Developmental expression of dynamin in the chick retinotectal system.

Dynamin I, a GTPase involved in the endocytic cycle of synaptic vesicle membranes, is believed to support axonal outgrowth and/or synaptogenesis. To explore the temporal and spatial patterns of dynamin I distribution in neuronal morphogenesis, we compared the developmental expression of dynamin with the expression of presynaptic membrane proteins such as SV2, synaptotagmin, and syntaxin in the chick primary visual pathway. Western blots of retina and tectum revealed a steady increase of synaptotagmin and syntaxin from embryonic Day 7 (E7) to E11, whereas for the same time frame no detectable increase of dynamin was found. Later stages showed increasing amounts of all tested proteins until the first postnatal week. Immunofluorescence revealed that SV2, synaptotagmin, and syntaxin are present in retinal ganglion cell axons from E4 on. In later stages, the staining pattern in the retina and along the visual pathway paralleled the formation and maturation of axons. In contrast, dynamin is not detectable by immunofluorescence in the developing retina and optic tectum before synapse formation. Our data indicate that, in contrast to the early expression of synaptotagmin, SV2, and syntaxin during axonal growth, dynamin is upregulated after synapse formation, suggesting its function predominantly during and after synaptogenesis but not in axonogenesis.(J Histochem Cytochem 47:1297-1306, 1999)     

Interactions between proteins implicated in exocytosis and voltage-gated calcium channels

Neurotransmitter release from synaptic vesicles is triggered by voltage-gated calcium influx through P/Q-type or N-type calcium channels. Purification of N-type channels from rat brain synaptosomes initially suggested molecular interactions between calcium channels and two key proteins implicated in exocytosis: synaptotagmin I and syntaxin 1. Co-immunoprecipitation experiments were consistent with the hypothesis that both N- and P/Q-type calcium channels, but not L-type channels, are associated with the 7S complex containing syntaxin 1, SNAP-25, VAMP and synaptotagmin I or II. Immunofluorescence confocal microscopy at the frog neuromuscular junction confirmed that calcium channels, syntaxin 1 and SNAP-25 are co-localized at active zones of the presynaptic plasma membrane where transmitter release occurs. Experiments with recombinant proteins were performed to map synaptic protein interaction sites on the alpha 1A subunit, which forms the pore of the P/Q-type calcium channel. In vitro-translated 35S-synaptotagmin I bound to a site located on the cytoplasmic loop linking homologous domains II and III of the alpha 1A subunit. This direct link would target synaptotagmin, a putative calcium sensor for exocytosis, to a microdomain of calcium influx close to the channel mouth. Cysteine string proteins (CSPs) contain a J-domain characteristic of molecular chaperones that cooperate with Hsp70. They are located on synaptic vesicles and thought to be involved in modulating the activity of presynaptic calcium channels. CSPs were found to bind to the same domain of the calcium channel as synaptotagmin, and also to associate with VAMP. CSPs may act as molecular chaperones in association with Hsp70 to direct assembly or dissociation of multiprotein complexes at the calcium channel.     

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.     

Rab3A is a new interacting partner of synaptotagmin I and may modulate synaptic membrane fusion through a competitive mechanism

Rab3 and synaptotagmin have been reported to be the key proteins that have opposite actions but cooperatively play critical regulatory roles in selecting and limiting the number of vesicles released at central synapses. However, the exact mechanism has not been fully understood. In this study, Rab3A and synaptotagmin I, the most abundant isoforms of Rab3 and synaptotagmin, respectively, in brain were for the first time demonstrated to directly interact with each other in a Ca²⁺-independent manner, and the KKKK motif in the C2B domain of synaptotagmin I was a key site for the Rab3A binding, which was further confirmed by the competitive inhibition of inositol hexakisphosphate. Further studies demonstrated that Rab3A competitively affected the synaptotagmin I interaction with syntaxin 1B that was involved in membrane fusion during the synaptic vesicle exocytosis. These data indicate that Rab3A is a new synaptotagmin I interacting partner and may participate in the regulation of synaptic membrane fusion and thus the vesicle exocytosis by competitively modulating the interaction of synaptotagmin with syntaxin of the t-SNARE complex in presynaptic membranes.     

SV2B regulates synaptotagmin 1 by direct interaction

SV2 proteins are abundant synaptic vesicle proteins expressed in two major (SV2A and SV2B) and one minor (SV2C) isoform. SV2A and SV2B have been shown to be involved in the regulation of synaptic vesicle exocytosis. Previous studies found that SV2A, but not SV2B, can interact with the cytoplasmic domain of synaptotagmin 1, a Ca2+ sensor for synaptic vesicle exocytosis. To determine whether SV2B can interact with full-length synaptotagmin 1, we performed immunoprecipitations from brain protein extracts and found that SV2B interacts strongly with synaptotagmin 1 in a detergent-resistant, Ca2+ -independent manner. In contrast, an interaction between native SV2A and synaptotagmin 1 was not detectable under these conditions. The SV2B-synaptotagmin 1 complex also contained the synaptic t-SNARE proteins, syntaxin 1 and SNAP-25, suggesting that SV2B may participate in exocytosis by modulating the interaction of synaptotagmin 1 with t-SNARE proteins. Analysis of retinae in SV2B knock-out mice revealed a strong reduction in the level of synaptotagmin 1 in rod photoreceptor synapses, which are unique in that they express only the SV2B isoform. In contrast, other synaptic vesicle proteins were not affected by SV2B knock out, indicating a specific role for SV2B in the regulation of synaptotagmin 1 levels at certain synapses. These experiments suggest that the SV2B-synaptotagmin 1 complex is involved in the regulation of synaptotagmin 1 stability and/or trafficking. This study has demonstrated a new role of SV2B as a regulator of synaptotagmin 1 that is likely mediated by direct interaction of these two synaptic proteins.     

Immunocytochemical Analysis of Axonal Outgrowth in Synaptotagmin Mutations

Abstract: 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. We have examined the possible requirement for synaptotagmin in axonal outgrowth by following neuronal development in Drosophila embryos deficient for the synaptotagmin gene. We find that synaptotagmin is expressed abundantly in axons and growth cones before synapse formation in wild-type embryos. Using antibodies to the intravesicular domain of synaptotagmin to label live embryos, we demonstrate that vesicle populations containing synaptotagmin actively undergo exocytosis during axonogenesis. We have used immunocytochemical techniques to examine the distribution of the axonal protein Fasciclin II, the presynaptic membrane protein syntaxin, and the synaptic vesicle protein cysteine string protein, in synaptotagmin null mutations. 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, we suggest that the molecular mechanisms underlying vesicular-mediated membrane expansion during axonal outgrowth are distinct from those required for synaptic vesicle fusion during neurotransmitter release.     

The Proteins Synaptotagmin and Syntaxin Are Not General Targets of Lambert-Eaton Myasthenic Syndrome Autoantibody

Abstract: Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune neuromuscular disease in which impairment of Ca²⁺ entry into the nerve ending and consequent impaired release of acetylcholine (ACh) results in muscle weakness. The identity of the primary antigenic target molecule(s) of the autoantibodies is uncertain. Electrophysiological studies and ⁴⁵Ca²⁺ uptake studies implicate a direct effect on the Ca²⁺ channel complex at the motor nerve terminal. Some recent studies, however, suggest a more indirect interference caused by binding of autoantibodies to synaptotagmin or syntaxin, molecules presumed to be involved in docking and/or coupling the synaptic vesicles to the Ca²⁺ channels in the active zone for vesicle exocytosis and transmitter release. Western blot analyses of rat and human brain membrane proteins and pure recombinant synaptotagmin and syntaxin were used to examine directly the targets of LEMS autoantibodies and determine specifically whether or not synaptotagmin and/or syntaxin were general targets in LEMS. IgG from 14 patients with LEMS was used to probe western blots of gels containing synaptotagmin, syntaxin, rat synaptosomal proteins, and human brain membrane proteins. Several similar immunoreactive bands were observed using both rat and human brain membranes. These include high-molecular-weight protein bands whose size would be consistent with being components of Ca²⁺ channels. No reactive component was observed against either syntaxin or synaptotagmin in IgG of the 14 LEMS patients. However, both human and rat brain membranes contain proteins recognized by antibodies directed against synaptotagmin or syntaxin, indicating their immunologic relatedness and evolutionary conservation. These results suggest that large-molecular-weight proteins consistent with being Ca²⁺ channel subunits rather than syntaxin and synaptotagmin are general targets of LEMS autoantibodies.     

PRIP (Phospholipase C-related but Catalytically Inactive Protein) Inhibits Exocytosis by Direct Interactions with Syntaxin 1 and SNAP-25 through Its C2 Domain

Membrane fusion for exocytosis is mediated by SNAREs, forming trans-ternary complexes to bridge vesicle and target membranes. There is an array of accessory proteins that directly interact with and regulate SNARE proteins. PRIP (phospholipase C-related but catalytically inactive protein) is likely one of these proteins; PRIP, consisting of multiple functional modules including pleckstrin homology and C2 domains, inhibited exocytosis, probably via the binding to membrane phosphoinositides through the pleckstrin homology domain. However, the roles of the C2 domain have not yet been investigated. In this study, we found that the C2 domain of PRIP directly interacts with syntaxin 1 and SNAP-25 but not with VAMP2. The C2 domain promoted PRIP to co-localize with syntaxin 1 and SNAP-25 in PC12 cells. The binding profile of the C2 domain to SNAP-25 was comparable with that of synaptotagmin I, and PRIP inhibited synaptotagmin I in binding to SNAP-25 and syntaxin 1. It was also shown that the C2 domain was required for PRIP to suppress SDS-resistant ternary SNARE complex formation and inhibit high K⁺-induced noradrenalin release from PC12 cells. These results suggest that PRIP inhibits regulated exocytosis through the interaction of its C2 domain with syntaxin 1 and SNAP-25, potentially competing with other SNARE-binding, C2 domain-containing accessory proteins such as synaptotagmin I and by directly inhibiting trans-SNARE complex formation.     

Coelomocyte numbers and expression of HSP70 in wounded sea stars during hypoxia

Hypoxia, mainly caused by eutrophication, is a common and growing problem on marine soft bottoms. Echinoderms are known for their ability to regenerate tissue after wounding but hypoxia has a negative influence on regeneration and also on reproduction in echinoderms. We have investigated the cellular and molecular responses to wounding stress and hypoxia in the sea star Asterias rubens by using the total coelomocyte count (TCC) and the expression of heat shock proteins (HSPs). As early as 1 h after wounding, sea stars under hypoxic conditions show significantly increased TCC and, after 6 h, cell numbers increase approximately two-fold. After a 3-h hypoxia exposure of wounded animals, Western blot analysis reveals highly elevated coelomocyte cytoplasmic HSP70 expression. Non-wounded sea stars exposed to hypoxia and wounded animals kept in normoxia show enhanced HSP70 expression only after 24 h. Immunocytochemical analysis has not demonstrated any translocation of HSP70 from the cytoplasm to the nucleus. We conclude that both wounding and hypoxia elicit a stress response in sea stars and that the combined stress produces synergistic effects that may inhibit the initial processes of wound healing and regeneration.     

Chronic electromagnetic field exposure decreases HSP70 levels and lowers cytoprotection

Electromagnetic field (EMF) exposures have been shown to induce heat shock proteins (HSPs), which help to maintain the conformation of cellular proteins during periods of stress. We have previously reported that short-term exposure of chick embryos to either 60 Hz (extremely low frequency: ELF), or radio-frequency (RF: 915 MHz) EMFs induce protection against hypoxia. Experiments presented in the current report are based on a study in which long-term (4 days), continuous exposure to ELF-EMFs decreased protection against ultraviolet radiation. Based on this result, it was hypothesized that de-protection against hypoxia should also occur following long-term, continuous, or daily, repeated exposures to EMFs. To test this hypothesis, chick embryos were exposed to ELF-EMFs (8 microT) continuously for 4 days, or to ELF or RF (3.5 mW incident power)-EMFs repeated daily (20, 30, or 60 min once or twice daily for 4 days). Several of the exposure protocols yielded embryos that had statistically significant decreases in protection against hypoxic stress (continuous and 30 or 60 min ELF twice daily; or 30 or 60 min once daily RF). This is consistent with our finding that following 4 days of ELF-EMF exposure, HSP70 levels decline by 27% as compared to controls. In addition, the superposition of ELF-EMF noise, previously shown to minimize ELF-EMF induced hypoxia protection, inhibited hypoxia de-protection caused by long term, continuous ELF or daily, repeated RF exposures. This EMF-induced decrease in HSP70 levels and resulting decline in cytoprotection suggests a mechanism by which daily exposure (such as might be experienced by mobile phone users) could enhance the probability of cancer and other diseases.     

Presynaptic trafficking of synaptotagmin I is regulated by protein palmitoylation

Protein palmitoylation plays a critical role in sorting and targeting of several proteins to pre- and postsynaptic sites. In this study, we have analyzed the role of palmitoylation in trafficking of synaptotagmin I and its modulation by synaptic activity. We found that palmitoylation of N-terminal cysteines contributed to sorting of synaptotagmin I to an intracellular vesicular compartment at the presynaptic terminal. Presynaptic targeting is a unique feature of N-terminal sequences of synaptotagmin I because the palmitoylated N terminus of synaptotagmin VII failed to localize to presynaptic sites. We also found that palmitate was stably associated with both synaptotagmin I and SNAP-25 and that rapid neuronal depolarization did not affect palmitate turnover on these proteins. However, long-term treatment with drugs that either block synaptic activity or disrupt SNARE complex assembly modulated palmitoylation and accumulation of synaptotagmin I at presynaptic sites. We conclude that palmitoylation is involved in trafficking of specific elements involved in transmitter release and that distinct mechanisms regulate addition and removal of palmitate on select neuronal proteins.     

The transmembrane domain of syntaxin 1A is critical for cytoplasmic domain protein-protein interactions

Assembly of the plasma membrane proteins syntaxin 1A and SNAP-25 with the vesicle protein synaptobrevin is a critical step in neuronal exocytosis. Syntaxin is anchored to the inner face of presynaptic plasma membrane via a single C-terminal membrane-spanning domain. Here we report that this transmembrane domain plays a critical role in a wide range of syntaxin protein-protein interactions. Truncations or deletions of the membrane-spanning domain reduce synaptotagmin, alpha/beta-SNAP, and synaptobrevin binding. In contrast, deletion of the transmembrane domain potentiates SNAP-25 and rbSec1A/nsec-1/munc18 binding. Normal partner protein binding activity of the isolated cytoplasmic domain could be "rescued" by fusion to the transmembrane segments of synaptobrevin and to a lesser extent, synaptotagmin. However, efficient rescue was not achieved by replacing deleted transmembrane segments with corresponding lengths of other hydrophobic amino acids. Mutations reported to diminish the dimerization of the transmembrane domain of syntaxin did not impair the interaction of full-length syntaxin with other proteins. Finally, we observed that membrane insertion and wild-type interactions with interacting proteins are not correlated. We conclude that the transmembrane domain, via a length-dependent and sequence-specific mechanism, affects the ability of the cytoplasmic domain to engage other proteins.     

Identification of SNAREs involved in synaptotagmin VII-regulated lysosomal exocytosis

Ca2+-regulated exocytosis of lysosomes has been recognized recently as a ubiquitous process, important for the repair of plasma membrane wounds. Lysosomal exocytosis is regulated by synaptotagmin VII, a member of the synaptotagmin family of Ca2+-binding proteins localized on lysosomes. Here we show that Ca2+-dependent interaction of the synaptotagmin VII C(2)A domain with SNAP-23 is facilitated by syntaxin 4. Specific interactions also occurred in cell lysates between the plasma membrane t-SNAREs SNAP-23 and syntaxin 4 and the lysosomal v-SNARE TI-VAMP/VAMP7. Following cytosolic Ca2+ elevation, SDS-resistant complexes containing SNAP-23, syntaxin 4, and TI-VAMP/VAMP7 were detected on membrane fractions. Lysosomal exocytosis was inhibited by the SNARE domains of syntaxin 4 and TI-VAMP/VAMP7 and by cleavage of SNAP-23 with botulinum neurotoxin E, thereby functionally implicating these SNAREs in Ca2+-regulated exocytosis of conventional lysosomes.     

Stonins—Specialized Adaptors for Synaptic Vesicle Recycling and Beyond?

Stonins are a small family of evolutionarily conserved clathrin adaptor complex AP-2μ-related factors that may act as cargo-specific sorting adaptors in endocytosis and perhaps beyond. Whereas little is known about the localization and function of stonin 1, recent work suggests that stonin 2 serves as a linker between the endocytic proteins AP-2 and Eps15 and the calcium-sensing synaptic vesicle (SV) protein synaptotagmin 1. The molecular determinants involved in the recognition of SV cargo by the μ-homology domain of stonin 2 are evolutionarily conserved from worm to man, thereby identifying stonin 2 and its invertebrate homologs uncoordinated (UNC)-41 and stoned B as endocytic adaptors dedicated to the retrieval of surface-stranded SV proteins, most notably synaptotagmin. In this review, we summarize the current state of knowledge about mammalian stonins with a special focus on the role of stonin 2 in SV recycling at presynaptic nerve terminals.     

Interactions between Presynaptic Calcium Channels and Proteins Implicated in Synaptic Vesicle Trafficking and Exocytosis

Monoclonal antibodies were generated by immunizing mice with chick brain synaptic membranes and screening for immunoprecipitation of solubilized conotoxin GVIA receptors (N-type calcium channels). Antibodies against two synaptic proteins (p35--syntaxin 1 and p58--synaptotagmin) were produced and used to purify and characterize a ternary complex containing N-type channels associated with these two proteins. These results provided the first evidence for a specific interaction between presynaptic calcium channels and SNARE proteins involved in synaptic vesicle docking and calcium-dependent exocytosis. Immunoprecipitation experiments supported the conclusion that syntaxin 1/SNAP-25/VAMP/synaptotagmin I or II complexes associate with N-type, P/Q-type, but not L-type calcium channels from rat brain nerve terminals. Immunofluorescent confocal microscopy at the frog neuromuscular junction was consistent with the co-localization of syntaxin 1, SNAP-25, and calcium channels, all of which are predominantly expressed at active zones of the presynaptic plasma membrane facing post-synaptic folds rich in acetylcholine receptors. The interaction of proteins implicated in calcium-dependent exocytosis with presynaptic calcium channels may locate the sensor(s) that trigger vesicle fusion within a microdomain of calcium entry.     

Specificity of Ca2+-dependent protein interactions mediated by the C2A domains of synaptotagmins

Synaptotagmins represent a family of neuronal proteins thought to function in membrane traffic. The best characterized synaptotagmin, synaptotagmin I, is essential for fast Ca2+-dependent synaptic vesicle exocytosis, indicating a role in the Ca2+ triggering of membrane fusion. Synaptotagmins contain two C2 domains, the C2A and C2B domains, which bind Ca2+ and may mediate their functions by binding to specific targets. For synaptotagmin I, several putative targets have been identified, including the SNARE proteins syntaxin and SNAP-25. However, it is unclear which of the many binding proteins are physiologically relevant. Furthermore, more than 10 highly homologous synaptotagmins are expressed in brain, but it is unknown if they execute similar binding reactions. To address these questions, we have performed a systematic, unbiased study of proteins which bind to the C2A domains of synaptotagmins I-VII. Although the various C2A domains exhibit similar binding activities for phospholipids and syntaxin, we found that they differ greatly in their protein binding patterns. Surprisingly, none of the previously characterized binding proteins for synaptotagmin I are among the major interacting proteins identified. Instead, several proteins that were not known to interact with synaptotagmin I were bound tightly and stoichiometrically, most prominently the NSF homologue VCP, which is thought to be involved in membrane fusion, and an unknown protein of 40 kDa. Point mutations in the Ca2+ binding loops of the C2A domain revealed that the interactions of these proteins with synaptotagmin I were highly specific. Furthermore, a synaptotagmin I/VCP complex could be immunoprecipitated from brain homogenates in a Ca2+-dependent manner, and GST-VCP fusion proteins efficiently captured synaptotagmin I from brain. However, when we investigated the tissue distribution of VCP, we found that, different from synaptic proteins, VCP was not enriched in brain and exhibited no developmental increase paralleling synaptogenesis. Moreover, binding of VCP, which is an ATPase, to synaptotagmin I was inhibited by both ATP and ADP, indicating that the native, nucleotide-occupied state of VCP does not bind to synaptotagmin. Together our findings suggest that the C2A-domains of different synaptotagmins, despite their homology, exhibit a high degree of specificity in their protein interactions. This is direct evidence for diverse roles of the various synaptotagmins in brain, consistent with their differential subcellular localizations. Furthermore, our results indicate that traditional approaches, such as affinity chromatography and immunoprecipitations, are useful tools to evaluate the overall spectrum of binding activity for a protein but are not sufficient to estimate physiological relevance.     

Differential Phosphorylation of Syntaxin and Synaptosome-Associated Protein of 25 kDa (SNAP-25) Isoforms

Abstract : The synaptic plasma membrane proteins syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) are central participants in synaptic vesicle trafficking and neurotransmitter release. Together with the synaptic vesicle protein synaptobrevin/vesicle-associated membrane protein (VAMP), they serve as receptors for the general membrane trafficking factors N -ethylmaleimide-sensitive factor (NSF) and soluble NSF attachment protein (α-SNAP). Consequently, syntaxin, SNAP-25, and VAMP (and their isoforms in other membrane trafficking pathways) have been termed SNAP receptors (SNAREs). Because protein phosphorylation is a common and important mechanism for regulating a variety of cellular processes, including synaptic transmission, we have investigated the ability of syntaxin and SNAP-25 isoforms to serve as substrates for a variety of serine/threonine protein kinases. Syntaxins 1A and 4 were phosphorylated by casein kinase II, whereas syntaxin 3 and SNAP-25 were phosphorylated by Ca²⁺ - and calmodulin-dependent protein kinase II and cyclic AMP-dependent protein kinase, respectively. The biochemical consequences of SNARE protein phosphorylation included a reduced interaction between SNAP-25 and phosphorylated syntaxin 4 and an enhanced interaction between phosphorylated syntaxin 1A and the synaptic vesicle protein synaptotagmin I, a potential Ca²⁺ sensor in triggering synaptic vesicle exocytosis. No other effects on the formation of SNARE complexes (comprised of syntaxin, SNAP-25, and VAMP) or interactions involving n-Sec1 or α-SNAP were observed. These findings suggest that although phosphorylation does not directly regulate the assembly of the synaptic SNARE complex, it may serve to modulate SNARE complex function through other proteins, including synaptotagmin I.     

Presence of syntaxin 1A in secretory granules of chromaffin cells and interaction with chromogranins A and B

Syntaxin 1A and synaptotagmin I are key participants of fusion complex formation during exocytotic processes, and syntaxin 1A is known to be present in the plasma membrane. Here, we show the presence of not only synaptotagmin I but also syntaxin 1A in secretory granules of bovine adrenal chromaffin cells by immunogold electron microscopy, and further demonstrate the interaction of these proteins with chromogranins A and B (CGA and CGB), two major proteins of secretory granules. Interaction between chromogranins and the components of fusion complex also suggests active participation of CGA and CGB in fusion complex formation and subsequent exocytosis.