumulation of all markers increased continuously. Retrograde accumulations also occurred, and the immunofluorescence for the retrograde component, indicating recycling, was weaker than that for the anterograde component, suggesting that more than half of the vaults are degraded within the nerve terminal. High resolution immunofluorescence revealed a granular structure-in accordance with the biochemical characteristics of vaults. Of interest was the observation that the increase of vault immunoreactivity proximal to the crush accelerated with time after crushing, while that of SV2-containing particles appeared to decelerate, indicating that the crush procedure with time may have induced perikaryal alterations in the production and subsequent export to the axon of synaptic vesicles and vault protein. Our data show that ribonucleoprotein-immunoreactive particles can be actively transported within axons in situ from the soma to the nerve terminal and back. The results suggest that the transport of vaults is driven by fast axonal transport motors like the SV2-containing vesicles and mitochondria. Vaults exhibit an anterograde and a retrograde transport component, similar to that observed for the vesicular organelles carrying SV2 and for mitochondria. Although the function of vaults is still unknown studies of the axonal transport of this organelle may reveal insights into the mechanisms of cellular transport of ribonucleoprotein particles in general.

A plethora of presynaptic proteins associated with ATP-storing organelles in cultured astrocytes.

Cultured astrocytes can release a variety of messenger substances via receptor-mediated mechanisms, implicating their potential for regulated exocytosis and the participation of proteins of the SNARE complex. Here we demonstrate the astrocytic expression and organellar association of a large variety of synaptic proteins (synaptobrevin II, synaptotagmin I, synaptophysin, rab3a, synapsin I, SNAP-25, and syntaxin I) and also of the ubiquitous cellubrevin. As revealed by immunoblotting the expression of synaptic proteins was highest within the first few days after plating. Synaptophysin and SNAP-25 showed the most significant decline with prolonged culture time. Rab3a and synaptobrevin II were retained at a high level and synaptotagmin I, synapsin I, and syntaxin I at a lower level until 20 DIV. The immunoreaction for cellubrevin was low at the beginning and increased with prolonged culture time. As revealed by light microscopical immunocytochemistry the proteins are expressed by GFAP-positive astrocytes and associated with organelles of varying size. Immunoelectron microscopical analysis allocates synaptobrevin II and synaptophysin to the membranes of vesicular organelles. Double labeling experiments for pairs of synaptic proteins reveal that individual synaptic proteins can be entirely colocalized or partly reside on different organelles. Subcellular fractionation of astrocyte cultures by sucrose density gradient centrifugation after 2, 6, 13, and 20 DIV showed that the proteins sediment with ATP containing organelles of a broad density range. Our data suggest that messenger substances may be released from cultured astrocytes via receptor-mediated, Ca2+-dependent exocytosis.

Recombinant major vault protein is targeted to neuritic tips of PC12 cells.

The major vault protein (MVP) is the predominant constituent of ubiquitous, evolutionarily conserved large cytoplasmic ribonucleoprotein particles of unknown function. Vaults are multimeric protein complexes with several copies of an untranslated RNA. Double labeling employing laser-assisted confocal microscopy and indirect immunofluorescence demonstrates partial colocalization of vaults with cytoskeletal elements in Chinese hamster ovary (CHO) and nerve growth factor (NGF)-treated neuronlike PC12 cells. Transfection of CHO and PC12 cells with a cDNA encoding the rat major vault protein containing a vesicular stomatitis virus glycoprotein epitope tag demonstrates that the recombinant protein is sorted into vault particles and targeted like endogenous MVPs. In neuritic extensions of differentiated PC12 cells, there is an almost complete overlap of the distribution of microtubules and vaults. A pronounced colocalization of vaults with filamentous actin can be seen in the tips of neurites. Moreover, in NGF-treated PC12 cells the location of vaults partially coincides with vesicular markers. Within the terminal tips of neurites vaults are located near secretory organelles. Our observations suggest that the vault particles are transported along cytoskeletal-based cellular tracks.

Peripheral synapses at identifiable mechanosensory neurons in the spider Cupiennius salei: synapsin-like immunoreactivity.

Indirect immunocytochemical tests were used at the light- and electron-microscopic levels to investigate peripheral chemical synapses in identified sensory neurons of two types of cuticular mechanosensors in the spider Cupiennius salei Keys.: (1) in the lyriform slit-sense organ VS-3 (comprising 7-8 cuticular slits, each innervated by 2 bipolar sensory neurons) and (2) in tactile hair sensilla (each supplied with 3 bipolar sensory cells). All these neurons are mechanosensitive. Application of a monoclonal antibody against Drosophila synapsin revealed clear punctate immunofluorescence in whole-mount preparations of both mechanoreceptor types. The size and overall distribution of immunoreactive puncta suggested that these were labeled presynaptic sites. Immunofluorescent puncta were 0.5-6.8 micrometer long and located 0.5-6.6 micrometer apart from each other. They were concentrated at the initial axon segments of the sensory neurons, while the somata and the dendritic regions showed fewer puncta. Western blot analysis with the same synapsin antibody against samples of spider sensory hypodermis and against samples from the central nervous system revealed a characteristic doublet band at 72 kDa and 75 kDa, corresponding to the apparent molecular mass of synapsin in Drosophila and in mammals. Conventional transmissionelectron-microscopic staining demonstrated that numerous chemical synapses (with at least 2 vesicle types) were present at these mechanosensory neurons and their surrounding glial sheath. The distribution of these synapses corresponded to our immunofluorescence results. Ultrastructural examination of anti-synapsin-stained neurons confirmed that reaction product was associated with synaptic vesicles. We assume that the peripheral synaptic contacts originate from efferents that could exert a complex modulatory influence on mechanosensory activity.

Formation and sequence analysis of secretoneurin, a neuropeptide derived from secretogranin II, in mammalian, bird, reptile, amphibian and fish brains.

Secretoneurin is a recently-characterized neuropeptide derived from secretogranin II, a protein belonging to the class of chromogranins. We investigated the phylogeny of this peptide by immunoblotting and gel-filtration high performance liquid chromatography followed by radioimmunoassay of brain extracts of various species including chicken, lizard, frog and fish. In addition the amino acid sequence of secretoneurin from pig, hamster, rabbit, guinea-pig and chicken was established by reverse transcriptase polymerase chain reaction. Secretoneurin is strongly conserved during evolution, it is not only expressed in various mammalian species but found also in the brain of birds, reptiles, amphibians and fish. In all these species a significant or near complete processing of secretogranin II to secretoneurin was observed. These data provide significant evidence for the neuropeptide nature of the novel functional peptide.

Major vault protein of electric ray is a phosphoprotein.

The major vault protein is the predominant member of a large cytosolic ribonucleoprotein particle, named vaults. Vaults are abundant in nerve terminals of the electric organ of Torpedo marmorata. Negative staining of isolated vaults reveals particle dimensions of 45x65 nm in size. Comparison of the major vault protein (MVP100) from the two electric ray species Torpedo marmorata and Discopyge ommata reveals few microheterogeneities in amino acid sequence. Potential phosphorylation sites for various protein kinases are highly conserved. Phosphorylation studies demonstrate that the major vault protein of Torpedo is a substrate of various protein kinases. MVP100 is phosphorylated by protein tyrosine kinase in vivo and protein kinase C and casein kinase II in vitro. Inhibitors and activators of protein kinases specifically modulate the phosphorylation of MVP100.

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.

Analysis of a cDNA encoding the major vault protein from the electric ray Discopyge ommata.

The major vault protein is the predominant constituent of vaults ubiquitous large cytosolic ribonucleoprotein particles. A cDNA clone encoding the 100-kDa major vault protein (MVP100) was isolated from an electric lobe library of Discopyge ommata. The complete nucleotide sequence was determined. Northern blot analysis revealed a 2.8-kb transcript with a high expression in neural tissue. Southern blot analysis indicates that the electric ray MVP100 is a single copy-gene with at least two introns. The primary structure of major vault proteins characterized in slime mold, ray, rat and human is evolutionary highly conserved.

Colocalization on the same synaptic vesicles of syntaxin and SNAP-25 with synaptic vesicle proteins: a re-evaluation of functional models required?

Synaptic vesicle docking and calcium dependent exocytosis are thought to require the specific interaction of proteins of the synaptic vesicle membrane (such as VAMP/synaptobrevin and synaptotagmin) and their plasma membrane-located counterparts (such as syntaxin and SNAP-25). When isolating synaptic vesicles by glycerol velocity gradient centrifugation we found cosedimentation of the presumptive presynaptic plasma membrane proteins syntaxin and SNAP-25 with synaptic vesicle membrane proteins. In order to further identify the antibody binding organelles we performed an immunoelectron microscopical analysis of synaptosomal profiles. Syntaxin and SNAP-25 were not only associated with the plasma membrane but to a large extent also with synaptic vesicle profiles. In order to answer the question whether the syntaxin and SNAP-25 containing vesicular compartment would also carry classical synaptic vesicle membrane markers we performed double labeling experiments using poly- and monoclonal antibodies. We found colocalization on the same vesicle not only of SNAP-25 and syntaxin but also of SNAP-25 with the synaptic vesicle membrane proteins SV2 and synaptotagmin and of syntaxin with the vesicular membrane protein synaptophysin. Our results demonstrate that syntaxin and SNAP-25 are colocalized with classical vesicle membrane proteins on the same vesicle and suggest that the functional models for the interaction of presynaptic proteins need to be re-evaluated.

The major vault protein (MVP100) is contained in cholinergic nerve terminals of electric ray electric organ.

A protein of Mr 100,000 (MVP100) is highly enriched in the electromotor system of electric rays. Biochemical analysis indicates that MVP100 is contained in the cholinergic nerve terminals of Torpedo electric organ as part of a large cytosolic complex. On sucrose density gradient centrifugation MVP100 comigrates with synaptic vesicles or synaptosomes. It can be partially separated from synaptic vesicles by gel filtration or glycerol velocity gradient centrifugation. Within the complex MVP100 behaves like a hydrophobic protein and is protected against proteolytic attack. MVP100 can be immunodetected by an antibody against phosphotyrosine, and it becomes phosphorylated on incubation with [gamma-32P]ATP. By screening an electric ray electric lobe cDNA library the primary structure of MVP100 was analyzed. MVP100 is highly homologous to the major vault proteins of slime mold and rat and to the human lung resistance-related protein. Compared with non-neural tissues the expression of MVP100 is highest in brain and enriched in the electric lobe that contains the somata of the electromotor neurons. Immunoelectron microscopic analysis reveals a close association of MVP100 and synaptic vesicles in the nerve terminals of the electric organ.

Two synpatic vesicle proteins of 25 kDa: a comparison of the molecular properties and tissue distribution of svp25 and o-rab3.

Two synaptic vesicle proteins of the electric ray Torpedo--svp25 and o-rab3--are compared with respect to their biochemical properties and tissue distribution. On SDS-PAGE both proteins migrate to the same position of about 25 kDa. As revealed by application of monospecific antibodies and subcellular fractionation both proteins comigrate and cofractionate with the synaptic vesicle compartment. o-Rab3 and svp25 can be separated by lectin chromatography; svp25 is highly glycosylated and binds to concanavalin A sepharose. Upon deglycosylation using glycopeptidase F and O-glycosidase its apparent molecular mass is reduced to about 14 kDa. Partial amino acid sequences obtained by direct microsequencing of purified and deglycosylated svp25 revealed that svp25 is a novel protein that has not yet been characterized in molecular terms. Whereas svp25 was detected in all brain areas investigated, the expression of o-rab3 was found to be restricted to specific regions. An immunoblot analysis demonstrates an exclusive association of both proteins with neural tissues. Our results suggest that cholinergic synaptic vesicles from electric ray electric organ contain at least two membrane-associated proteins of an apparent molecular mass of 25 kDa, the membrane associated o-rab3 and the membrane integral protein svp25. The two proteins can be separated by lectin chromatography for assessment of their biochemical properties.