Insects are an ancient group of animals, the first of which probably appeared 360 to 400 million years ago. Analyses of insect genomes and proteomes provide a unique opportunity to compare evolution between the model organism D. melanogaster and numerous additional insect genomes. The insects whose genomes were sequenced ensure coverage of a valuable phylogenetic breadth, spanning the fruit fly (D. melanogaster(, the honey bee (Apis mellifera), the red flour beetle (Tribolium castaneum), the mosquitoes (Anopheles gambiae and Aedes aegypti), the silk worm (Bombyx mori) and the wasp (Nasonia vitripennis). All together, about 330,000 protein sequences from insects are currently available in public protein databases, which already include 12 additional Drosophila genomes. A current list of insect genome projects is accessible in Additional data file 1. In the present study we refer only to representative genomes that are substantially divergent and include the beetle, honeybee, mosquito, and fly (with D. melanogaster being the reference). We focus on establishing a functional synapse whose molecular assembly governs learning and memory as well as the complex behavior of the organism.

We compiled an extended catalog of mammalian presynaptic proteins based on the detailed anatomy of the synaptic vesicle 18, data from functional annotations by Gene Ontology (GO) 26, and a manual collection of genes of presynaptic function 27. This collection is compared with insect proteomes. A summary of the sequence conservation of each gene (a total of 120 representative genes) with the insect proteome is shown in Table 1. Analyzing this catalog (PS120 - presynaptic 120 genes) revealed that 50% are well conserved and have a sequence similarity in excess of 65% for most of the sequence. Among them, 60% are at a similarity level in excess of 75% for most of the sequence. Thus, the majority of proteins that participate in human presynaptic structures are extremely well conserved.

Most of the PS120 proteins belong to gene families, with some of the families being very large. For example, synaptotagmins and Rabs have numerous alternative spliced variants in addition to their large number of genes (17 and 60, respectively). For most instances, the size of the gene family in insects is smaller and on average is only 40% when compared with human. To exemplify this observation, we investigate the syntaxin family. There are 12 genes in human (and additional variants) that can be divided into subfamilies. The human subfamily of syntaxin 1, which functions as the t-SNARE in synaptic vesicle fusion (including Stx1, Stx2, Stx3, Stx4, and Stx11), is represented by only two genes in the fly (namely dStx1 and dStx4) 1 and in the other insects. However, in general, there are more gene variants that result from alternatively splicing events in the fly genome relative to the other insects.

A search of insect homologs for the PS120 clearly shows that even within the most conserved set between human and insects (60 genes), there are 12 genes for which there is no clear homolog in the current protein databases in at least one of the insect representatives (honeybee, beetle, mosquito, and fly). The same applies to about 30 additional proteins from the remainder of the PS120 gene list. Additional information on protein partners and protein length, and detailed information on the levels of sequence conservation is provided in Additional data files 2.

The completion of genomes for at least four insect representatives and the additional information from partially assembled genomes (Additional data file 1) makes it possible to revisit some of the apparently missed genes (Table 1 and Additional data file 2). Evidently, comparing related genomes enhances the quality of in silico genome annotations 28. A search in the public non-redundant database revealed that about one-third of the PS120 homologous sequences were missing in at least one of the insect representatives (Table 1). Moreover, for a small number of genes, no homologs were detected in any of the insects. In cases in which significant sequence similarity in all four insect representatives is absent, we strongly argue that these genes are genuinely absent in insects. This is supported by a lack of significant similarity in additional fly genomes, and in the silkworm and the wasp genomes (Additional data file 3).

Additional data file 3 provides information on apparently missing genes that are not apparent from protein databases (see Materials and methods, below). For 70% significant similarity in the genome-assembled sequences was identified. This high similarity is often supported by the existence of an expressed mRNA. For a few genes, only limited evidence on transcription levels exists. More importantly, for 11 genes no homologs were detected in insects by searching protein data against translated insect genomes. Among these genes are growth-associated protein (GAP)-43, which is implicated in cytoskeleton and protein kinase C signaling during synapse establishment 29, and two large proteins that shape the cytoskeletal mesh at the active zone: bassoon (about 3,900 amino acids) and piccolo (about 5,100 amino acids) 30. In addition, the SNARE regulator complexin 4, the syntaxin-tubulin binding protein syntabulin (FLJ20366), and SNAP-25-interacting protein are not detected in insects. Although most proteins of the synaptic vesicle membranes are strongly conserved, we were unable to detect SV31 (also called TMEM163; a genuine protein of the synaptic vesicle (SV) membranes) 31 or synaptophysin (one of the most abundant proteins in mammalian synaptic vesicles). Furthermore, no sequence similarity was noted for the syntaxin regulators amisyn (STXBP6) 32 and syntaphilin 33. Syntaphilin, which has been implicated in regulation of exocytosis and endocytosis 34, is conserved from human to pufferfish and zebrafish but was lost in the branch of the frogs and insects. A borderline similarity to dynactin and α-liprin suggests that the function in cytoskeletal remodeling and in cell-matrix interactions may be taken over by other proteins. Interestingly, many of the genes that are not conserved from human to insects are functionally related to active zone architecture and specifically to the underlying cytoskeleton mesh of the synapse.

In the PS120 gene list, rather close conservation is evident between insect and human genes (measured by a similarity >75% throughout the sequence) for 16 genes. This small set includes the v-SNARE VAMP2, the t-SNARE syntaxin 1A, and a few small GTP proteins (Ral, Rab3A, ARF1, and ARF6). In addition, this set includes essential components of the endocytic machinery (dynamin 1, AP2, AP3, EHD1, and clathrin) and proteins that activate transduction pathways (calmodulin and 14-3-3). That the function of these gene products is indispensable was expected, but proteins that coordinate synaptic vesicles with the active zone are also included in this selected list, namely cytohesin-1 35 and Mals-1 36. Both of these proteins share a function in determining the size of the readily releasable pool of synaptic vesicles and are critical for replenishing this pool.

In an attempt to gain new information on the structure and function of presynaptic proteins, we applied a comparative view and conducted multiple sequence alignment (MSA) analysis of human and insects for representatives of the exocytotic machinery, VAMP-2, and synaptotagmin 1 (Figure 1). VAMP-2 is a short, evolutionary conserved protein of 120 to 220 amino acids with a SNARE-interacting domain and a single transmembrane domain (TMD) that crosses the synaptic vesicle membrane. Short signatures in VAMP's sequence that serve as recognition sites for tetanus and botulinum toxins 37 and the amino acids that are critical for VAMP targeting 38 are conserved from human to insects (Figure 1a). The sequence difference in the MSA is restricted to VAMP2 protein tails. A short proline-rich region that is responsible for VAMP2 interaction with synaptophysin 39 is not conserved. This is in accordance with the lack of synaptophysin in insect synaptic vesicles 40 (Table 1). On the other hand, a short region facing the synaptic vesicle lumen is highly conserved among all insects. Interestingly, there are two VAMP variants in honeybee that differ only in their luminal domain, enforcing a functional difference between these two variants (Figure 1). The possibility that a functional binding domain is located in the luminal domain is consistent with findings for other synaptic vesicle proteins, including synaptotagmin 41 and SV2 42.

MSA of highly conserved sequences from human to insects was also performed for synaptotagmin (Figure 1b). Synaptotagmins belong to a large and diverse gene family that coordinate multiple signals with trafficking and with membrane fusion 54344. In the mammalian synapse, synaptotagmin 1 (and 2) is a genuine synaptic vesicle protein that serves as the calcium sensor and interacts with SNAREs as well as with the calcium channel 45. In addition, synaptotagmin is a linker to the endocytotic adaptor protein AP2 46. The overall similarity of synaptotagmin between mammals and insects is high throughout the cytoplasmic region, but this similarity does not extend to the luminal region. In the cytoplasmic region, the domain that was postulated to interact with AP2 and with neurexin is strongly conserved, suggesting that not only is the main function of the protein conserved but also is its engagement in a rich protein interaction network.

Because endocytosis and membrane recycling are integral processes in presynaptic function, we compared stoned B (STNB) between human and insects 46 (Additional data file 4). Stoned genes (in insects StnA and StnB) are part of the protein lattice network that is involved in clathrin-mediated endocytosis at synapses. The conservation level of human stoned B (called SALF) is rather low (<50% sequence similarity). Several short signatures along the proteins act in the binding of AP2 subunits (for example, AP50 for StnB and α-adaptin for StnA). The number and the positions of these short signatures are not conserved in vertebrates and insects (Additional data file 4). In addition to the binding of AP2 by StnB, it binds to synaptotagmin 1 within the 300 amino acids in the carboxyl-terminal in the fly and human homologs. Stoned proteins may support synaptotagmin 1 recycling by mediating the association with the AP2 complex. Based on the MSA analysis, additional strongly conserved sequences are suggested (syntaxin; Additional data file 5). These sequences are probably essential in interactions between yet undefined partners that are common to mammals and insects. Most MSAs of PS120 show that the level of conservation is much higher among the insect sequences as compared with human or other organisms. We emphasize that MSA from insects to human for strongly conserved proteins (synaptotagmin, syntaxin 1A, and VAMP2) and for much less conserved genes (stoned B, SCAMP1, and synapsin 1) is instrumental in detecting overlooked sequences that may be important for protein interactions, protein modifications, and regulatory functions. The MSA for syntaxin 1 and synapsin 1 is included in Additional data file 5.

We tested whether the comparative genomics perspective is informative in studying the evolution of physical and functional complexes in exocytosis and trafficking. To this end, we tested the conservation levels for the various components of the exocyst.

The exocyst is a large complex that was initially identified at the tip of the yeast bud. It participates in tethering vesicles to the plasma membrane. It coordinates exocytosis with small G-protein signalling molecules such as Ral-A, Arf6, and Rab11 47. The exocyst is composed of eight subunits that are denoted EXOC1 to EXOC8 (Figure 2a) and are homologs of the yeast Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84 genes 48. The level of conservation of the various subunits between human and fly range from 30% to 50% sequence identity (50% to 70% sequence similarity; Figure 2). The homologous relationship is evident and is supported by alignments that cover the entire protein length. However, among the insects, the mutual sequence conservation for EXOC8 is rather low (Figure 2a), because the honeybee and beetle homologs for EXOC8 are further diverged; hence, an apparent homology could not be assigned. Because the function of the exocyst relies on coordination of its subunits, we anticipated that EXOC8 would be missed during the task of genome annotation. This is further supported by the observation that several interacting proteins of the exocyst such as Ral-A 47 and septin 5 49 are strongly conserved in all insects (Figure 2b). A search for sequence similarity in the honeybee and beetle genomes identified a supported mRNA for EXOC8 in honeybee and an apparently unprocessed sequence in the beetle genome (for details, see Additional data file 3). We conclude that physical complexes co-evolved because of similar evolutionary constraints.

Coatomer protein (COP)-1 vesicles are principally involved in transport of cargo between the endoplasmic reticulum (ER) and early Golgi 5051. Specifically, they mediate both the anterograde flow of cargo through the Golgi to the cell surface and the retrograde retrieval of recycling proteins from late to early Golgi compartments. COP-1 is composed of 7 genes (α, β, β ', γ, δ, ε, and ζ subunits, additional genes resulting from duplication events, γ2 and ζ2) that are different in sequence and length. For example, whereas COPA (human homolog of α) is composed of 1,200 amino acid, COPZ (human homolog of ζ) consists of only 200 amino acids. Figure 3a shows the sequence identity of COP-1 components relative to human, for all four insect representatives. As may be observed, in eight of the nine genes the degree of conservation between human and insects varies little across insect species. An exception is COPE (εCOP-1), which, in addition to being the least conserved in the fly and mosquito, exhibits a large variation in the levels of conservation among insects. The honeybee COPE is significantly more conserved than that of the fly, mosquito or beetle homologs. We anticipate that COPE may display a different pace of evolutionary change that may be a result of its specific role in the COP-1 complex. Indeed, a role for this component in stabilizing rather than in the assembly of the COP-1 complex has been proposed 52.

The synapse is a compact structure with multiple organelles, including transport vesicles, early and late endosomes, lysosomes, and peroxisomes. Indeed, many of the PS120 representatives function in vesicle trafficking and sorting. Snapin (SNAPAP) is among the genes that are missing in some but not all insects. Snapin was initially identified as a SNAP-25 binding protein and a regulator of the interaction of synaptotagmin with the SNAREs 53. The relevance of snapin in neurotransmitter release regulation was questioned 54, and instead it was postulated to be part of the biogenesis of lysosome-related organelles complex (BLOC) 5556. We compared the conservation of the subunits of BLOC-1 in human and insect (Figure 3b). The BLOC-1 complex is composed of eight short proteins (12 to 15 kDa) that are rich in helical structures. The composition of BLOCs is based on biochemical purifications and on localization studies 57, but the function of the individual subunits of BLOC-1 remains elusive.

A human homolog of snapin (SNAPAP; Table 1) is detected in honeybee, fly, and mosquito, but cannot be detected in the beetle genome (see Additional data files 2 and 3). BLOC1S2 and cappuccino are also missing in the beetle proteome, whereas BLOC1S3 is missing in all insects (Figure 3b). A detailed pair-wise interaction analysis showed that BLOC1S3 is peripheral and its interaction with other BLOC-1 subunits is only through BLOC1S2 57. Another component of BLOC-1 is dysbindin (DTNBP1). DTNBP1 is weakly conserved in insects (identity 26% to 28% from human to fly and beetle), and the fact that it is missing in both mosquitoes (Anopheles and Aedes) indicates that this is probably not due to annotation mistakes (Figure 3b). Interestingly, defects in DTNBP1 and other BLOC-1 components are linked to severe pathologies in humans 58. Our findings are consistent with the notion that BLOC-1 is functional despite some missing components and suggest that there is some level of redundancy among BLOC-1 subunits in insects.

The analysis of BLOC-1, COP-1, and the exocyst complexes (Figures 2 and 3) implies that the conservation levels for most subunits are similar within each complex and functional group. To test the generality of this observation along the evolutionary tree, we quantified the level of sequence identity in proteins that function in trafficking complexes and organelle biogenesis. The pair-wise sequence identity serves to reflect the conservation index. We tested the following organisms relative to human: mouse (Mus musculus), chicken (Gallus gallus) bony fish (Zebrafish; Danio rerio), frog (Xenopus laevis) and fly (D. malanogaster). Figure 4 shows the conservation relative to human proteins (measured as the percentage identity) for vesicle trafficking and organelle biogenesis complexes. We tested the presynaptic site protein complexes (exocyst and COP-1) and organelle biogenesis sets (BLOC-1 and peroxin biogenesis [PEX] genes, which participate in peroxisome biogenesis) and complexes from the postsynaptic site: the dystrophin glycoprotein complex (DGC), a complex that serves as a link between the cytoskeleton and the extracellular matrix in skeletal muscle cells 59; and the active signaling complex of the metabotropic glutamate receptor (mGC), which includes glutamate receptors and their partners, such as cytoskeletal and post-translational modification enzymes.

In general, for the four sets of presynaptic sites, all tested species maintain a conservation index in a rather tight range, in which each complex exhibits a unique profile along the evolutionary tree. Specifically, the conservation of fly to human is in accordance with a high degree of coordination among these four complexes. The exocyst and COP-1 are the least diverged whereas the organelle biogenesis complexes (PEX and BLOC-1) exhibit a more active evolutionary divergence for at least some of their components (Figure 4). Although the components of COP-1 and BLOC-1 physically interact, the PEXs (peroxisome-related proteins) are a dynamic group of proteins with 14 gene products that function in executing the peroxisomal life cycle 6061. Each PEX protein is unique in length, structure, and function. The evolutionary conservation pattern is preserved across the five species included in this analysis, throughout the various components of the complexes. Presumably, the shared functions of the different components lead to their co-evolution.

To explore whether the coordination within complexes and functional groups along the evolutionary tree holds for other physical or functional complexes, we examined the DGC 59 and the active signaling complex of the mGC from the postsynaptic membrane 62. Among the various proteins of these postsynaptic complexes, each species exhibits a different level of conservation relative to human. For example, DGC, the frog DMD (dystrophin), and UTRN (utrophin) 63 are almost identical to human, whereas SGCB and SGCD (β-sarcoglycan and δ-sarcoglycan) are poorly conserved. On the other hand, in zebrafish utrophin is poorly conserved whereas β-sarcoglycan and δ-sarcoglycan are more similar to human. A similar uncoordinated profile for conservation was shown for the mGC. We propose that for biogenesis, exocytosis, and trafficking complexes of the presynaptic sites (but not for postsynaptic signaling complexes), evolutionary constraints have led to co-evolution of the components.

Sequential protein interactions are fundamental to the lifecycle of the synaptic vesicle and to trafficking and organelle biogenesis in synapses. This leads to proteins that are engaged in multiple protein interactions. For example, more than 60 different interactions have been reported for syntaxin 1 and tens of interactions for synaptotagmin. Although some findings may result from spurious interactions, many have been experimentally confirmed and others are yet to be discovered. We illustrate this via VAMP2 as a prototype for an extremely conserved protein from human to insects. Figure 5 shows a graph centered on human VAMP2 along with 20 of its high-fidelity interacting proteins. Nineteen of these (excluding only synaptophysin) are conserved in all insects, and conservation for most of them (18/19) is very high (network conservation is 0.86). Another extreme case is that of the Rab3A protein (Figure 5b). Although the valence of Rab3A is very high (19), the properties of the two graphs are substantially different (Figure 5). Few Rab3A partners are missing in all insects and additional ones are missing in some insects, leading to a low conservation (network conservation 0.3).

The fraction of connecting edges in the graph relative to the maximal possible edges is a measure of the connectivity among interacting proteins. Density values for the interacting proteins of VAMP2 and RAB3A are 16.8% and 6.4%, respectively. Figure 5 illustrates proteins of the presynaptic apparatus that differ substantially in their valence, network conservation score, and density value.

We illustrate the properties of protein-protein interaction graphs for several representative proteins from the PS120 set (Figure 6; gene names are according to official symbols; see Additional data file 2). The protein interaction networks are supported by evidence from the literature, experimental data, and strong homology. Only high confidence interactions are shown (see Materials and methods, below). These proteins are as follows: VAMP8, a synaptic vesicle and exocytosis related protein; neurexin-1 (NRXN1), which acts in synaptogenesis and in the pre-post synaptic junctions; synaptojanin 1 (SYNJ1) and dynamin 1 (DNM1), which are endocytotic proteins that function in synaptic vesicle recovery and in clathrin-based endocytosis, respectively; and Rim-1 (RIMS) and Cast (ERC2), which are two active zone organizers.

The protein valence (defined as the number of direct edges from the vertex representing the protein) ranges from seven for ERC2 to 23 for DNM1. The graphs of RIMS, ERC2, and NRXN1 have relatively low connectivity. Specifically, in the NRXN1 graph there are only 14 edges, and for RIMS (with a valence of 15) just 29 connecting edges are observed. The density of the different graphs and their network conservation scores are marked (Figure 6). Note that for some of the interacting proteins no insect homologs are known (marked by a yellow circle; Figure 6). The low connectivity graphs are characteristic for additional proteins of the active zone and for some master regulators such as RAB3A (Figure 5) and LIN7A (Additional data file 6). DMN1, which is one of the central proteins of endocytosis, exhibits a mixed property in the protein interaction graph. Most edges are of low connectivity, but about one-third of the edges are highly connected. DMN1 and SYNJ1 valence is rather high (23 and 20, respectively) with only an intermediate network conservation score of 0.42 and 0.43, respectively. Note that for exocytosis proteins (namely VAMP2 and VAMP8), both the network conservation score and the density values are higher (Figures 5 and 6).

The interaction graphs for VAMP2 (Figure 5), VAMP8 (Figure 6), α-SNAP (SNAPA) and synaptotagmin 1 (SYT1; Additional data file 6), syntaxin 1 (STX1), and SNAP25 (not shown) are characterized by relatively high conservation and by high density values. The properties of the graphs for DNM1 and SYNJ1 are valid for numerous endocytotic proteins, including AP2A (Additional data file 6), clathrin, and amphiphysin (not shown).

Almost all proteins in the PS120 gene list are engaged in multiple protein interactions (Additional data file 2). Interactions between proteins in the synapse occur throughout the processes of endocytosis, membrane fusion, protein recruitment, transport of vesicles, and organelle biogenesis. We tested whether conservation (as reflected by percentage sequence identity from insects to human) and the valence of all proteins are correlated. We considered only interactions with high confidence (see Materials and methods, below) and tested the two quantitative measures. Figure 7a shows the sequence similarity of all PS120 proteins as a function of the valence of human proteins. It is evident that the two quantities are positively correlated. Among the PS120 proteins, some exhibit extremely high numbers of interacting proteins (up to 50), as evident for signal transduction proteins, whereas others have no interacting partners. The latter could also be due to lack of current knowledge. Detailed information on PS120 protein conservation and valence is available in Additional data file 7. Note that correlation between conservation and protein valence is strongly associated with sequences that share greater than 50% identity (Figure 7). The positive correlation between conservation and protein valence is based on the full PS120 list. A similar analysis of components of functional complexes in trafficking (COP-1 and PEX; Figure 7b) is in accordance with the overall trend observed for PS120. (Note that these additional 25 proteins of COP-1 and PEX are not included in PS120.) We conclude that despite a relatively narrow range of sequence conservation among the components of each complex (Figures 2 and 3), a greater conservation score between insect and human is correlated with higher valence.

The next question that was addressed is whether the subsets of proteins that function primarily in endocytosis and in exocytosis exhibit different properties of sequence conservation and protein valence. To this end, we compiled two nonoverlapping subsets for endocytosis and exocytosis (24 proteins each; see Materials and methods, below). These two lists excluded proteins that are bona fide participants of both processes (for instance, DOC2, UNC13, PSCD1, and RIMS) or are connected to cytoskeleton modulation (such as ADD2, bassoon, CADPS, DLG1, KIF1A, and MYRIP). In the set of exocytotic and endocytotic proteins, the average protein valences are 10 and 7.9, respectively (P value for being identical = 0.423; Additional data file 8). However, for the exocytotic set the average valence for proteins with low conservation (<50% sequence identity) is 4.5, and 13.9 for those that share greater than 50% sequence identity. This result is significant (P = 0.034). In the endocytotic set, the valences of low and highly conserved proteins (<50% and >50% identity) were not significantly different (P = 0.605) and measured to be 7.2 and 8.6, respectively (Additional data file 7).

We observed that the protein interaction graphs differ substantially with regard to their properties between essential proteins of the exocytotic and endocytotic sets (Additional data file 8). Specifically, several of the endocytotic proteins interact with proteins that are less conserved between insects and human (Figure 6). We tested the underlying molecular architecture of exocytotic and endocytotic proteins. Figure 8 shows a set of 15 proteins from each of the exocytotic and endocytotic sets. On average the endocytotic proteins are longer: 850 and 340 amino acids for the endocytotic and exocytotic proteins, respectively.

Additional features that were tested include the fraction of low complexity and coiled coil regions. It was shown that in both protein sets approximately 10% of the sequences are occupied by low complexity regions. However, coiled coil domains are detected mostly in endocytotic proteins. The most obvious difference is in the architecture of the exocytotic and endocytotic proteins, in that most endocytotic proteins are multi-domain proteins, some of them repeating several times within a protein and across the protein set. Examples are clathrin (CLTC; clathrin domains repeat seven times) and intersectin (ITSN2; with multiple SH3 domains). (For consistency, we applied the domain assignment according to Pfam 64.) Such features are rare among the exocytotic protein set (Figure 8). Some highly represented domains in the endocytotic protein set are short (Figure 8). These domains coordinate interactions with either short signatures (SH3), lipid moieties (PX and PH), or signaling ions (EF-hand). These are abundant domains that appear thousands of times in the animal kingdom and are used in a variety of cellular contexts, including outside the synapse.

The abundance of short domains with a broad specificity is rare in the exocytotic context. The domains marked as v-SNARE and t-SNARE, Synaptobrevin and Sec1 (Figure 8) are engaged in protein-protein interactions. Specificity is often achieved through elongated interfaces in helical regions 65. An exception is the C2 domain, which appears in synaptotagmin. Although synaptotagmin is a genuine protein of exocytosis, it has some resemblance in its architecture to endocytotic proteins. Specifically, the C2 domain is broadly used in signaling proteins that are regulated by calcium in a context of membrane interactions. Many C2-containing proteins in the synapse are actually linkers of exocytosis and endocytosis 66, including BAIAP3, SYTL4, RIMS, and MUNC13 (Additional data file 2 [part b]). Roles of synaptotagmin and synaptotagmin-like proteins as linkers between exocytosis and endocytosis 46 and the cytoskeleton 67 have been proposed.

Mammalian synaptic vesicles were characterized by three major proteins 71: SV2, synaptotagmin, and synaptophysin. Many of the synaptic vesicle membranous proteins appeared to be regulators of SNAREs and thus control neurotransmitter release. Examples are the transporter-like SV2, which controls synaptoagmin 1 72, and synaptophysin, which controls VAMP-2 73. Based on a recent study on the composition of a generic mammalian synaptic vesicle 18, it was calculated that VAMP2 and synaptophysin are at a 2:1 molar ratio and both are the most abundant proteins of the synaptic vesicle membrane. In synaptophysin knockout mice, no changes in synapse function and brain morphology have been detected, but VAMP2 concentration on the synaptic vesicle membrane was shown to be markedly altered 74. Along this line, the absence of synaptophysin gene in insects is intriguing (Table 1). Synaptophysin is part of a small family of four-transmembrane proteins that includs synaptogyrin, pantophysin, and synaptoporin, all of which are missing in insects. A genomic search revealed a weak similarity to synaptogyrin in honeybee supported by a transcript (XR_015081.1; hypothetical LOC552402 having mRNA of 704 nucleotides) and a weak homology in beetle for Synaptoporin (XM_967892.1; similar to synaptoporin, LOC661749). Despite its abundance in mammalian synaptic vesicles, Synaptophysin and the other members of the four-transmembrane protein set must be dispensable for the functionality of synaptic vesicles. This is in agreement with the findings of a recent study on C. elegans 75, in which complete removal of the synaptophysin gene family resulted in normal synaptic properties, synaptogenesis, and neuronal architecture. It is plausible that in insects VAMP accessibility is regulated by an alternative regulator. The extension of VAMP's luminal domain in insects is consistent with the possibility that such a domain is used for regulation (Figure 1