Membrane Protein Of Synaptic Vesicles Research Articles

Structure and Organization of the DrosophilaCholinergic Locus

The Drosophila cholinergic locus is composed of two distinct genetic functions: choline acetyltransferase (ChAT; EC 2.3.1.6), the enzyme catalyzing biosynthesis of neurotransmitter acetylcholine (ACh), and the vesicular ACh transporter (VAChT), the synaptic vesicle membrane protein which pumps transmitter into vesicles. Both genes share a common first exon and the remainder of the VAChT gene contains a single coding exon residing entirely within the first intron of ChAT. RNase protection analysis indicates that all Drosophila VAChT specific transcripts contain the shared first exon and suggests common transcriptional control for ChAT and VAChT. Similar types of genomic organization have been evolutionarily conserved for cholinergic loci in nematodes and vertebrates, and may operate to ensure coordinate expression of these functionally related genes in the same cells. The relative levels of Drosophila ChAT and VAChT mRNA differ, however, in different tissues or in Cha mutants, indicating that independent regulation of ChAT and VAChT transcripts may occur post-transcriptionally. The predicted Drosophila VAChT protein is composed of 578 amino acids and contains 12 conserved putative transmembrane domains. Full-length VAChT cDNA is 7.2 kilobase long and has unusually long 5'- and 3'-untranslated regions (UTR). The 5'-UTR contains a GTG ChAT translational initiation codon along with three other potential ATG initiation codons. These features of the VAChT 5'-UTR region suggest that a ribosome scanning model may not be used for VAChT translation initiation.

Mechanisms of Protein Secretion in Endocrine and Exocrine Cells

Publisher Summary This chapter describes that dense core vesicles (DCVs) exocytosis in exocrine cells proceeds by mechanisms that are less clearly understood and involves proteins or protein isoforms different from those employed in neural/endocrine secretion. Biochemical and molecular biological studies have resulted in the characterization of at least 25 types of synaptic vesicle (SV) membrane proteins that may mediate aspects of SV function including calcium-dependent exocytosis, endocytosis, and neurotransmitter loading. In neuronal cells, there are important physiological differences between the exocytosis of SVs that contain neurotransmitters such as glutamate and the exocytosis of larger DCVs that contain neuropeptides, including calcium sensitivity and speed. Microinjection and genetic studies have documented the importance of SNARE proteins and synaptotagmin in neurosecretion without revealing their sites of action and mechanisms. Of the other isoforms of synaptotagmin characterized, synaptotagmin III has the most appealing characteristics as a calcium sensor for DCV exocytosis. Expression of synaptotagmin isoforms in the acinar pancreas has not been reported, so it is uncertain whether this protein family plays a role in calcium sensing for zymogen granule exocytosis.

Regulation of AP-2-Synaptotagmin Interaction by Inositol High Polyphosphates

The inositol high-polyphosphate series (IHPS) inhibits neurotransmission through binding to the second C2 domain of synaptotagmins I and II(Syt), synaptic vesicle membrane proteins. We have revealed that several proteins, including α adaptins which are specific subunits of clathrin assembly protein, AP2, were eluted from mouse brain by affinity elution chromatography from the C2 domains of Syt II-immobilized Sepharose using 50 μM of InsP6. The interaction between Syt II and AP2 was more markedly inhibited by IHPS than by the same concentration of InsP3. Limited digestion of mouse crude synaptosomal fractions with trypsin revealed different cleavage patterns in the presence and absence of 50 μM InsP6. These results suggest that IHPS-binding to the C2B domain of synaptotagmin alters the state of protein–protein interaction including the synaptotagmin-AP2 interaction, possibly resulting in the inhibition of events involved in the synaptic vesicle trafficking.

Genetic divergence at the synaptophysin (Syp I) locus among Norwegian coastal and north‐east Arctic populations of Atlantic cod

Several nuclear RFLP loci have been discovered recently that exhibit extensive allele frequency variation among Norwegian coastal and north‐east Arctic populations of Atlantic cod Gadus morhua. One of these polymorphisms was detected by hybridizing an anonymous cDNA clone (GM798) against genomic DNA digested with the restriction enzyme DraI. This cDNA clone has now been sequenced and identified as synaptophysin (Syp I), an integral synaptic vesicle membrane protein. Primers were constructed that amplify an intron of the Syp I gene that is polymorphic for the DraI site, thus making it possible to use a PCR‐based assay to score the polymorphism. A total of 965 individuals sampled from the Barents Sea, coastal areas and fjords in northern Norway have been analysed for this polymorphism. The results confirm that highly significant differences exist between NE Arctic and coastal cod at the Syp I locus. A cluster analysis revealed a deep split between coastal and Arctic populations and hierarchical F‐statistics indicated that about 40% of the total variation was attributable to differences between Arctic and coastal groups. The temporal stability of allele frequencies was assessed by comparing Syp I allele frequencies among samples of juveniles (0 group) captured at specific locations in fjords in consecutive years and among samples of adults and juveniles collected from the same fjord. Samples of juveniles collected in 1994 and 1995 in Malangen were genetically indistinguishable whereas juveniles sampled from Dønnesfjord and Ullsfjorden over the same 2‐year period exhibited significant differences. Adults and 0‐group individuals collected from the same fjord were found to be genetically indistinguishable in Malangen, but not in Balsfjorden. In addition to detecting large differences among Arctic and coastal groups, the Syp I locus suggests that genetic heterogeneity exists among resident populations of cod in different fjords and that gene flow among populations throughout northern Norway may be considerably lower than previously believed.

Reduction of synaptophysin immunoreactivity in the prefrontal cortex of subjects with schizophrenia. Regional and diagnostic specificity.

Multiple lines of evidence indicate that the prefrontal cortex is a site of dysfunction in schizophrenia. However, the apparent absence of gross structural abnormalities in this area suggests that the pathophysiological characteristics of schizophrenia may involve more subtle disturbances in prefrontal cortical circuitry, such as alterations in synaptic connectivity and transmission. In this study, immunoreactivity for synaptophysin, an integral membrane protein of small synaptic vesicles, was used to assess the integrity of cortical synaptic circuitry in schizophrenia. Using immunocytochemical techniques and adjusted optical density measurements, we examined synaptophysin immunoreactivity in prefrontal cortical areas 9 and 46 and in area 17 (the primary visual cortex) from 10 pairs of case subjects with schizophrenia and control subjects matched on a pairwise basis for age, sex, race, and postmortem interval, and in 5 matched pairs of nonschizophrenic psychiatric case subjects and normal control subjects. Compared with levels found in matched control subjects, synaptophysin immunoreactivity in areas 46 and 9 was significantly decreased (P < .001 and P < .008, respectively) across all cortical layers in the case subjects with schizophrenia. In contrast, no differences were observed in area 17. In addition, levels of synaptophysin immunoreactivity in areas 46, 9, and 17 did not differ between 5 nonschizophrenic psychiatric case subjects and their matched controls, suggesting that decreased synaptophysin levels in the prefrontal cortex of patients with schizophrenia may be specific to that disorder. Additional studies are required to determine if the decrease in levels of synaptophysin immunoreactivity is caused by a decrease in the number or size of presynaptic terminals, a decrease in the number of synaptic vesicles per terminal, or a decrease in the expression of synaptophysin. However, all of these potential explanations are consistent with a disturbance in synaptic transmission in the prefrontal cortex of patients with schizophrenia.

Direct interaction of the calcium sensor protein synaptotagmin I with a cytoplasmic domain of the alpha1A subunit of the P/Q-type calcium channel.

Synaptotagmins are synaptic vesicle proteins containing two calcium-binding C2 domains which are involved in coupling calcium influx through voltage-gated channels to vesicle fusion and exocytosis of neurotransmitters. The interaction of synaptotagmins with native P/Q-type calcium channels was studied in solubilized synaptosomes from rat cerebellum. Antibodies against synaptotagmins I and II, but not IV co-immunoprecipitated [125I]omega-conotoxin MVIIC-labelled calcium channels. Direct interactions were studied between in vitro-translated [35S]synaptotagmin I and fusion proteins containing cytoplasmic loops of the alpha1A subunit (BI isoform). Gel overlay revealed the association of synaptotagmin I with a single region (residues 780-969) located in the intracellular loop connecting homologous domains II and III. Saturable calcium-independent binding occurred with equilibrium dissociation constants of 70 nM and 340 nM at 4 degrees C and pH 7.4, and association was blocked by addition of excess recombinant synaptotagmin I. Direct synaptotagmin binding to the pore-forming subunit of the P/Q-type channel may optimally locate the calcium-binding sites that initiate exocytosis within a zone of voltage-gated calcium entry.

Reduction of synaptophysin immunoreactivity in the prefrontal cortex of subjects with schizophrenia. Regional and diagnostic specificity.

Multiple lines of evidence indicate that the prefrontal cortex is a site of dysfunction in schizophrenia. However, the apparent absence of gross structural abnormalities in this area suggests that the pathophysiological characteristics of schizophrenia may involve more subtle disturbances in prefrontal cortical circuitry, such as alterations in synaptic connectivity and transmission. In this study, immunoreactivity for synaptophysin, an integral membrane protein of small synaptic vesicles, was used to assess the integrity of cortical synaptic circuitry in schizophrenia. Using immunocytochemical techniques and adjusted optical density measurements, we examined synaptophysin immunoreactivity in prefrontal cortical areas 9 and 46 and in area 17 (the primary visual cortex) from 10 pairs of case subjects with schizophrenia and control subjects. matched on a pairwise basis for age, sex, race, and postmortem interval, and in 5 matched pairs of nonschizophrenic psychiatric case subjects and normal control subjects. Compared with levels found in matched control subjects, synaptophysin immunoreactivity in areas 46 and 9 was significantly decreased (P < .001 and P < .008, respectively) across all cortical layers in the case subjects with schizophrenia. In contrast, no differences were observed in area 17. In addition, levels of synaptophysin immunoreactivity in areas 46, 9, and 17 did not differ between 5 nonschizophrenic psychiatric case subjects and their matched controls, suggesting that decreased synaptophysin levels in the prefrontal cortex of patients with schizophrenia may be specific to that disorder. Additional studies are required to determine if the decrease in levels of synaptophysin immunoreactivity is caused by a decrease in the number or size of presynaptic terminals, a decrease in the number of synaptic vesicle per terminal, or a decrease in the expression of synaptophysin. However, all of these potential explanations are consistent with a disturbance in synaptic transmission in the prefrontal cortex of patients with schizophrenia.

Differential Involvement of Synaptic Vesicle and Presynaptic Plasma Membrane Proteins in Alzheimer's Disease

Alzheimer's disease (AD) is characterized by progressive cognitive decline. Recent studies have shown that synaptic loss in the cortex is the major correlate of cognitive decline in AD. In the present study we assessed synaptic proteins such as synaptobrevin, synaptophysin, synaptotagmin, synaptosomal-associated protein 25 (SNAP-25), and syntaxin1/HPC-1 in control and AD brains to determine whether synaptic proteins are equally or differentially affected in AD. Western analysis showed that in AD levels of synaptobrevin and synaptophysin were decreased by some 30% from amounts in controls, while those of synaptotagmin, SNAP-25, and syntaxin 1/HPC-1 were decreased by only about 10%. As synaptobrevin and synaptophysin are localized mainly in transmitter-containing synaptic vesicles while SNAP-25 and syntaxin 1/HPC-1 are found in presynaptic plasma membranes, these results suggest differential involvement of synaptic components in AD.

Brain myosin V is a synaptic vesicle-associated motor protein: evidence for a Ca2+-dependent interaction with the synaptobrevin-synaptophysin complex.

Brain myosin V is a member of a widely distributed class of unconventional myosins that may be of central importance to organelle trafficking in all eukaryotic cells. Molecular constituents that target this molecular motor to organelles have not been previously identified. Using a combination of immunopurification, extraction, cross-linking, and coprecipitation assays, we demonstrate that the tail domain of brain myosin V forms a stable complex with the synaptic vesicle membrane proteins, synaptobrevin II and synaptophysin. While myosin V was principally bound to synaptic vesicles during rest, this putative transport complex was promptly disassembled upon the depolarization-induced entry of Ca2+ into intact nerve endings. Coimmunoprecipitation assays further indicate that Ca2+ disrupts the in vitro binding of synaptobrevin II to synaptophysin in the presence but not in the absence of Mg2+. We conclude that hydrophilic forces reversibly couple the myosin V tail to a biochemically defined class of organelles in brain nerve terminals.

Association of syntaxin with SNAP‐25 and VAMP (synaptobrevin) during axonal transport

Two proteins of the presynaptic plasma membrane, syntaxin and SNAP-25, and a synaptic vesicle membrane protein, VAMP/synaptobrevin, form stable protein complexes that are involved in the docking and fusion of synaptic vesicles at the presynaptic membrane. These protein complexes have also been described in a homogeneous population of cholinergic synaptosomes purified from Torpedo electric organ. In the present study, we performed similar experiments combining velocity sedimentation and immunoprecipitation on control or ligated electric nerves and found it was possible to distinguish syntaxin that is in the axonal plasma membrane from syntaxin that is transported by the fast anterograde axonal flow. Although syntaxin that is resident in axonal membranes is associated with SNAP-25 but not with VAMP, syntaxin that accumulates proximally to a ligature is associated with both SNAP-25 and VAMP in a stoichiometry very similar to that of the nerve terminal complex. In control nerves, lower amounts of syntaxin from a complex with VAMP, in proportion to syntaxin, which is conveyed by the axonal flow. Because added VAMP was unable to associate with syntaxin in solubilized control nerves and because neither solubilization by SDS nor dilution to the nanomolar range of syntaxin and VAMP concentrations before solubilization change the stoichiometry between the immunoprecipitated proteins, this complex appears to be both formed prior to solubilization and stable thereafter. Hence, heterotrimeric complexes containing syntaxin, SNAP-25, and VAMP are already formed during fast anterograde axonal transport, before reaching the nerve endings. J. Neurosci. Res. 48:313–323, 1997. © 1997 Wiley-Liss, Inc.

Association of syntaxin with SNAP-25 and VAMP (synaptobrevin) during axonal transport

Two proteins of the presynaptic plasma membrane, syntaxin and SNAP-25, and a synaptic vesicle membrane protein, VAMP/synaptobrevin, form stable protein complexes that are involved in the docking and fusion of synaptic vesicles at the presynaptic membrane. These protein complexes have also been described in a homogeneous population of cholinergic synaptosomes purified from Torpedo electric organ. In the present study, we performed similar experiments combining velocity sedimentation and immunoprecipitation on control or ligated electric nerves and found it was possible to distinguish syntaxin that is in the axonal plasma membrane from syntaxin that is transported by the fast anterograde axonal flow. Although syntaxin that is resident in axonal membranes is associated with SNAP-25 but not with VAMP, syntaxin that accumulates proximally to a ligature is associated with both SNAP-25 and VAMP in a stoichiometry very similar to that of the nerve terminal complex. In control nerves, lower amounts of syntaxin form a complex with VAMP, in proportion to syntaxin, which is conveyed by the axonal flow. Because added VAMP was unable to associate with syntaxin in solubilized control nerves and because neither solubilization by SDS nor dilution to the nanomolar range of syntaxin and VAMP concentrations before solubilization change the stoiechiometry between the immunoprecipitated proteins, this complex appears to be both formed prior to solubilization and stable thereafter. Hence, heterotrimeric complexes containing syntaxin, SNAP-25, and VAMP are already formed during fast anterograde axonal transport, before reaching the nerve endings.

Unforgettable tetanus

Unforgettable tetanus

104 Functional diversity of C2 domains of synaptotagmin family

We isolated a binding protein for inositol 1,3,4,5-tetrakisphosphate (InsP4) from detergent-solubilized mouse cerebellar membrane fractions by sequential column chromatographies. Partial amino acid sequencing of the purified sample revealed that the protein is essentially identical to rat synaptotagmin II, an integral membrane protein of synaptic vesicles. Immunoprecipitation experiment of [3H]lnsP4 binding activity of the purified protein using polyclonal antibody against the C2A domain of rat synaptotagmin II also revealed that mouse synaptotagmin II is the InsP4 binding protein (IP4BP). Scatchard analysis of InsP4 binding to the IP4BP/synaptotagmin indicates a single binding site with a Kd of 30 nM. The present finding that InsP4 binds strongly to synaptotagmin II suggests an important role for inositol polyphosphates in the regulation of neurotransmitter release.

Trafficking of Cell-Surface β-Amyloid Precursor Protein: Evidence that a Sorting Intermediate Participates in Synaptic Vesicle Recycling

We recently demonstrated that the Alzheimer's beta-amyloid precursor protein (APP) is internalized from the axonal cell surface. In this study, we use biochemical and cell biological methods to characterize endocytotic compartments that participate in trafficking of APP in central neurons. APP is present in presynaptic clathrin-coated vesicles purified from bovine brain, together with the recycling synaptic vesicle integral membrane proteins synaptophysin, synaptotagmin, and SV2. In contrast, APP is largely excluded from synaptic vesicles purified from rat brain. In primary cerebellar macroneurons, cell-surface APP is internalized with recycling synaptic vesicle integral membrane proteins but is subsequently sorted away from synaptic vesicles and transported retrogradely to the neuronal soma. Internalized APP partially co-localizes with rab5a-containing compartments in axons and with V-ATPase-containing compartments in both axons and neuronal soma. These results provide direct biochemical evidence that an obligate sorting compartment participates in the regeneration of synaptic vesicles during exo/endocytotic recycling at nerve terminals but do not preclude concurrent "kiss-and-run" recycling. Moreover, APP is now, to our knowledge, the first demonstrated example of an axonal cell-surface protein that is internalized with recycling synaptic vesicle membrane proteins but is subsequently sorted away from synaptic vesicles.

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

Synaptic vesicle proteins in cells of the sympathoadrenal lineage

The cells of sympathoadrenal lineage display different characteristics after differentiation, although they stem from the same neural crest precursor during embryonic development. In the present study we compared the distribution patterns of several synaptic vesicle proteins in the superior cervical ganglion (SCG) and the adrenal medulla. Using indirect immunofluorescence combined with confocal laser scanning microscopy, it was observed that antisera against integral synaptic vesicle membrane proteins (SV2, synaptotagmin I, synaptobrevin II and synaptophysin) induced strong immunoreactivities in these cells, but anti-synaptobrevin I caused only a faint fluorescence. Immunoreactivities of the synaptic vesicle-associated proteins Rab3a and SNAP25 were also observed in the cells. Synapsin-Ia-reactive material appeared absent from chromaffin cells but present in small amounts in sympathetic neurons in the SCG and iris terminals. On the other hand, synapsin IIa immunoreactive material was strong in most SCG neurons and in adrenergic iris terminals. The neural specific clatrin light chain was detected in the SCG cells and in ganglion cells of the adrenal, but only weak traces could be observed in chromaffin cells. One of the vesicular monoamine transmitter transporters, VMAT2, which is expressed in catecholamine neurons in the brain stem, was observed in most cells in the SCG and also in groups of cells in the adrenal medulla, where the VMAT2-positive small chromaffin cells were PNMT-negative. SIF cells in the SCG contained most of the synaptic vesicle proteins investigated. The results show that after differentiation, sympathetic neurons, SIF cells and adrenal chromaffin cells still share many vesicle proteins even though their physiology is different.

Calcium-dependent switching of the specificity of phosphoinositide binding to synaptotagmin.

The synaptic vesicle membrane protein synaptotagmin (tagmin) is essential for fast, calcium-dependent, neurotransmitter release and is likely to be the calcium sensor for exocytosis, because of its many calcium-dependent properties. Polyphosphoinositides are needed for exocytosis, but it has not been known why. We now provide a possible connection between these observations with the finding that the C2B domain of tagmin I binds phosphatidylinositol-4,5-bisphosphate (PIns-4,5-P2), its isomer phosphatidylinositol-3,4-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate (PIns-3,4,5-P3). Calcium ions switch the specificity of this binding from PIns-3,4,5-P3 (at calcium concentrations found in resting nerve terminals) to PIns-4,5-P2 (at concentration of calcium required for transmitter release). Inositol polyphosphates, known blockers of neurotransmitter release, inhibit the binding of both PIns-4,5-P2 and PIns-3,4,5-P3 to tagmin. Our findings imply that tagmin may operate as a bimodal calcium sensor, switching bound lipids during exocytosis. This connection to polyphosphoinositides, compounds whose levels are physiologically regulated, could be important for long-term memory and learning.

Complete genomic sequence and analysis of 117 kb of human DNA containing the gene BRCA1.

Over 100 distinct disease-associated mutations have been identified in the breast-ovarian cancer susceptibility gene BRCA1. Loss of the wild-type allele in > 90% of tumors from patients with inherited BRCA1 mutations indicates tumor suppressive function. The low incidence of somatic mutations suggests that BRCA1 inactivation in sporadic tumors occurs by alternative mechanisms, such as interstitial chromosomal deletion or reduced transcription. To identify possible features of the BRCA1 genomic region that may contribute to chromosomal instability as well as potential transcriptional regulatory elements, a 117,143-bp DNA sequence encompassing BRCA1 was obtained by random sequencing of four cosmids identified from a human chromosome 17 specific library. The 24 exons of BRCA1 span an 81-kb region that has an unusually high density of Alu repetitive DNA (41.5%), but relatively low density (4.8%) of other repetitive sequences. BRCA1 intron lengths range in size from 403 bp to 9.2 kb and contain the intragenic microsatellite markers D17S1323, D17S1322, and D17S855, which localize to introns 12, 19, and 20, respectively. In addition to BRCA1, the contig contains two complete genes: Rho7, a member of the rho family of GTP binding proteins, and VAT1, an abundant membrane protein of cholinergic synaptic vesicles. Partial sequences of the 1A1-3B B-box protein pseudogene and IFP 35, an interferon induced leucine zipper protein, reside within the contig. An L21 ribosomal protein pseudogene is embedded in BRCA1 intron 13. The order of genes on the chromosome is: centromere-1FP 35-VAT1-Rho7-BRCA1-1A1-3B-telomere.

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.

Adrenal chromaffin cells contain functionally different SNAP-25 monomers and SNAP-25/syntaxin heterodimers

Syntaxin and SNAP-25 (synaptosome-associated protein of 25 kDa), associated with the neuronal plasmalemma, and synaptobrevin, a membrane protein of synaptic vesicles, are essential components of the exocytotic apparatus of synaptic vesicles. All three can be proteolytically cleaved by tetanus and/or botulinum neurotoxins. As a consequence of their cleavage, exocytosis of neurotransmitters is blocked. In adrenal chromaffin cells botulinum neurotoxin A only incompletely inhibits exocytosis. This incomplete inhibition of exocytosis is associated with only partial cleavage of SNAP-25 by the toxin, indicating that distinct pools of SNAP-25 may exist in chromaffin cells which differ in their sensitivities to botulinum neurotoxin A. In line with this result we localized SNAP-25 by immunogold electron microscopy not only to the plasmalemma but also to the chromaffin vesicle membrane. Moreover, in addition to SNAP-25 monomers, stable SNAP-25/syntaxin heterodimers were found in chromaffin cells. Subfractionation studies revealed the presence of SNAP-25/syntaxin heterodimers in an enriched fraction of chromaffin vesicles. This complex proved to be stable in SDS, and SNAP-25 within heterodimers was resistant to proteolytic attack by botulinum neurotoxin A. We suggest that these preexisting heterodimers may serve as receptors of soluble NSF attachment proteins (SNAP receptors) during chromaffin vesicle exocytosis.