Syntaxin 1A

Protein Interactions (part 1/3)

Synaptotagmin: a Syntaxin binding detector of cellular Ca2+ levels

Synaptotagmin is a synaptic vesicle specific protein that binds calcium and phospholipids in vitro and is required for calcium-regulated fusion of synaptic vesicles with the presynaptic membrane. The possible requirement for synaptotagmin in axonal outgrowth has been examined by following neuronal development in Drosophila embryos deficient for the synaptotagmin gene. In wild-type embryos, synaptotagmin is expressed abundantly in axons and growth cones before synapse formation. Using antibodies to the intravesicular domain of synaptotagmin to label live embryos, it has been demonstrated that vesicle populations containing Synaptotagmin actively undergo exocytosis during axonogenesis. In synaptotagmin null mutations, immunocytochemical techniques were used to examine the distribution of the axonal protein Fasciclin II, the presynaptic membrane protein Syntaxin, and the synaptic vesicle protein Cysteine string protein. The distribution of these proteins is similar in wild-type and synaptotagmin mutant embryos, suggesting that synaptotagmin is not required for axonogenesis in the CNS or PNS. Based on these findings, it is suggested that the molecular mechanisms underlying vesicular-mediated membrane expansion during axonal outgrowth are distinct from those required for synaptic vesicle fusion during neurotransmitter release (Littleton, 1995).

Synaptotagmin serves as the major Ca2+ sensor for regulated exocytosis from neurons. While the mechanism by which synaptotagmin regulates membrane fusion remains unknown, studies using Drosophila indicate that the molecule functions as a multimeric complex and that its second C2 domain is essential for efficient excitation-secretion coupling. Biochemical data is described that may account for these phenomena. Ca2+ causes synaptotagmin to oligomerize, primarily forming dimers, via its second C2 domain. This effect is specific for divalent cations that can stimulate exocytosis of synaptic vesicles (Ca2+ >> Ba2+, Sr2+ >> Mg2+) and occurs with an EC50 value of 3-10 microM Ca2+. In contrast, a separate Ca2+-dependent interaction between synaptotagmin and syntaxin, a component of the fusion apparatus, occurs with an EC50 value of approximately 100 microM Ca2+ and involves the synergistic action of both C2 domains of synaptotagmin. It is proposed that Ca2+ triggers two consecutive protein-protein interactions: the formation of synaptotagmin dimers at low Ca2+ concentrations followed by the association of synaptotagmin dimers with syntaxin at higher Ca2+-concentrations. These findings, in conjunction with physiological studies, indicate that the Ca2+-induced dimerization of synaptotagmin is important for the efficient regulation of exocytosis by Ca2+ (Chapman, 1996).

Since the demonstration that Ca2+ influx into the presynaptic terminal is essential for neurotransmitter release, there has been much speculation about the Ca2+ receptor responsible for initiating exocytosis. Numerous experiments have shown that the protein, or protein complex, binds multiple Ca2+ ions, resides near the site of Ca2+ influx, and has a relatively low affinity for Ca2+. Synaptotagmin is an integral membrane protein of synaptic vesicles that contains two copies of a domain known to be involved in Ca(2+)-dependent membrane interactions. Synaptotagmin has been shown to bind Ca2+ in vitro with a relatively low affinity. In addition, Synaptotagmin has been shown to bind indirectly to Ca2+ channels, positioning the protein close to the site of Ca2+ influx. Recently, a negative regulatory role for Synaptotagmin has been proposed, in which it functions as a clamp to prevent fusion of synaptic vesicles with the presynaptic membrane. Release of the clamp would allow exocytosis. Genetic and electrophysiological evidence is presented that Synaptotagmin forms a multimeric complex that can function as a clamp in vivo. However, upon nerve stimulation and Ca2+ influx, all synaptotagmin mutations dramatically decrease the ability of Ca2+ to promote release, suggesting that Synaptotagmin probably plays a key role in activation of synaptic vesicle fusion. This activity cannot simply be attributed to the removal of a barrier to secretion, as the increase in rate of spontaneous vesicle fusion can be electrophysiologically separated from the decrease in evoked response. Some syt mutations, including those that lack the second Ca(2+)-binding domain, decrease the fourth-order dependence of release on Ca2+ by approximately half, consistent with the hypothesis that a Synaptotagmin complex functions as a Ca2+ receptor for initiating exocytosis (Littleton, 1998).

Nerve terminal specializations include mechanisms for maintaining a subpopulation of vesicles in a docked, fusion-ready state. The relationship between synaptotagmin and the number of morphologically docked vesicles has been investigated by an electron microscopic analysis of Drosophila synaptotagmin (syt) mutants. The overall number of synaptic vesicles in a terminal is reduced, although each active zone continues to have a cluster of vesicles in its vicinity. In addition, there is an increase in the number of large vesicles near synapses. Examining the clusters, it was found that the pool of synaptic vesicles immediately adjacent to the presynaptic membrane, the pool that includes the docked population, is reduced to 24% of control in the syt null mutation. To separate contributions of overall vesicle depletion and increased spontaneous release from direct effects of synaptotagmin on morphological docking, syt mutants were examined in an altered genetic background. Recombining syt alleles onto a second chromosome bearing an as yet uncharacterized mutation results in the expected decrease in evoked release but suppresses the increase in spontaneous release frequency. Motor nerve terminals in this genotype contain more synaptic vesicles than control, yet the number of vesicles immediately adjacent to the presynaptic membrane near active zones is still reduced (33% of control). These findings demonstrate that there is a decrease in the number of morphologically docked vesicles seen in syt mutants. The decreases in docking and evoked release are independent of the known increase in spontaneous release that occurs in syt mutants. These results support the hypothesis that synaptotagmin stabilizes the docked state (Reist, 1998).

The Drosophila stoned locus encodes two novel gene products termed stonedA and stonedB, which possess sequence motifs shared by proteins involved in intracellular vesicle traffic. A specific requirement for stoned in the synaptic vesicle cycle has been suggested by synthetic genetic interactions between stoned and shibire, a gene essential for synaptic vesicle recycling. A synaptic role for stoned gene products is also suggested by altered synaptic transients in electroretinograms recorded from stoned mutant eyes. The StonedA protein is highly enriched at Drosophila nerve terminals. Mutant alleles that affect stonedA disrupt the normal regulation of synaptic vesicle exocytosis at neuromuscular synapses in Drosophila. Spontaneous neurotransmitter release is enhanced dramatically, and evoked release is reduced substantially in such stoned mutants. Ultrastructural studies reveal no evidence of major disorganization at stoned mutant nerve terminals. Thus, these data indicate a direct role for stonedA in regulating synaptic vesicle exocytosis. However, genetic and morphological observations suggest additional, subtle effects of stoned mutations on synaptic vesicle recycling. Remarkably, almost all phenotypes for stoned mutants are similar to those for mutants of synaptotagmin, a protein postulated to regulate both exocytosis and the recycling of synaptic vesicles. A model is proposed in which stonedA functions together with synaptotagmin to regulate synaptic vesicle cycling (Stimson, 1998).

StonedA is not the first protein for which dual roles in synaptic vesicle exocytosis and endocytosis have been proposed. Both biochemical and genetic studies suggest that, in addition to its role in regulating synaptic vesicle fusion, synaptotagmin may regulate synaptic vesicle recycling. Although the possible role of synaptotagmin in synaptic vesicle recycling has not been addressed in studies of Drosophila synaptotagmin mutants, Caenorhabditis elegans synaptotagmin (snt-1) mutants exhibit phenotypes consistent with impaired synaptic vesicle recycling. For example, the synaptic vesicle protein synaptobrevin exhibits an abnormally diffuse distribution in the snt-1 nerve cord, suggesting an accumulation and lateral spreading of this protein within neuronal plasma membrane. This altered distribution of synaptobrevin in snt-1 nerve cord is qualitatively similar to the altered distributions of synaptotagmin and csp in stoned boutons. Thus, parallels between stoned mutants and synaptotagmin mutants suggest that stonedA and synaptotagmin may share functions in synaptic vesicle recycling as well as in synaptic vesicle fusion. If stonedA does in fact regulate general synaptic vesicle endocytosis, could defects in transmitter release at stonedts2 and stnc synapses be secondary to a primary defect in synaptic vesicle recycling? This is thought to be unlikely for the following reasons: (1) EM studies show that any recycling defects at stnts2 and stnc mutant synapses must be very subtle; (2) partial depletion of synaptic vesicles, which may be achieved by stimulating shabirets1 mutants at nonpermissive temperature, does not result in the specific phenotypes observed at stoned mutant synapses. The elevated mini frequency and the reduced evoked release are unique phenotypes of stoned mutants and thus probably reflect a specific function of StonedA in regulating Ca2+-dependent neurotransmitter release. The simplest interpretation of the data is that stonedts2 and stonednc mutants have independent defects in synaptic vesicle fusion and synaptic vesicle recycling (Stimson, 1998 and references).

Synaptotagmin (p65) is an abundant synaptic vesicle protein that contains two copies of a sequence that is homologous to the regulatory region of protein kinase C. Full length cDNAs encoding human and Drosophila synaptotagmins were characterized to study its structural and functional conservation in evolution. The deduced amino acid sequences for human and rat synaptotagmins show 97% identity, whereas Drosophila and rat synaptotagmins are only 57% identical but exhibit a selective conservation of the two internal repeats that are homologous to the regulatory region of protein kinase C (78% invariant residues in all three species). The two internal repeats of synaptotagmin are only slightly more homologous to each other than to protein kinase C, and the differences between the repeats are conserved in evolution, suggesting that they might not be functionally equivalent. The cytoplasmic domains of human and Drosophila synaptotagmins produced as recombinant proteins in Escherichia coli specifically bind phosphatidylserine similar to rat synaptotagmin. They also hemagglutinate trypsinized erythrocytes at nanomolar concentrations. Hemagglutination is inhibited both by negatively charged phospholipids and by a recombinant fragment from rat synaptotagmin that contains only a single copy of the two internal repeats. Together these results demonstrate that synaptotagmin is highly conserved in evolution compatible with a function in the trafficking of synaptic vesicles at the active zone. The similarity of the phospholipid binding properties of the cytoplasmic domains of rat, human, and Drosophila synaptotagmins and the selective conservation of the sequences that are homologous to protein kinase C suggest that these are instrumental in phospholipid binding. The human gene for synaptotagmin was mapped by Southern blot analysis of DNA from somatic cell hybrids to chromosome 12 region cen-q21, and the Drosophila gene by in situ hybridization to 23B (Perin, 1991).

Synaptotagmin is a synaptic vesicle-specific integral membrane protein that has been suggested to play a key role in synaptic vesicle docking and fusion. By monitoring Synaptotagmin's cellular and subcellular distribution during development, it is possible to study synaptic vesicle localization and transport, and synapse formation. The study of Synaptotagmin's expression during Drosophila neurogenesis has been initiated in order to follow synaptic vesicle movement prior to and during synapse formation, as well as to localize synaptic sites in Drosophila. In situ hybridizations to whole-mount embryos show that synaptotagmin message is present in the cell bodies of all peripheral nervous system neurons, in mature neurons, and in many, if not all, central nervous system neurons during neurite outgrowth and synapse formation.. Immunocytochemical staining with antisera specific to Synaptotagmin indicates that the protein is present at all stages of the Drosophila life cycle following germ band retraction. In embryos, Synaptotagmin is only transiently localized to the cell body of neurons and is transported rapidly along axons during axonogenesis. After synapse formation, Synaptotagmin accumulates in a punctate pattern at all identifiable synaptic contact sites, suggesting a general role for Synaptotagmin in synapse function. In embryos and larvae, the most intense staining is found along two broad longitudinal tracts on the dorsal side of the ventral nerve cord and the brain, and at neuromuscular junctions in the periphery. In the adult head, Synaptotagmin localizes the discrete regions of the neuropil where synapses are predicted to occur. These data indicate that synaptic vesicles are present in axons before synapse formation, and become restricted to synaptic contact sites after synapses are formed. Since a similar expression pattern of Synaptotagmin has been reported in mammals, it is proposed that the function of Synaptotagmin and the mechanisms governing localization of the synaptic vesicle before and after synapse formation are conserved in invertebrate and vertebrate species. The ability to mark synapses in Drosophila should facilitate the study of synapse formation and function, providing a new tool to dissect the molecular mechanisms underlying these processes (Littleton, 1993a).

Synaptotagmin (Syt), a synaptic vesicle-specific protein known to bind Ca2+ in the presence of phospholipids, has been proposed to mediate Ca(2+)-dependent neurotransmitter release. The role of Syt in neurotransmitter release in vivo has beed addressed by generating mutations in synaptotagmin in the fruitfly and assaying the subsequent effects on neurotransmission. Most embryos that lack syt fail to hatch and exhibit very reduced, uncoordinated muscle contractions. Larvae with partial lack-of-function mutations show almost no evoked excitatory junctional potentials (EJPs) in 0.4 mM Ca2+ and a 15-fold reduction in EJP amplitude in 1.0 mM Ca2+ when compared with heterozygous controls. In contrast, an increase in the frequency of spontaneous miniature EJPs is observed in the mutants. These results provide in vivo evidence that Syt plays a key role in Ca2+ activation of neurotransmitter release and indicate the existence of separate pathways for evoked and spontaneous neurotransmitter release (Littleton, 1993b).

Synaptotagmin is one of the major integral membrane proteins of synaptic vesicles. It has been postulated to dock vesicles to their release sites, to act as the Ca2+ sensor for the release process, and to be a fusion protein during exocytosis. To clarify the function of this protein, a genetic analysis of the synaptotagmin gene in Drosophila was undertaken. Five lethal alleles of synaptotagmin were identified, at least one of which lacks detectable protein. Surprisingly, however, many embryos homozygous for this null allele hatch and, as larvae, crawl, feed, and respond to stimuli. Electrophysiological recordings in embryonic cultures confirm that synaptic transmission persists in the null allele. Therefore, synaptotagmin is not absolutely required for the regulated exocytosis of synaptic vesicles. The lethality of synaptotagmin in late first instar larvae is probably due to a perturbation of transmission that leaves the main apparatus for vesicle docking and fusion intact (DiAntonio, 1993a).

Synaptotagmin is a synaptic vesicle protein implicated in neurotransmitter release. Molecular characterization of four mutant alleles of this protein in Drosophila has permitted an investigation of Synaptotagmin's role in synaptic physiology and of some of the structural requirements for its function. Reduced levels of Synaptotagmin resulted in a substantial alteration in synaptic function in the eye and at larval neuromuscular junctions. Decreased neurotransmitter release cause smaller evoked synaptic potentials. However, the frequency, but not the size, of spontaneous quantal events is simultaneously increased. These abnormalities do not appear to be secondary to a detectable morphological change in the arborization of the synapse. The increased frequency of spontaneous events is insufficient to deplete significantly the vesicle supply and thereby account for reduced transmission (DiAntonio, 1994).

Synaptotagmin is an integral synaptic vesicle protein proposed to be involved in Ca(2+)-dependent exocytosis during synaptic transmission. Null mutations in synaptotagmin have been made in Drosophila, and the protein's in vivo function has been assayed at the neuromuscular synapse. In the absence of synaptotagmin, synaptic transmission is dramatically impaired but is not abolished. In null mutants, evoked vesicle release is decreased by a factor of 10. Moreover, the fidelity of excitation-secretion coupling is impaired so that a given stimulus generates a more variable amount of secretion. However, this residual evoked release shows Ca(2+)-dependence similar to normal release, suggesting either that Synaptotagmin is not the Ca2+ sensor or that a second, independent Ca2+ sensor exists. While evoked transmission is suppressed, the rate of spontaneous vesicle fusion is increased by a factor of 5. It is concluded that Synaptotagmin is not an absolutely essential component of the Ca(2+)-dependent secretion pathway in synaptic transmission but is necessary for normal levels of transmission. These data support a model in which Synaptotagmin functions as a negative regulator of spontaneous vesicle fusion and acts to increase the efficiency of excitation-secretion coupling during synaptic transmission (Broadie, 1994).

The stoned locus of Drosophila encodes two novel proteins, stonedA (STNA) and stonedB (STNB), both of which are expressed in the nervous system. Flies with defects at the stoned locus have abnormal behavior and altered synaptic transmission. Genetic interactions, in particular with the shibire (dynamin) mutation, indicated a presynaptic function for stoned and suggested an involvement in vesicle cycling. Immunological studies revealed colocalization of the Stoned proteins at the neuromuscular junction with the integral synaptic vesicle protein Synaptotagmin (SYT). stoned interacts genetically with synaptotagmin to produce a lethal phenotype. The STNB protein is found by co-immunoprecipitation to be associated with synaptic vesicles, and glutathione S-transferase pull-downs demonstrate an in vitro interaction between the µ2-homology domain of STNB and the C2B domain of the SYTI isoform. The STNA protein is also found in association with vesicles, and it too exhibits an in vitro association with SYTI. However, the bulk of STNA is in a nonmembranous fraction. By using the shibire mutant to block endocytosis, STNB has been shown to be present on some synaptic vesicles before exocytosis. However, STNB is not associated with all synaptic vesicles. It is hypothesized that STNB specifies a subset of synaptic vesicles with a role in the synaptic vesicle cycle that is yet to be determined (Phillips, 2000).

The Drosophila dicistronic stoned locus encodes two distinctive presynaptic proteins, Stoned A (StnA) and Stoned B (StnB); StnA is a novel protein without homology to known synaptic proteins, and StnB contains a domain with homology to the endocytotic protein AP50. Both Stoned proteins colocalize precisely with endocytotic proteins including the clathrin-associated coated pit adaptor protein complex AP2 and Dynamin in the 'lattice network' characteristic of endocytotic domains in Drosophila presynaptic terminals. FM1-43 dye uptake studies in stoned mutants demonstrate a striking decrease in the size of the endo-exo-cycling synaptic vesicle pool and loss of spatial regulation of the vesicular recycling intermediates. Mutant synapses display a significant delay in vesicular membrane retrieval after depolarization and neurotransmitter release. These studies suggest that the Stoned proteins play a role in mediating synaptic vesicle endocytosis. A highly specific synaptic mislocalization and degradation of Synaptotagmin I has been documented in stoned mutants. Transgenic overexpression of Synaptotagmin I rescues stoned embryonic lethality and restores endocytotic recycling to normal levels. Furthermore, overexpression of Synaptotagmin I in otherwise wild-type animals results in increased synaptic dye uptake, indicating that Synaptotagmin I directly regulates the endo-exo-cycling synaptic vesicle pool size. In parallel with recent biochemical studies, this genetic analysis strongly suggests that Stoned proteins regulate the AP2-Synaptotagmin I interaction during synaptic vesicle endocytosis. It is concluded that Stoned proteins control synaptic transmission strength by mediating the retrieval of Synaptotagmin I from the plasma membrane (Fergestad, 2001a).

While recent studies have focused on the proteins involved in exocytosis, it is not clear whether (or to what extent) the vesicle membrane recycles via clathrin-coated vesicles, or whether the membrane is directly retrieved by a fast endocytotic process. Biochemical studies lead to the proposal that the AP2 complex, a heterotetramer containing α-adaptin (See Drosophila α-Adaptin), plays an essential role in orchestrating different steps of endocytosis at the synapse. AP2 is able to bind to the cytoplasmic tail of a number of membrane receptors including synaptotagmin, a transmembrane protein that controls the Ca²⁺-dependent membrane fusion during exocytosis. Synaptotagmin interaction with AP2 is consistent with its proposed function during endocytosis, which is based on a synaptotagmin endocytotic mutant phenotype. This would argue that synaptotagmin plays a dual role in the vesicle cycle by acting in the final step of exocytosis and the initial step of endocytosis, thereby coupling the two processes at the plasma membrane. Once recruited to the inner surface of the plasma membrane, AP2 is likely to initiate the formation of clathrin-coated pits by triggering the assembly of clathrin triskelion subunits into a polygonal lattice that causes a bending of the membrane into the coated pit structure. Clathrin-coated pits detach from the plasmalemma by a GTP-dependent fission reaction that is mediated by the GTPase dynamin, and the resulting coated membrane vesicles become internalized. Dynamin was shown to bind the AP2 complex in vitro, and to be functionally required for the detachment of the clathrin-coated vesicles from the membrane. After internalization, the clathrin-coated vesicles shed their coats, a process that involves a number of proteins, including auxilin, Hsp-70, and the cysteine string protein (CSP), which may function in a chaperone-like manner to unfold the clathrin lattice at the outer surface (González-Gaitán, 1997 and references therein).

One can envision a scenario where the recruitment of the AP2 complex to the plasma membrane is a rate-limiting step, which in turn could be controlled by membrane-associated AP2 receptors that are released from exocytotic vesicles. Such a molecular link between exocytosis and endocytosis events would guarantee exocytosis-dependent membrane retrieval, as suggested by the temporal link of exocytosis and endocytosis and by the in vitro interaction between AP2 and synaptotagmin. The results of this study show, however, that synaptotagmin does not colocalize with a-Adaptin in shi mutants. This suggests that the role of synaptotagmin and AP2 association is more likely to serve the recycling of synaptotagmin from the membrane, returning it to the cytoplasmic pool of synaptic vesicles to take part in the subsequent exocytosis event. Alternatively, synaptotagmin could be one of several functionally redundant receptors to anchor the AP2 complex to the membrane to initiate a new vesicle cycle (González-Gaitán, 1997).

Calcium sensitive interaction of Syntaxin with Synaptotagmins I and IV

At nerve terminals, a focal and transient increase in intracellular Ca(2+) triggers the fusion of neurotransmitter-filled vesicles with the plasma membrane. The most extensively studied candidate for the Ca(2+)-sensing trigger is synaptotagmin I, whose Ca(2+)-dependent interactions with acidic phospholipids and syntaxin have largely been ascribed to its C(2)A domain, although the C(2)B domain also binds Ca(2+). Genetic tests of synaptotagmin I have been equivocal as to whether it is the Ca(2+)-sensing trigger of fusion. Synaptotagmin IV, a related isoform that does not bind Ca(2+) in the C(2)A domain, might be an inhibitor of release. An essential aspartate of the Ca(2+)-binding site of the synaptotagmin I C(2)A domain was mutated and expressed in Drosophila lacking synaptotagmin I. Despite the disruption of the binding site, the Ca(2+)-dependent properties of transmission were not altered. Similarly, synaptotagmin IV could substitute for synaptotagmin I. It is concluded that the C(2)A domain of synaptotagmin is not required for Ca(2+)-dependent synaptic transmission, and that synaptotagmin IV promotes rather than inhibits transmission (Robinson, 2002).

Drosophila Synaptotagmin is homologous and highly similar to the mammalian synaptotagmins, and possesses similar biochemical properties. Aspartate residues 223, 229, 282, 284 and 290 in Drosophila (hereafter D1-D5) correspond to those that coordinate Ca2+ binding in rat synaptotagmin I. D2 and D3 form the high-affinity Ca1 binding site, and mutations of D2 and D3 are the most deleterious to the Ca2+-dependent interactions of the C₂A domain. Mutating D2 or D3 to asparagine abolishes Ca2+-dependent binding to syntaxin and phospholipids. Furthermore, the synaptotagmin IV isoform has a serine in the D3 position and consequently does not bind either phospholipids or syntaxin in a Ca2+-dependent manner (Robinson, 2002).

To test the functional significance of Ca2+ binding to the C₂A domain, an asparagine was substituted for the D2 residue and the Ca2+ dependence of syntaxin binding was assayed. In the absence of Ca2+, little binding was observed of syntaxin to a fusion protein of glutathione S-transferase (GST) and the C₂A domain; in 1 mM Ca2+, binding was greatly enhanced. The D2N mutation prevents this binding even in the presence of Ca2+; the syntaxin bound by the mutated C₂A is similar to that of GST alone. When the synaptotagmin construct includes C₂A and C₂B, the syntaxin-synaptotagmin I interaction is independent of Ca2+ and persists in synaptotagmin I D2N. Binding of phospholipid to the wild-type Drosophila C₂A domain is dependent on Ca2+ and is abrogated by the D2N mutation. The D2N mutation reduces, but does not entirely block, Ca2+-dependent binding of phospholipids to a synaptotagmin I construct containing both C2 domains. Thus, mutating this aspartate drastically alters the Ca2+-dependent interactions of the C₂A domain of synaptotagmin I, and reveals a residual Ca2+ dependence on some interactions that is likely to reside in the C₂B domain (Robinson, 2002).

Although the equivalence of the Drosophila and mammalian synaptotagmins can not be presumed from sequence alone, the D2 residue of the Drosophila protein is necessary for conferring Ca2+ sensitivity on protein-protein and protein-phospholipid interactions of the C₂A domain of synaptotagmin I. The use of this mutation in this study does not depend on the total ablation of all Ca2+ binding to this domain. It rests instead on the biochemical and structural evidence that the mutation causes substantial changes to the site. If the C₂A domain binds the Ca2+ ions that trigger membrane fusion, and if these protein-protein or protein-lipid interactions are related to the initiation of fusion, a dramatic alteration in Ca2+ affinity should be mirrored in a profound alteration in the ability of Ca2+ to stimulate transmitter release (Robinson, 2002).

To test the functional competence of synaptotagmin I^D2N, it and wild-type Synaptotagmin I were epitope tagged, placed under the control of an upstream activating sequence (UAS) promoter, and reintroduced into the fly (P[UAS-HA-syt] and P[UAS-D2N]). The expression of each construct was driven by the heat-shock-dependent driver hsp70-Gal4. This expression system approximately doubled the amount of Synaptotagmin I protein present in the adult head compared with synaptotagmin I (sytI) heterozygotes, although some of this protein may reside in non-neuronal tissues. Each synaptotagmin transgene and driver was crossed into a sytI-null background. The sytI gene encodes the major synaptotagmin isoform at neuromuscular junctions and its absence greatly reduces transmitter release. Thus these null genotypes provide a suitable background for judging the efficacy of a synaptotagmin in which Ca2+ binding has been altered (Robinson, 2002).

Both control and D2N transgenes rescue the synaptic phenotype in a heat-shock-dependent manner. The amplitude of excitatory junctional potentials (EJPs) in the P[UAS-HA-syt] larvae fell within the range observed in the syt+ control, although they were on average smaller, suggesting that the heat-shock-driven wild-type transgene is not a perfect substitute for the normal gene. The P[UAS-D2N] transgene also confers robust EJPs. In a second set of experiments, an elav-Gal4 driver was used to induce neuronal expression of P[UAS-D2N]; under this circumstance the response was fast, tightly coupled to nerve stimulation, and very similar in time course to those of the wild type. Thus, despite mutation of the essential aspartate, the transgene can alleviate most of the disruption of synaptic transmission caused by removing synaptotagmin I (Robinson, 2002).

The Ca2+ dependence of transmission mediated by the synaptotagmin I^D2N construct was examined by looking at the apparent cooperativity (N) of release from the slope of double-log plots of quantal content versus extracellular Ca2+ concentration. This study shows that a major alteration of the C₂A Ca2+-binding site has a negligible effect on the Ca2+-dependent properties of the synapse. This result stands in contrast to a previous study that looked at minor perturbations of Ca2+ binding in the C₂A domain and observed a decrease in release probability that was interpreted as a correlation of those changes (Robinson, 2002).

These observations prompted a reinvestigation of the function of synaptotagmin IV, the isoform whose sequence contains a serine substitution for the D3 aspartate and whose inability to bind Ca2+ in the C₂A domain causes it to act as an inhibitor of release. To this end, the UAS-sytIV transgene, with elav-Gal4 to activate neuronal expression, was crossed into the sytI-null background. Larvae lacking synaptotagmin I typically die at early stages, but, if separated from their heterozygous siblings, they can survive to adulthood. Nonetheless, transmission at this synapse is nearly absent. When sytIV was expressed in the sytI^AD4/AD4 background, transmission was restored to near-control levels. Consistent with previous studies, a modest increase in the frequency, but not the amplitude, of spontaneous miniature EJPs was observed in sytI-null larvae. This phenomenon was reversed by the expression of synaptotagmin IV. Thus, despite the inability of the C₂A domain of synaptotagmin IV to bind Ca2+ and the reported interference of this protein with Ca2+-dependent biochemical processes, synaptotagmin IV can substitute efficiently for synaptotagmin I in synaptic transmission (Robinson, 2002).

The synaptotagmin I^D2N mutation afforded a stringent test of the hypothesis that the C₂A domain of synaptotagmin I is the Ca2+ sensor that transduces the change in cytosolic Ca2+ into a signal for exocytosis. This transgene restored fast, Ca2+-dependent transmission with properties extremely similar to those of wild-type animals and sytI-null animals that had been rescued with a wild-type transgene. Similarly, although synaptotagmin IV does not bind Ca2+ in its C₂A domain, it efficiently supports transmission in a sytI-null background. These findings are not easily reconciled with the hypothesis that the C₂A domain of synaptotagmin is the Ca2+-sensing trigger. The release observed was dependent on the induction of the transgenes, so transmission in these animals should predominantly reflect the properties of the transgene and not those of any minor second synaptotagmin gene that might be present. If the mutated Ca2+-binding site was indeed the trigger for fusion, alterations in the Ca2+ dependence of transmission should have been observed. It cannot be ruled out that some residual Ca2+ binding takes place in the D2N mutation, although biochemical assays indicate it must be very slight, but the profound reduction in Ca2+ binding that occurred should have been mirrored by a sharp decrease in synaptic function (Robinson, 2002).

The apparent cooperativity of Ca2+ action in the nerve terminal is often used to characterize the sensor. This cooperativity has been inferred from the non-linearity of secretion to changes in extracellular Ca2+, which should, at low Ca2+ concentrations, be proportional to local change in cytosolic Ca2+. The ability of synaptotagmin to bind multiple Ca2+ ions has been put forward as a potential correlate for the multiple predicted Ca2+ ions that trigger exocytosis. This parameter should be a sensitive reporter of changes in the triggering site, including changes in ions bound per site or the removal of one of several Ca2+-binding steps. Thus, the observation that this property is indifferent to the D2N mutation is a further indication that the C₂A domain is not involved in the triggering of exocytosis. Similarly, the range of Ca2+ concentrations that evoke release is unaffected, suggesting that the Michaelis constant (Km) for the sensor had not been greatly altered (Robinson, 2002).

The finding that Ca2+ binding by the C₂A domain is not essential for release raises the possibility that the C₂B domain fulfils this function or that the two domains share the task. Yet, if Ca2+ sensing for fusion were accomplished by Ca2+ binding to both C₂A and C₂B, then the mutations in this study should have affected transmission significantly, and the apparent cooperativity of fusion should have been reduced by the impairment of C₂A function. Functions of C₂B that are independent of C₂A should be addressed directly with further mutations (Robinson, 2002).

In Drosophila, C. elegans and mice, some synaptic transmission persists in the absence of synaptotagmin I, and this transmission is dependent on Ca2+ concentration, similar to what has been observed in wild type. The present observation that synaptotagmin IV can participate in fast, Ca2+-dependent transmission suggests that synaptotagmin IV or another isoform could mediate the residual release in sytI-null mutants. In contrast, the fact that Ca2+-dependent properties of transmission are constant in the face of the different Ca2+-binding properties of either synaptotagmin IV or I^D2N suggests that Ca2+ binding, at least by the C₂A domain, is not the primary means by which synaptotagmin promotes fusion. Decreased release, which was consistently observed in synaptotagmin I mutations, could instead be accounted for by a defect in any of several steps in nerve terminal function, including a decrease in the pool of releasable docked vesicles or a decrease in the probability of fusion of a docked vesicle. This model would be consistent with the interactions of synaptotagmin with plasma membrane proteins and the AP-2 complex and with ultrastructural studies of mutants. The Ca2+-binding site in the C2 domains of synaptotagmins may permit Ca2+ to modulate vesicle docking or endocytosis, and thereby have subtler synaptic effects that were not assayed in this study (Robinson, 2002).

This study reopens the question of the nature of the Ca2+ sensor that triggers release. In addition to the C₂B domain of synaptotagmin, many other candidates have been identified, and multiple sensors may exist to account for the steep dependence of fusion on Ca2+ concentration. These alternatives now merit closer examination (Robinson, 2002).

Synaptotagmin-1 docks secretory vesicles to syntaxin-1/SNAP-25 acceptor complexes

Docking, the initial association of secretory vesicles with the plasma membrane, precedes formation of the SNARE complex, which drives membrane fusion. For many years, the molecular identity of the docked state, and especially the vesicular docking protein, has been unknown, as has the link to SNARE complex assembly. This study, using adrenal chromaffin cells, identifies the vesicular docking partner as synaptotagmin-1, the calcium sensor for exocytosis, and SNAP-25 as an essential plasma membrane docking factor, which, together with the previously known docking factors Munc18-1 and syntaxin, form the minimal docking machinery. Moreover, the requirement for Munc18-1 in docking, but not fusion, can be overcome by stabilizing syntaxin/SNAP-25 acceptor complexes. These findings, together with cross-rescue, double-knockout, and electrophysiological data, led to a proposal that vesicles dock when synaptotagmin-1 binds to syntaxin/SNAP-25 acceptor complexes, whereas Munc18-1 is required for the downstream association of synaptobrevin to form fusogenic SNARE complexes (de Wit, 2009).

These data identify two genes, Snap-25 and synaptotagmin-1, that, together with two previously characterized genes, munc18-1 and syntaxin-1, are required for docking of secretory vesicles. This study addressed the involvement of the syntaxin-1/SNAP-25 acceptor complex and found that two conditions that favor the formation of syntaxin-1/SNAP-25 acceptor complexes rescue the docking defects in munc18-1 null mutants: SNAP-25 overexpression and expression of truncated synaptobrevin. Furthermore, null mutations for SNAP-25 and the vesicular protein synaptotagmin-1 abolish docking, and SNAP-25 no longer rescues docking in synaptotagmin-1/munc18-1 double-null mutants. By using synaptotagmin-1 and SNAP-25 mutations that affect their interaction, both proteins were confirmed to act in concert for correct anchoring of secretory vesicles to fusion sites. Moreover, the rescue of docking, but not fusion, after expression of SNAP-25 or the synaptobrevin-2 C-terminal fragment on the munc18-1 null background indicates that Munc18-1 is not an essential constituent of the docking complex itself, but plays an essential downstream role. Together, the null mutation and (cross-) rescue experiments indicate that the corresponding four proteins work together to dock vesicles and at the same time suggest that Munc18-1 plays a unique, orchestrating role. While docking is established between syntaxin-1/SNAP-25 acceptor complexes at the target membrane and synaptotagmin-1 on the vesicle membrane, Munc18-1 promotes the formation or stability of the correct acceptor SNARE complexes (de Wit, 2009).

Munc18-1 can interact with both 'closed' and 'open' syntaxin-1, but it is unclear which binding mode is essential to perform its function in docking. Munc18-1 binding to 'open' syntaxin-1 involves an interaction with the N-terminal H(abc) domain of syntaxin-1 and the four-helical bundle of the assembled SNARE complex. It has been shown that N-terminal interaction is not sufficient for docking, since a docking phenotype similar to syntaxin-1 and munc18-1 null was observed in chromaffin cells from knockin mice that express a mutant syntaxin-1 that only allows N-terminal interaction. In addition, when the well-characterized D34N/M38V double mutant of Munc18-1 that is known to perturb the interaction with 'closed' syntaxin was expressed, it was observed that docking was not restored in munc18-1 null chromaffin cells. Other studies have shown that Munc18-1 binding to 'open' syntaxin is essential to execute fusion. In the present study, docking and fusion phenotypes were experimentally separated in munc18-1 null chromaffin cells. The observations that SNAP-25 and SybCT overexpression, which both increase the number of syntaxin-1/SNAP-25 dimers, restore docking implies that Munc18-1 promotes the existence/stability of intermediate syntaxin-1/SNAP-25 dimers at the target membrane and therefore probably binds to these intermediate complexes. This increased number of acceptor complexes is not sufficient to restore fusion in the absence of Munc18-1, which firmly establishes a postdocking role for Munc18-1 in SNARE-dependent fusion. Currently, it is unclear whether Munc18-1's function downstream of docking requires either binding to intermediate syntaxin-1/SNAP-25 dimers alone or also binding to assembled SNARE complexes (containing synaptobrevin-2) to promote fusion as shown previously in vitro. In addition, these experiments with synaptotagmin-1 and SNAP-25 mutations, which have been shown to impair secretion, show that in the presence of Munc18-1 a correlation exists between mutations that impair secretion and those that impair docking. This is not the case in the absence of Munc18-1, emphasizing its postdocking role in SNARE-dependent fusion (de Wit, 2009).

This study identifies synaptotagmin-1 as a vesicular docking factor that binds to the assembled docking acceptor discussed above and has the capacity to anchor vesicles to the target membrane. This docking role of synaptotagmin-1 is consistent with previous findings in invertebrate synapses, which, however, have not been specifically interpreted in terms of docking because of additional phenotypes in these synapses: large effects on undocked vesicle populations near the active zone, which has been related to the increased mini rate observed in these mutant synapses, and/or impaired recruitment. Interestingly, a mutation used in the latter study is in an area of the molecule that was later identified to interact with SNAP-25(de Wit, 2009).

The docking role of synaptotagmin-1 proposed in this study does not conflict with its well-established role in fusion. However, while its role in fusion is strictly Ca²⁺ dependent, its role in docking is probably Ca²⁺ independent, since resting chromaffin cells have a strong docking phenotype in the absence of synaptotagmin-1 and its Ca²⁺ affinity is insufficient to be activated by resting Ca²⁺ levels in the cytosol. This is in line with a Ca²⁺-independent, upstream role previously suggested in rescue experiments in fly neuromuscular junction (Loewen, 2006). It is tempting to speculate that on top of this principally Ca²⁺-independent docking role, synaptotagmins may also contribute to the well-known but incompletely understood Ca²⁺-dependent acceleration of vesicle recruitment/docking/priming (de Wit, 2009).

Secretory systems typically express multiple synaptotagmins. In chromaffin cells, synaptotagmin-7 can partially compensate for the loss of synaptotagmin-1, but the secretion phenotype of the synaptotagmin-1 null cells is still drastic. In analogy, the docking phenotype in synaptotagmin-1 null cells is also drastic, but still slightly less severe than the munc18-1 null phenotype. This may be explained by a partial compensation by other synaptotagmins. The presence of multiple synaptotagmins, with different Ca²⁺ sensitivities and the new evidence that they are not only involved in fusion (and endocytosis), but also in docking, may require reinterpretation of previous studies on these proteins. Most studies assess upstream processes by measuring the final one (fusion) and thereby sample a composite measure of the combined effects of experimental manipulations on all upstream steps. For these combined effects to be dissected, new methodologies may be required to directly assess these upstream steps and to go beyond what current secretion assays have revealed about the complexity of the secretory pathway (de Wit, 2009).

Invertebrate synapses, docking phenotypes for Munc18-1, syntaxin-1, SNAP-25, and synaptotagmin-1 have not been described or are at least less evident. It is possible that these proteins are dispensable for synaptic vesicle docking and that distinct mechanisms dock vesicles in synapses. However, it seems more likely that docking principles are conserved among secretory systems. This idea is strongly supported by the fact that docking phenotypes have been observed in invertebrate synapses upon mutations in three of the four genes. However, these phenotypes are generally subtle and sometimes require advanced methodology and new docking definitions to become evident. In the case of synaptotagmin, invertebrate phenotypes are robust, but additional phenotypes were observed that prevented a specific interpretation in terms of docking. It is likely that docking phenotypes are less evident in vertebrate synapses either because of redundancy arising from the expression of multiple isoforms for some of the docking genes identified here or because structurally unrelated proteins that are not expressed in chromaffin cells restrict undocking of synaptic vesicles even when essential docking factors are not expressed. Finally, it is plausible that undocking and docking phenotypes are simply not as evident in the densely packed nerve terminal (de Wit, 2009).

With the currently identified four genes for docking and the link to SNARE complex assembly, a consistent (minimal) working model for the exocytotic pathway from the initial docking step until the final fusion reaction can now be synthesized for the first time, proposing the following four steps: First, Munc18-1 binds the closed conformation of syntaxin-1. Munc18-1 interacts with two epitopes in syntaxin-1, the Habc domain, and the N-terminal domain. Second, SNAP-25 binds the syntaxin-1/Munc18-1 heterodimer. Third, secretory vesicles reach the target membrane area and associate via synaptotagmin-1 to this trimeric syntaxin-1/Munc18-1/SNAP-25 complex, which effectuates docking. This binding requires the C2B domain of synaptotagmin-1, and recent studies suggest that Munc18-1's function here is to further help stabilize the syntaxin-1/SNAP-25 (1:1) acceptor complex for subsequent binding of synaptobrevin-2. In addition, since only vesicles docked in the presence of Munc18-1 are able to fuse, Munc18-1 might help restrict fusion to specific sites on the plasma membrane. By attaching the vesicle to the plasma membrane, the calcium sensor for exocytosis (synaptotagmin-1) has the additional function of localizing vesicles close to calcium channels. Fourth, synaptobrevin-2 then binds to the synaptotagmin-1/syntaxin-1/Munc18-1/SNAP-25 complex and the four helical SNARE bundle forms, which subsequently allows complexins to associate with the four helical SNARE bundle, and ultimately the vesicle fuses upon Ca²⁺ entry. It has been proposed that synaptobrevin-2 replaces Munc18-1, but, given the proposed fusion-promoting actions of Munc18-1 while associated to SNARE complexes, Munc18-1 may also continue to associate with the ternary SNARE complex until fusion is triggered (de Wit, 2009).

Synaptobrevin - also known as VAMP or vesicle-associated membrane protein

Syntaxin Protein Interactions part 2/3 | part 3/3

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