The synaptophysin-synaptobrevin complex is developmentally upregulated in cultivated neurons but is absent in neuroendocrine cells.

Regulated secretion requires the formation of a fusion complex consisting of synaptobrevin, syntaxin and SNAP 25. One of these key proteins, synaptobrevin, also complexes with the vesicle protein synaptophysin. The fusion complex and the synaptophysin-synaptobrevin complex are mutually exclusive. Using a combination of immunoprecipitation and crosslinking experiments we report here that the synaptophysin-synaptobrevin interaction in mouse whole brain and defined brain areas is upregulated during neuronal development as previously reported for rat brain. Furthermore the synaptophysin-synaptobrevin complex is also upregulated within 10-12 days of cultivation in mouse hippocampal neurons in primary culture. Besides being constituents of small synaptic vesicles in neurons synaptophysin and synaptobrevin also occur on small synaptic vesicle analogues of neuroendocrine cells. However, the synaptophysin-synaptobrevin complex was not found in neuroendocrine cell lines and more importantly it was also absent in the adrenal gland, the adenohypophysis and the neurohypophysis although the individual proteins could be clearly detected. In the rat pheochromocytoma cell line PC 12 complex formation between synaptophysin and synaptobrevin could be initiated by adult rat brain cytosol. In conclusion, the synaptophysin-synaptobrevin complex is upregulated in neurons in primary culture but is absent in the neuroendocrine cell lines and tissues tested. The complex may provide a reserve pool of synaptobrevin during periods of high synaptic activity. Such a reserve pool probably is less important for more slowly secreting neuroendocrine cells and neurons. The synaptophysin on small synaptic vesicle analogues in these cells appears to resemble the synaptophysin of embryonic synaptic vesicles since complex formation can be induced by adult brain cytosol.     

The synaptophysin/synaptobrevin interaction critically depends on the cholesterol content

Synaptophysin interacts with synaptobrevin in membranes of adult small synaptic vesicles. The synaptophysin/synaptobrevin complex promotes synaptobrevin to built up functional SNARE complexes thereby modulating synaptic efficiency. Synaptophysin in addition is a cholesterol-binding protein. Depleting the membranous cholesterol content by filipin or beta-methylcyclodextrin (beta-MCD) decreased the solubility of synaptophysin in Triton X-100 with less effects on synaptobrevin. In small synaptic vesicles from rat brain the synaptophysin/synaptobrevin complex was diminished upon beta-MCD treatment as revealed by chemical cross-linking. Mice with a genetic mutation in the Niemann-Pick C1 gene developing a defect in cholesterol sorting showed significantly reduced amounts of the synaptophysin/synaptobrevin complex compared to their homo- or heterozygous littermates. Finally when using primary cultures of mouse hippocampus the synaptophysin/synaptobrevin complex was down-regulated after depleting the endogenous cholesterol content by the HMG-CoA-reductase inhibitor lovastatin. Alternatively, treatment with cholesterol up-regulated the synaptophysin/synaptobrevin interaction in these cultures. These data indicate that the synaptophysin/synaptobrevin interaction critically depends on a high cholesterol content in the membrane of synaptic vesicles. Variations in the availability of cholesterol may promote or impair synaptic efficiency by interfering with this complex.     

Synaptobrevin binding to synaptophysin: a potential mechanism for controlling the exocytotic fusion machine.

The synaptic vesicle protein synaptobrevin (VAMP) has recently been implicated as one of the key proteins involved in exocytotic membrane fusion. It interacts with the synaptic membrane proteins syntaxin I and synaptosome-associated protein (SNAP)-25 to form a complex which precedes exocytosis [Söllner et al. (1993b) Cell, 75, 409-418]. Here we demonstrate that the majority of synaptobrevin is bound to the vesicle protein synaptophysin in detergent extracts. No syntaxin I was found in this complex when synaptophysin-specific antibodies were used for immunoprecipitation. Conversely, no synaptophysin was associated with the synaptobrevin-syntaxin I complex when syntaxin-specific antibodies were used for immunoprecipitation. Thus, the synaptobrevin pool bound to synaptophysin is not available for binding to syntaxin I and SNAP-25, and vice versa. Synaptobrevin-synaptophysin binding was also demonstrated by chemical cross-linking in isolated nerve terminals. Furthermore, recombinant synaptobrevin II efficiently bound synaptophysin and its isoform synaptoporin, but not the more distantly related synaptic vesicle protein p29. Recombinant synaptobrevin I bound with similar efficiency, whereas the non-neuronal isoform cellubrevin displayed a lower affinity towards synaptophysin. Treatment with high NaCl concentrations resulted in a dissociation of the synaptobrevin-synaptophysin complex. In addition, the interaction of synaptobrevin with synaptophysin was irreversibly abolished by low amounts of SDS, while the interaction with syntaxin I was enhanced. We conclude that synaptophysin selectively interacts with synaptobrevin in a complex which excludes the t-SNAP receptors syntaxin I and SNAP-25, suggesting a role for synaptophysin in the control of exocytosis.     

Fluorescence resonance energy transfer detection of synaptophysin I and vesicle-associated membrane protein 2 interactions during exocytosis from single live synapses

To investigate the molecular interactions of synaptophysin I and vesicle-associated membrane protein 2 (VAMP2)/synaptobrevin II during exocytosis, we have used time-lapse videomicroscopy to measure fluorescence resonance energy transfer in live neurons. For this purpose, fluorescent protein variants fused to synaptophysin I or VAMP2 were expressed in rat hippocampal neurons. We show that synaptophysin I and VAMP2 form both homo- and hetero-oligomers on the synaptic vesicle membrane. When exocytosis is stimulated with alpha-latrotoxin, VAMP2 dissociates from synaptophysin I even in the absence of appreciable exocytosis, whereas synaptophysin I oligomers disassemble only upon incorporation of the vesicle with the plasma membrane. We propose that synaptophysin I has multiple roles in neurotransmitter release, regulating VAMP2 availability for the soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex and possibly participating in the late steps of exocytosis.     

Mice lacking synaptophysin reproduce and form typical synaptic vesicles

Synaptophysin is one of the major integral membrane proteins of the small (30–50 nm diameter) electron-translucent transmitter-containing vesicles in neurons and of similar vesicles in neuroendocrine cells. Since its expression is tightly linked to the occurrence of these vesicle types, we mutated the X-chromosomally located synaptophysin gene in embryonic stem cells for the generation of synaptophysin-deficient mice in order to study the consequence of synaptophysin ablation for the formation and function of such vesicles in vivo. the behavior and appearance of mice lacking synaptophysin was indistinguishable from that of their litter mates and reproductive capacity was comparable to normal mice. Furthermore, no drastic compensatory changes were noted in the expression of several other neuronal polypeptides or in the mRNA levels of synaptophysin isoforms, the closely related neuronal synaptoporin/synaptophysinII, and the ubiquitous pantophysin. Immunofluorescence microscopy of several neuronal and neuroendocrine tissues showed that overall tissue architecture was maintained in the absence of synaptophysin, and that the distribution of other synaptic vesicle components was not visibly affected. In electron-microscopic preparations, large numbers of vesicles with a diameter of 39.9 nm and an electron-translucent interior were seen in synaptic regions of synaptophysin-deficient mice; these vesicles could be labeled by antibodies against synaptic vesicle proteins, such as synaptobrevin 2.This research was supported by the DFG-SFB 317     

Synaptophysin binds to physophilin, a putative synaptic plasma membrane protein

We have developed procedures for detecting synaptic vesicle-binding proteins by using glutaraldehyde-fixed or native vesicle fractions as absorbent matrices. Both adsorbents identify a prominent synaptic vesicle-binding protein of 36 kD in rat brain synaptosomes and mouse brain primary cultures. The binding of this protein to synaptic vesicles is competed by synaptophysin, a major integral membrane protein of synaptic vesicles, with half-maximal inhibition seen between 10(-8) and 10(-7) M synaptophysin. Because of its affinity for synaptophysin, we named the 36-kD synaptic vesicle-binding protein physophilin (psi nu sigma alpha, greek = bubble, vesicle; psi iota lambda os, greek = friend). Physophilin exhibits an isoelectric point of approximately 7.8, a Stokes radius of 6.6 nm, and an apparent sedimentation coefficient of 5.6 S, pointing to an oligomeric structure of this protein. It is present in synaptic plasma membranes prepared from synaptosomes but not in synaptic vesicles. In solubilization experiments, physophilin behaves as an integral membrane protein. Thus, a putative synaptic plasma membrane protein exhibits a specific interaction with one of the major membrane proteins of synaptic vesicles. This interaction may play a role in docking and/or fusion of synaptic vesicles to the presynaptic plasma membrane.     

The expression pattern and assembly profile of synaptic membrane proteins in ribbon synapses of the developing mouse retina

In the present study, we generated a systematic overview of the expression pattern and assembly profile of synaptic membrane proteins in ribbon synapses of the developing mouse retina. Using indirect immunofluorescence microscopy, we analyzed the spatial and temporal distribution of 11 important membrane and membrane-associated synaptic proteins (syntaxin 1/3, SNAP-25, synaptobrevin 2, synaptogyrin, synaptotagmin I, SV2A, SV2B, Rab3A, clathrin light chains, CSP and neuroligin I) during synaptogenesis. The temporospatial distribution of these synaptic proteins was "normalized" by the simultaneous visualization of the synaptic vesicle protein synaptophysin, which served as an internal reference protein. We found that expression of various synaptic membrane proteins started at different time points and changed progressively during development. At early stages of development synaptic vesicle membrane proteins at extrasynaptic locations did not always colocalize with synaptophysin, indicating that these proteins probably do not reside in the same transport vesicles. Despite a non-synchronized onset of protein expression, clustering and colocalization of all synaptic membrane proteins at ribbon synapses roughly occurred in the same time window (between day 4 after birth, P4, and P5). Thus, the basic synaptic membrane machinery is already present in ribbon synapses before the well-known complete morphological maturation of ribbon synapses between P7 and P12. We conclude that ribbon synapse formation is a multistep process in which the concerted recruitment of synaptic membrane proteins is a relatively early event and clearly not the final step.     

Activity-dependent changes of the presynaptic synaptophysin-synaptobrevin complex in adult rat brain

The vesicular protein synaptobrevin contributes to two mutually exclusive complexes in mature synapses. Synaptobrevin tightly interacts with the plasma membrane proteins syntaxin and SNAP 25 forming the SNARE complex as a prerequisite for exocytotic membrane fusion. Alternatively, synaptobrevin binds to the vesicular protein synaptophysin. It is unclear whether SNARE complex formation is diminished or facilitated when synaptobrevin is bound to synaptophysin. Here we show that the synaptophysin-synaptobrevin complex is increased in adult rat brain after repeated synaptic hyperactivity in the kindling model of epilepsy. Two days after the last kindling-induced stage V seizure the relative amount of synaptophysin-synaptobrevin complex obtained by co-immunoprecipitation from cortical and hippocampal membranes was increased twofold compared to controls. By contrast the relative amounts of various synaptic proteins as well as that of the SNARE complex did not change in membrane preparations from kindled rats compared to controls. The increased amount of synaptophysin-synaptobrevin complex in kindled rats supports the idea that this complex represents a reserve pool for synaptobrevin enabling synaptic vesicles to adjust to an increased demand for synaptic efficiency. We conclude that the synaptophysin-synaptobrevin interaction is involved in activity-dependent plastic changes in adult rat brain.     

Fusion of synaptic vesicles and plasma membrane in the presence of synaptosomal soluble proteins

Fusion between synaptic vesicles and plasma membranes isolated from rat brain synaptosomes is regarded as a model of neurosecretion. The main aim of current study is to investigate whether the synaptosomal soluble proteins are essential members of Ca(2+)-triggered fusion examined in this system. Fusion experiments were performed using fluorescent dye octadecylrhodamine B, which was incorporated into synaptic vesicle membranes at self-quenching concentration. The fusion of synaptic vesicles, containing marker octadecylrhodamine B, with plasma membranes was detected by dequenching of the probe fluorescence. Membrane fusion was not found in Ca(2+)-supplemented buffer solution, but was initiated by the addition of the synaptosomal soluble proteins. When soluble proteins were treated with trypsin, they lost completely the fusion activity. These experiments confirmed that soluble proteins of synaptosomes are sensitive to Ca(2+) signal and essential for membrane fusion. The experiments, in which members of fusion process were treated with monoclonal antibodies raised against synaptotagmin and synaptobrevin, have shown that antibodies only partially inhibited fusion of synaptic vesicles and plasma membranes in vitro. These results indicate that other additional component(s), which may or may not be related to synaptobrevin or synaptotagmin, mediate this process. It can be assumed that fusion of synaptic vesicles with plasma membranes in vitro depends upon the complex interaction of a large number of protein factors.     

Clathrin-coated vesicles in nervous tissue are involved primarily in synaptic vesicle recycling

The recycling of synaptic vesicles in nerve terminals is thought to involve clathrin-coated vesicles. However, the properties of nerve terminal coated vesicles have not been characterized. Starting from a preparation of purified nerve terminals obtained from rat brain, we isolated clathrin-coated vesicles by a series of differential and density gradient centrifugation steps. The enrichment of coated vesicles during fractionation was monitored by EM. The final fraction consisted of greater than 90% of coated vesicles, with only negligible contamination by synaptic vesicles. Control experiments revealed that the contribution by coated vesicles derived from the axo-dendritic region or from nonneuronal cells is minimal. The membrane composition of nerve terminal-derived coated vesicles was very similar to that of synaptic vesicles, containing the membrane proteins synaptophysin, synaptotagmin, p29, synaptobrevin and the 116-kD subunit of the vacuolar proton pump, in similar stoichiometric ratios. The small GTP-binding protein rab3A was absent, probably reflecting its dissociation from synaptic vesicles during endocytosis. Immunogold EM revealed that virtually all coated vesicles carried synaptic vesicle proteins, demonstrating that the contribution by coated vesicles derived from other membrane traffic pathways is negligible. Coated vesicles isolated from the whole brain exhibited a similar composition, most of them carrying synaptic vesicle proteins. This indicates that in nervous tissue, coated vesicles function predominantly in the synaptic vesicle pathway. Nerve terminal-derived coated vesicles contained AP-2 adaptor complexes, which is in agreement with their plasmalemmal origin. Furthermore, the neuron-specific coat proteins AP 180 and auxilin, as well as the alpha a1 and alpha c1-adaptins, were enriched in this fraction, suggesting a function for these coat proteins in synaptic vesicle recycling.     

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

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

The synaptophysin/synaptobrevin complex dissociates independently of neuroexocytosis

Synaptophysin is one of the most abundant membrane proteins of small synaptic vesicles. In mature nerve terminals it forms a complex with the vesicular membrane protein synaptobrevin, which appears to modulate synaptobrevin's interaction with the plasma membrane-associated proteins syntaxin and SNAP25 to form the SNARE complex as a prerequisite for membrane fusion. Here we show that synaptobrevin is preferentially cleaved by tetanus toxin while bound to synaptophysin or when existing as a homodimer. The synaptophysin/synaptobrevin complex is, however, not affected when neuronal secretion is blocked by botulinum A toxin which cleaves SNAP25. Excessive stimulation with alpha-latrotoxin or Ca(2+)-ionophores dissociates the synaptophysin/synaptobrevin complex and increases the interaction of the other SNARE proteins. The stimulation-induced dissociation of the synaptophysin/synaptobrevin complex is not inhibited by pre-incubating neurones with botulinum A toxin, but depends on extracellular calcium. However, the synaptophysin/synaptobrevin complex cannot be directly dissociated by calcium alone or in combination with magnesium. The dissociation of synaptobrevin from synaptophysin appears to precede its interaction with the other SNARE proteins and does not depend on the final fusion event. This finding further supports the modulatory role the synaptophysin/synaptobrevin complex may play in mature neurones.     

Biogenesis of synaptic vesicles in vitro

Synaptic vesicles are synthesized at a rapid rate in nerve terminals to compensate for their rapid loss during neurotransmitter release. Their biogenesis involves endocytosis of synaptic vesicle membrane proteins from the plasma membrane and requires two steps, the segregation of synaptic vesicle membrane proteins from other cellular proteins, and the packaging of those unique proteins into vesicles of the correct size. By labeling an epitope-tagged variant of a synaptic vesicle protein, VAMP (synaptobrevin), at the cell surface of the neuroendocrine cell line PC12, synaptic vesicle biogenesis could be followed with considerable precision, quantitatively and kinetically. Epitope-tagged VAMP was recovered in synaptic vesicles within a few minutes of leaving the cell surface. More efficient targeting was obtained by using the VAMP mutant, del 61-70. Synaptic vesicles did not form at 15 degrees C although endocytosis still occurred. Synaptic vesicles could be generated in vitro from a homogenate of cells labeled at 15 degrees C. The newly formed vesicles are identical to those formed in vivo in their sedimentation characteristics, the presence of the synaptic vesicle protein synaptophysin, and the absence of detectable transferrin receptor. Brain, but not fibroblast cytosol, allows vesicles of the correct size to form. Vesicle formation is time and temperature-dependent, requires ATP, is calcium independent, and is inhibited by GTP-gamma S. Thus, two key steps in synaptic vesicle biogenesis have been reconstituted in vitro, allowing direct analysis of the proteins involved.     

Synaptic vesicle proteins and early endosomes in cultured hippocampal neurons: differential effects of Brefeldin A in axon and dendrites

The pathways of synaptic vesicle (SV) biogenesis and recycling are still poorly understood. We have studied the effects of Brefeldin A (BFA) on the distribution of several SV membrane proteins (synaptophysin, synaptotagmin, synaptobrevin, p29, SV2 and rab3A) and on endosomal markers to investigate the relationship between SVs and the membranes with which they interact in cultured hippocampal neurons developing in isolation. In these neurons, SV proteins are detected as punctate immunoreactivity that is concentrated in axons but is also present in perikarya and dendrites. In the same neurons, the transferrin receptor, a well established marker of early endosomes, is selectively concentrated in perikarya and dendrites. In the perikaryal- dendritic region, BFA induced a dramatic tubulation of transferrin receptors as well as a cotubulation of the bulk of synaptophysin. Synaptotagmin, synaptobrevin, p29 and SV2 immunoreactivities retained a primarily punctate distribution. No tubulation of rab3A was observed. In axons, BFA did not produce any obvious alteration of the distribution of SV proteins, nor of peroxidase- or Lucifer yellow- labeled early endosomes. The selective effect of BFA on dendritic membranes suggests the existence of functional differences between the endocytic systems in dendrites and axons. Cotubulation of transferrin receptors and synaptophysin in the perikaryal-dendritic region is consistent with a functional interconnection between the traffic of SV proteins and early endosomes. The heterogeneous effects of BFA on SV proteins in this cell region indicates that SV proteins are differentially sorted upon exit from the TGN and are coassembled into SVs at the cell periphery.     

The C-terminal transmembrane region of synaptobrevin binds synaptophysin from adult synaptic vesicles

Synaptophysin and synaptobrevin are abundant membrane proteins of neuronal small synaptic vesicles. In mature, differentiated neurons they form the synaptophysin/synaptobrevin (Syp/Syb) complex. Synaptobrevin also interacts with the plasma membrane-associated proteins syntaxin and SNAP25, thereby forming the SNARE complex necessary for exocytotic membrane fusion. The two complexes are mutually exclusive. Synaptobrevin is a C-terminally membrane-anchored protein with one transmembrane domain. While its interaction with its SNARE partners is mediated exclusively by its N-terminal cytosolic region it has been unclear so far how binding to synaptophysin is accomplished. Here, we show that synaptobrevin can be cleaved in its synaptophysin-bound form by tetanus toxin and botulinum neurotoxin B, or by botulinum neurotoxin D, leaving shorter or longer C-terminal peptide chains bound to synaptophysin, respectively. A recombinant, C-terminally His-tagged synaptobrevin fragment bound to nickel beads specifically bound synaptophysin, syntaxin and SNAP25 from vesicular detergent extracts. After cleavage by tetanus toxin or botulinum toxin D light chain, the remaining C-terminal fragment no longer interacted with syntaxin or SNAP 25. In contrast, synaptophysin was still able to bind to the residual C-terminal synaptobrevin cleavage product. In addition, the His-tagged C-terminal synaptobrevin peptide 68-116 was also able to bind synaptophysin in detergent extracts from adult brain membranes. These data suggest that synaptophysin interacts with the C-terminal transmembrane part of synaptobrevin, thereby allowing the N-terminal cytosolic chain to interact freely with the plasma membrane-associated SNARE proteins. Thus, by binding synaptobrevin, synaptophysin may positively modulate neurotransmission.     

Biogenesis of synaptic vesicle-like structures in a pheochromocytoma cell line PC-12

The presence of unique proteins in synaptic vesicles of neurons suggests selective targeting during vesicle formation. Endocrine, but not other cells, also express synaptic vesicle membrane proteins and target them selectively to small intracellular vesicles. We show that the rat pheochromocytoma cell line, PC12, has a population of small vesicles with sedimentation and density properties very similar to those of rat brain synaptic vesicles. When synaptophysin is expressed in nonneuronal cells, it is found in intracellular organelles that are not the size of synaptic vesicles. The major protein in the small vesicles isolated from PC12 cells is found to be synaptophysin, which is also the major protein in rat brain vesicles. At least two of the minor proteins in the small vesicles are also known synaptic vesicle membrane proteins. Synaptic vesicle-like structures in PC12 cells can be shown to take up an exogenous bulk phase marker, HRP. Their proteins, including synaptophysin, are labeled if the cells are surface labeled and subsequently warmed. Although the PC12 vesicles can arise by endocytosis, they seem to exclude the receptor-mediated endocytosis marker, transferrin. We conclude that PC12 cells contain synaptic vesicle-like structures that resemble authentic synaptic vesicles in physical properties, protein composition and endocytotic origin.     

Synaptophysin I selectively specifies the exocytic pathway of synaptobrevin 2/VAMP2

Biogenesis and recycling of synaptic vesicles are accompanied by sorting processes that preserve the molecular composition of the compartments involved. In the present study, we have addressed the targeting of synaptobrevin 2/VAMP2 (vesicle-associated membrane protein 2), a critical component of the synaptic vesicle--fusion machinery, in a heterotypic context where its sorting is not confounded by the presence of other neuron-specific molecules. Ectopically expressed synaptophysin I interacts with VAMP2 and alters its default surface targeting to a prominent vesicular distribution, with no effect on the targeting of other membrane proteins. Protein-protein interaction is not sufficient for the control of VAMP2 sorting, which is mediated by the C-terminal domain of synaptophysin I. Synaptophysin I directs the sorting of VAMP2 to vesicles before surface delivery, without influencing VAMP2 endocytosis. Consistent with this, dynamin and alpha-SNAP (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein) mutants which block trafficking at the plasma membrane do not abrogate the effect of synaptophysin I on VAMP2 sorting. These results indicate that the sorting determinants of synaptic vesicle proteins can operate independently of a neuronal context and implicate the association of VAMP2 with synaptophysin I in the specification of the pathway of synaptic vesicle biogenesis.     

Synaptobrevin transmembrane domain dimerization-revisited

Synaptobrevin is a membrane-spanning soluble N-ethyl maleimid-sensitive factor (NSF) attachment protein receptor (SNARE) protein of synaptic vesicles that is essential for neurotransmitter release. Various lines of evidence indicate that it exists alternatively as a monomer, as a homodimer, as a heterodimer with synaptophysin, or as a ternary complex with other SNAREs at the various stages of the synaptic vesicle cycle. Homodimerization of synaptobrevin was previously shown by different authors to depend on its single transmembrane segment, and the crucial residues forming the helix-helix interface have been mapped. Since another recent study challenged these results, we reinvestigated this issue. Here, we show that native synaptobrevin can be cross-linked in synaptic vesicle membranes to a homodimer by disulfide bond formation between cysteine residues of the transmembrane segment. Further, we demonstrate that determination of synaptobrevin transmembrane segment interactions in membranes or in detergent solution requires careful control of experimental conditions. Thus, our present results corroborate that homodimerization of synaptobrevin is mediated by its transmembrane segment.     

Targeting of the Synaptic Vesicle Protein Synaptobrevin in the Axon of Cultured Hippocampal Neurons: Evidence for Two Distinct Sorting Steps

Synaptic vesicles are concentrated in the distal axon, far from the site of protein synthesis. Integral membrane proteins destined for this organelle must therefore make complex targeting decisions. Short amino acid sequences have been shown to act as targeting signals directing proteins to a variety of intracellular locations. To identify synaptic vesicle targeting sequences and to follow the path that proteins travel en route to the synaptic vesicle, we have used a defective herpes virus amplicon expression system to study the targeting of a synaptobrevin-transferrin receptor (SB-TfR) chimera in cultured hippocampal neurons. Addition of the cytoplasmic domain of synaptobrevin onto human transferrin receptor was sufficient to retarget the transferrin receptor from the dendrites to presynaptic sites in the axon. At the synapse, the SB-TfR chimera did not localize to synaptic vesicles, but was instead found in an organelle with biochemical and functional characteristics of an endosome. The chimera recycled in parallel with synaptic vesicle proteins demonstrating that the nerve terminal efficiently sorts transmembrane proteins into different pathways. The synaptobrevin sequence that controls targeting to the presynaptic endosome was not localized to a single, 10– amino acid region of the molecule, indicating that this targeting signal may be encoded by a more distributed structural conformation. However, the chimera could be shifted to synaptic vesicles by deletion of amino acids 61–70 in synaptobrevin, suggesting that separate signals encode the localization of synaptobrevin to the synapse and to the synaptic vesicle.