Syntaxin 1A


Protein Interactions (part 3/3)

NSF, a Syntaxin-binding protein

Several lines of investigation have now converged to indicate that the neurotransmitter release apparatus is formed by assembly of cytosolic proteins with proteins of the synaptic vesicle and presynaptic terminal membranes. A genetic approach has been undertaken in Drosophila to investigate the functions of two types of cytosolic proteins thought to function in this complex: N-ethylmaleimide-sensitive fusion protein (NSF) and the soluble NSF attachment proteins (SNAPs). Drosophila homologs of the vertebrate and yeast NSF and SNAP genes have been identified. Both Drosophila genes encode polypeptides that closely resemble their vertebrate counterparts and are expressed in the nervous system; neither appears to be in a family of closely related Drosophila genes. These results indicate that the Drosophila NSF and SNAP genes are excellent candidates for mutational analysis of neurotransmitter release (Ordway, 1994).

The N-ethylmaleimide-sensitive fusion protein (NSF) is a cytoplasmic protein implicated in the fusion of intracellular transport vesicles with their target membranes. NSF is thought to function in the fusion of essentially all types of vesicles, including endoplasmic reticulum, Golgi, and endocytic vesicles, as well as secretory vesicles undergoing regulated fusion. However, little experimental evidence exists to address the possibility that organisms might have multiple NSF proteins serving distinct functions in the same or different cells. A neurally expressed Drosophila homolog (dNSF-1/comatose) has been cloned, and mutations have been identified in this gene that confer an apparent failure of synaptic transmission at elevated temperature. Drosophila contains a second NSF homolog, termed dNSF-2, that exhibits 84% amino acid identity to dNSF-1. dNSF-1 and dNSF-2 display overlapping but different temporal expression, and multiple transcripts are derived from the dNSF-2 gene. These findings raise the possibility that different NSF gene products serve distinct or overlapping functions with the organism (Pallanck, 1995).

N-Ethylmaleimide-sensitive fusion protein (NSF) is an ATPase known to have an essential role in intracellular membrane transport events. cDNA clones encoding a Drosophila melanogaster homolog of this protein, named dNSF, were characterized and found to be expressed in the nervous system. A second homolog of NSF, called dNSF-2, has been identified and it is a ubiquitous and widely utilized fusion protein belonging to a multigene family. The predicted amino acid sequence of dNSF-2 is 84.5% identical to dNSF (hereafter named dNSF-1); 59% identical to NSF from Chinese hamster, and 38.5% identical to the yeast homolog SEC18. The highest similarity was found in a region of dNSF-2 containing one of two ATP-binding sites; this region is most similar to members of a superfamily of ATPases. dNSF-2 is localized to a region between bands 87F12 and 88A3 on chromosome 3, and in situ hybridization techniques reveal expression in the nervous system during embryogenesis and in several imaginal discs and secretory structures in the larvae. Developmental modulation of dNSF-2 expression suggests that quantitative changes in the secretory apparatus are important in histogenesis (Boulianne, 1995).

The neuronal SNARE complex is formed via the interaction of synaptobrevin with syntaxin and SNAP-25. Purified SNARE proteins assemble spontaneously, while disassembly requires the ATPase NSF. Cycles of assembly and disassembly have been proposed to drive lipid bilayer fusion. However, this hypothesis remains to be tested in vivo. A Drosophila temperature-sensitive paralytic mutation in syntaxin has been isolated that rapidly blocks synaptic transmission at nonpermissive temperatures. This paralytic mutation specifically and selectively decreases binding to synaptobrevin and abolishes assembly of the 7S SNARE complex. Temperature-sensitive paralytic mutations in NSF (comatose) also block synaptic transmission, but over a much slower time course and with the accumulation of syntaxin and SNARE complexes on synaptic vesicles. These results provide in vivo evidence that cycles of assembly and disassembly of SNARE complexes drive membrane trafficking at synapses (Littleton, 1998).

The SNARE hypothesis has been proposed to explain both constitutive and regulated vesicular transport in eukaryotic cells, including release of neurotransmitter at synapses. According to this model, a vesicle targeting/docking complex consisting primarily of vesicle- and target-membrane proteins, known as SNAREs, serves as a receptor for the cytosolic N-ethylmaleimide-sensitive fusion protein (NSF). NSF-dependent hydrolysis of ATP disassembles the SNARE complex in a step postulated to initiate membrane fusion. While features of this model remain tenable, recent studies have challenged fundamental aspects of the SNARE hypothesis, indicating that further analysis of these components is needed to fully understand their roles in neurotransmitter release. This issue was addressed by using the temperature-sensitive Drosophila NSF mutant comatose (comt) to study the function of NSF in neurotransmitter release in vivo. Synaptic electrophysiology and ultrastructure in comt mutants have recently defined a role for NSF after docking in the priming of synaptic vesicles for fast calcium-triggered fusion. An SDS-resistant neural SNARE complex, composed of the SNARE polypeptides syntaxin, n-synaptobrevin, and SNAP-25, accumulates in comt mutants at restrictive temperature. Subcellular fractionation experiments indicate that these SNARE complexes are distributed predominantly in fractions containing plasma membrane and docked synaptic vesicles. Together with the electrophysiological and ultrastructural analyses of comt mutants, these results indicate that NSF functions to disassemble or otherwise rearrange a SNARE complex after vesicle docking and that this rearrangement is required to maintain the readily releasable pool of synaptic vesicles (Tolar, 1998).

N-Ethylmaleimide-sensitive fusion protein (NSF) is a cytosolic protein thought to play a key role in vesicular transport in all eukaryotic cells. Although NSF was proposed to function in the trafficking of synaptic vesicles responsible for neurotransmitter release, only recently have in vivo experiments begun to reveal a specific function for NSF in this process. Mutations in a Drosophila NSF gene, dNSF1, are responsible for the temperature-sensitive paralytic phenotype in comatose (comt) mutants. In this study, electrophysiological and ultrastructural analyses were performed in three different comt alleles to investigate the function of dNSF1 at native synapses in vivo. Electrophysiological analysis of postsynaptic potentials and currents at adult neuromuscular synapses reveal that in the absence of repetitive stimulation, comt synapses exhibit wild-type neurotransmitter release at restrictive (paralytic) temperatures. In contrast, repetitive stimulation at restrictive temperatures reveals a progressive, activity-dependent reduction in neurotransmitter release in comt but not in wild type. These results indicate that dNSF1 does not participate directly in the fusion of vesicles with the target membrane but rather functions in maintaining the pool of readily releasable vesicles competent for fast calcium-triggered fusion. To define dNSF1 function further, transmission electron microscopy was used to examine the distribution of vesicles within synaptic terminals, and a marked accumulation of docked vesicles at restrictive temperatures was observed in comt. Together, the results define a role for dNSF1 in the priming of docked synaptic vesicles for calcium-triggered fusion (Kawasaki, 1998).

The N-ethylmaleimide sensitive fusion protein (NSF) was originally identified as a cytosolic factor required for constitutive vesicular transport and later implicated in synaptic vesicle trafficking as well. Work at neuromuscular synapses in the temperature-sensitive NSF mutant comatose (comt) has shown that the comt gene product, dNSF1, functions after synaptic vesicle docking in the priming of vesicles for fast calcium-triggered fusion. Whether dNSF1 performs a similar function at central synapses associated with the well-characterized giant fiber neural pathway was investigated. These include a synapse within the giant fiber pathway, made by the peripherally synapsing interneuron (PSI), as well as synapses providing input to the giant fiber pathway. The latency (delay) between stimulation and a resulting muscle action potential was used to assess the function of each class of synapses. Repetitive stimulation of the giant fiber pathway in comt produces wild-type responses at both 20 and 36 degrees C, exhibiting a characteristic and constant latency between stimulation and the muscle response. In contrast, stimulation of presynaptic inputs to the giant fiber (referred to as the 'long latency pathway') reveals a striking difference between wild type and comt at 36 degrees C. Repetitive stimulation of the long latency pathway leads to a progressive, activity-dependent increase in the response latency in comt, but not in wild type. Thus the giant fiber pathway, including the PSI synapse, appears to function normally in comt, whereas the presynaptic inputs to the giant fiber pathway are disrupted. Several aspects of the progressive latency increase that are observed in the long latency pathway can be understood in the context of the activity-dependent reduction in neurotransmitter release observed at neuromuscular synapses. These results suggest that repetitive stimulation causes a progressive reduction in neurotransmitter release by presynaptic inputs to the giant fiber neuron, resulting in an increased latency preceding a giant fiber action potential. Thus synapses presynaptic to the giant fiber appear to utilize dNSF1 in a manner similar to the neuromuscular synapse, whereas the PSI chemical synapse may differ with respect to the expression or activity of dNSF1 (Kawasaki, 1999).

The wing of Drosophila has long been used as a model system to characterize intermolecular interactions important in development. Implicit in an understanding of developmental processes is the proper trafficking and sorting of signaling molecules, although the precise mechanisms that regulate membrane trafficking in a developmental context are not well studied. The Drosophila wing was used to assess the importance of SNARE-dependent membrane trafficking during development. N-Ethylmaleimide-sensitive fusion protein (NSF) is a key component of the membrane-trafficking machinery and a mutant form of NSF was constructed whose expression was directed to the developing wing margin. This resulted in a notched-wing phenotype, the severity of which was enhanced when combined with mutants of VAMP/Synaptobrevin or Syntaxin, indicating that it results from impaired membrane trafficking. Importantly, the phenotype is also enhanced by mutations in genes for wingless and components of the Notch signaling pathway, suggesting that these signaling pathways were disrupted. Finally, this phenotype was used to conduct a screen for interacting genes, uncovering two Notch pathway components that had not previously been linked to wing development. It is concluded that SNARE-mediated membrane trafficking is an important component of wing margin development and that dosage-sensitive developmental pathways can act as a sensitive reporter of partial membrane-trafficking disruption (Stewart, 2001).

The Syntaxin, VAMP, and SNAP-25 families of proteins are proposed to target and fuse transport vesicles with specific membrane compartments. The SNARE complex is a parallel four-helix bundle with one helix contributed by each of Syntaxin and VAMP and two contributed by SNAP-25 (Sutton, 1998). The formation of a trans-membrane complex, with VAMP on the transport vesicle and Syntaxin and SNAP-25 on the target membrane, is thought to lead to the fusion of the two membranes, resulting in a cis-membrane complex. It follows that the cis-residing protein complexes need to be broken apart to make those proteins available for further trans-complex formation. This complex breakdown occurs under the action of N-ethylmaleimide-sensitive fusion protein (NSF), an ATPase. NSF contains two nucleotide binding domains and demonstrable ATPase activity. Structural analyses have shown that NSF forms a hexamer in vivo. NSF is a homolog of the yeast gene SEC18 and analysis of SEC18 function also reveals its requirement for intracellular membrane transport. NSF-dependent ATP hydrolysis is required to disassemble SNARE complexes, although it is not required for the fusion step. Thus the role of NSF in vesicular transport appears to be primarily one of priming vesicles for fusion and dissociation of SNARE complexes to permit their recycling (Stewart, 2001).

In Drosophila there are two homologs of NSF: dNSF1 and dNSF2 (NEM-sensitive fusion protein 2). dNSF1 is the gene product of comatose and is primarily found in neurons, whereas dNSF2, in addition to being neuronally expressed, is broadly expressed within imaginal discs, salivary glands, and the ring gland (Boulianne, 1995). Thus, dNSF2 is the most likely isoform to contribute to intracellular trafficking in nonneuronal tissue. Despite their proposed role in most intracellular trafficking events, in vivo studies of SNARE proteins have concentrated on two main systems: the budding yeast and calcium-triggered exocytosis in neurons. Relatively little attention has been given to other in vivo contexts in which the SNARE proteins are likely to have important roles. For example, in signaling pathways it is self-evident that transmembrane receptors and ligands need to be delivered to the plasma membrane, although few studies have been devoted to specifically studying the role of SNARE proteins in this process and their potential influence on the strength of intracellular signaling (Stewart, 2001).

To investigate the role of SNARE proteins within a defined developmental process, advantage was taken of the key role of NSF in membrane-transport processes. Specifically, a dominant negative form of dNSF2 was expressed in wing imaginal discs and this was shown to disrupt proper wing margin formation. This phenotype is enhanced in trans-heterozygous combinations of mutant alleles of the SNARE proteins syntaxin or synaptobrevin, further supporting a role for SNAREs in this process. Using genetic and immunocytochemical analysis it has been shown that this phenotype can be attributed to a failure in the signaling pathways that normally govern wing margin development. Thus, SNARE-dependent transport mechanisms are critical to wing formation and their manipulation may provide new insights into the mechanisms controlling developmentally important signaling pathways (Stewart, 2001).

To investigate the function of SNARE-dependent transport mechanisms in Drosophila point mutants in the ATP-binding region of the D1 domain of dNSF2 were constructed. Each nucleotide-binding subdomain of NSF contains consensus ATP-binding domains known as the Walker A and Walker B motifs. The DEAD box of the Walker B motif is conserved in a large number of ATP-dependent enzymes and was first identified in RNA helicases that use ATP hydrolysis to unwind RNA prior to translation. This motif binds the Mg2+ ion that coordinates the phosphates of ATP for hydrolysis. In RNA helicases replacement of the glutamate residue within the modified DEAD box (DEID) eliminates ATP hydrolysis without affecting ATP binding). In mammalian NSF a similar substitution within that protein’s DEID box, E329Q, reduces ATPase activity and NSF-dependent Golgi transport activity. NSF has been shown to form hexamers and, when mixed with wild-type protein NSFE329Q, forms hexamers that also lack ATPase activity, leading to a dominant negative effect. Drosophila NSF2 shows 59% overall amino acid identity with CHO NSF and nearly 100% conservation within the ATP-binding p-loop and DEID box of the D1 domain. Thus the structural and functional properties of the dNSF2 ATPase domains are very likely to be identical to those previously defined in RNA helicases and mammalian NSF, and mutation of the glutamate residue with the Drosophila DEID box motif should also impair the ATPase activity of the protein (Stewart, 2001).

A NSFE/Q construct was created with a glutamate-to-glutamine substitution at position 326 of the dNSF2 D1 domain. In two separate ATPase assays it was found that the NEM-sensitive ATPase activity of NSFE/Q is 47.5% and 57.1% that of dNSF2WT. The mean ATPase activity is 15.2 nmol Pi/microg/h for the wild-type protein and 7.8 nmol Pi/microg/h for the mutant protein. The remaining ATPase activity in NSFE/Q may be attributable to the second ATPase site within the D2 domain of the protein (Stewart, 2001).

To express the mutant dNSF2 transgenic flies were created carrying UAS-NSFE/Q and UAS-NSFWT constructs for use in the Gal4-UAS expression system. C96-Gal4 is expressed in developing wing discs in a pattern that is similar to, though slightly broader than, wing margin proteins such as Wingless. When UAS-NSFE/Q is driven by C96-Gal4, loss of wing margin is observed. The expression of NSFWT does not cause any visible phenotype, indicating that simple overexpression of dNSF2 in the wing margin is not a cause of the phenotype (Stewart, 2001).

The observation that NSFE/Q causes loss of wing margin implies that SNARE-dependent transport is important for wing margin formation. To test this further mutant alleles of synaptobrevin and syntaxin, two well-characterized SNARE proteins, were used to determine whether they would enhance the wing phenotype. Indeed, all trans-heterozygous combinations of NSFE/QC96 with synaptobrevin or syntaxin loss-of-function alleles enhance the wing margin phenotype, thus providing further evidence of the involvement of SNARE proteins in wing margin development (Stewart, 2001).

The wing phenotype observed is similar to that observed with mutant alleles of Notch and Wingless signaling pathway genes. To determine whether components of these pathways could be contributing to the NSFE/QC96 wing phenotype the protein pattern of Wingless in third-instar imaginal wing discs was first examined and a striking effect on the distribution of Wingless was observed. In control discs Wingless appears as a three- to four-cell-wide stripe across the wing disc, whereas in discs expressing the mutant dNSF2 Wingless appears very narrow and patchy. Wg expression was then examined using a Wg-lacZ reporter construct and an incomplete pattern of Wingless expression was found, as was observed for the Wingless protein (Stewart, 2001).

Because Wg is a secreted protein Wg was examined under higher magnification using confocal microscopy to determine directly whether Wg secretion was impaired. In control discs there is punctate Wg staining, indicative of Wg secretion, in the tissue surrounding the narrow stripe of wing margin cells. In the regions of the mutant discs that are immunoreactive for Wg, punctate staining is seen surrounding the positive cells. However, the Wg signal is much stronger in those cells and confocal sectioning of the cells has revealed the accumulation of Wg at the apical region of the wing margin cells. These data indicate that mutant NSFE/Q impairs, but does not eliminate, Wingless secretion. Because Wingless expression is impaired and its activation is under the control of Notch signaling, the distribution patterns of other proteins involved in the Notch pathway were examined. Notch protein distribution was examined directly using a monoclonal antibody that recognizes the extracellular domain of Notch. At low magnification there is no major difference between mutant and control samples, with the antibody labeling the cell membranes in the wing pouch. However, at higher magnification, in addition to the membrane staining, immunoreactive puncta were also observed within the cells of the mutant wing disc that were not readily observed in the control discs. These puncta likely represent improperly sorted Notch proteins (Stewart, 2001).

The distribution of Cut, Delta, and Achaete, coded for by genes that are downstream of Notch activation in the wing margin signaling pathway, was examined -- all of these markers were disrupted in NSFE/QC96 larval wing discs. Cut is normally found in a pattern that overlaps with Wg along the presumptive wing margin, whereas in the mutant discs it appears in a broken pattern similar to that of Wg. Delta is normally expressed in two parallel bands along the D/V boundary and this pattern is thought to be the result of the downregulation of Delta in boundary cells by Cut and the upregulation of Delta in flanking cells by Wingless. In NSFE/QC96 wing discs the expression of Delta is reduced and the two parallel bands appear to be collapsed into a single band along the boundary. Achaete is normally expressed in two broad bands parallel to the D/V boundary in the anterior compartment of the wing disc defining a proneural cluster. In the NSFE/QC96 discs this pattern is severely disrupted: the number of Achaete-expressing cells is reduced and there is complete absence of Achaete in some areas (Stewart, 2001).

A similar pattern of disruption was found when lacZ reporter constructs were used to examine the expression of neuralized and vestigial, two other genes in the Notch pathway. neuA101-lacZ is normally detected in sensory organ precursors (SOPs) located in two rows of single cells parallel to the D/V boundary in the anterior compartment of late third-instar wing discs. In the mutant discs this pattern is disrupted and lacking in some areas along the wing margin, while SOPs elsewhere in the disc are unaffected. Similarly, vgBE-lacZ expression is disrupted. In wild-type discs vgBE-lacZ expression is seen in the D/V and anterior/posterior (A/P) boundaries, whereas in the mutant discs the expression in the D/V boundary is disrupted (Stewart, 2001).

Interestingly, expression in the A/P boundary remains, although the C96-Gal4 expression pattern overlaps this region. Taken together these results demonstrate that NSFE/Q affects the distribution and expression of several downstream components of the Notch signaling pathway. To confirm the effect of NSFE/Q on Notch signaling loss-of-function alleles of several genes in the Notch and Wingless pathways were examined for their ability to enhance the adult wing phenotype caused by NSFE/Q expression. In that Notch signaling is known to be highly sensitive to haploinsufficiency of interacting gene products, it was reasoned that these loss-of-function alleles should show genetic interaction. Two alleles of Notch and one each of Delta, Serrate, wingless, and fringe were examined and it was found that they all enhanced the wing phenotype in transheterozygous combination with NSFE/QC96. The severity of the phenotype produced by each allele was similar, although Df(1)N8, a null allele of Notch, did produce a more severe phenotype than did Nnd-3, a hypomorphic allele. With the exception of Df(1)N8, none of these mutants produces a wing-nicking phenotype when examined alone as heterozygotes. Thus, the enhancement of the adult wing phenotype by mutants in the Notch pathway supports the conclusion that NSFE/Q expression causes a defect in wing margin signaling pathways (Stewart, 2001).

Finally, the ability of UAS constructs of Notch, Delta, and Serrate to rescue the wing phenotype generated by NSFE/QC96 were tested. Complete rescue could be obtained with both Notch and Delta constructs. Serrate generally appears to rescue less well than do the other constructs because minor nicks in the distal wing persist. Furthermore, no rescue effect was seen when crosses were made to UAS-lacZ lines, indicating that competition for Gal4 protein is not responsible for rescue of the phenotype. The observation that UAS-Notch and UAS-Delta can completely rescue the NSFE/Q wing phenotype further indicates that the mutation affects intracellular transport and does not create a cell-lethal phenotype because cell lethality should not be rescued by Notch or Delta (Stewart, 2001).

Having established that NSFE/Q disrupts signaling at the wing margin in a SNARE-dependent manner, and that enhancement of the phenotype can be attributed to haploinsufficiency of known genes, it was asked whether the wings of the NSFE/QC96 flies could be used as a sensitized background to find novel genes involved in wing margin formation. To this end a small-scale screen was conducted for enhancers and suppressors of the phenotype. In the first set of experiments specific alleles of two genes were tested: big brain and porcupine. These have been shown to be important in Notch and Wingless signaling in other developmental contexts but have not previously been known to be important for wing margin development. In the NSFE/QC96 background it was found that both mutant alleles of these genes enhance the NSFE/QC96 wing margin phenotype. This result is the first report of the involvement of these two genes in wing margin development and suggests that NSFE/QC96 wings provide an ideal sensitized background for conducting forward genetic screens to identify novel genes involved in wing margin development (Stewart, 2001).

In the second set of experiments a test was performed for genetic interactions with deficiencies that uncover most of the Drosophila genome. Of the deficiencies tested, 33 interacting lines were identified that enhanced or suppressed the wing margin phenotype. The further characterization of these loci may reveal novel components of the SNARE or Notch and Wg signaling pathways (Stewart, 2001).

In view of current membrane-trafficking models, it is expected that expressing NSFE/Q impairs the ability of NSF to dissociate cis-SNARE complexes, making fewer SNARE proteins available for functional transmembrane complex formation and thus reducing intracellular transport. These results provide solid evidence that SNARE proteins are important in wing margin formation. This implies that the mutant NSF must suppress but not block all membrane traffic. The disruption of molecular markers, such as Wg, Delta, Achaete, Cut, Vestigial, and Neuralized, indicates that the NSFE/Q wing phenotype observed is the result of impaired signaling at the developing wing margin. This is consistent with data presented in other studies that manipulated the signaling pathway directly. For example, reduction of Notch activity with Nts alleles can lead to reduced and patchy Wingless expression. Wingless and Cut expression is also reduced and patchy in Notch mutant wing discs. Stripes of Delta and Serrate that normally flank the D/V boundary collapse into a single stripe along the margin in Nts alleles exposed to restrictive temperature. In NSFE/QC96 wing discs changes in Wingless, Cut, and Delta patterns were observed that are similar to those that occur when Notch activity is directly manipulated; therefore, it seems that NSFE/Q expression phenocopies genetic mutants of Notch (Stewart, 2001).

Because the Notch and Wingless signaling pathways are so intertwined in controlling wing margin development it is difficult to determine whether the dNSF2 mutants cause a primary defect in one or the other of these proteins, although it seems likely that there are parallel effects on both. The experiments show not only a direct impairment of Wingless trafficking but also that Wg-lacZ expression is disrupted. The latter suggests that an upstream activator of Wingless expression is impaired (although this could be Wingless itself). It is found that Notch subcellular localization is disrupted and that a Wg-independent target of Notch signaling, the vestigial boundary enhancer, is also disrupted. Because this vestigial enhancer element is thought to be under the sole control of Notch this supports the idea that NSFE/Q has a direct effect on Notch signaling. Thus vgBE-lacZ expression the data strongly suggest direct effects on both Wg and Notch. Moreover, because these molecules are at the top of the hierarchy controlling signaling at the wing margin this provides the likely explanation for the disruption of downstream targets of these genes (Stewart, 2001).

The molecular and genetic interactions that regulate developmentally important signaling pathways are important for defining the final outcome of the signaling cascade. For example, previous studies have identified several molecules, including Fringe, Big Brain, and Numb, that are proposed to influence Notch signals. Because the SNARE proteins interact with many protein partners, some of which are proposed to regulate their availability (e.g., Syntaxin’s interaction with rop/nsec-1), these data indicate that regulation of SNARE-dependent transport steps may represent an additional mechanism by which signal transduction pathways can be modulated during development (Stewart, 2001).

ROP, a Syntaxin-binding protein

The product of the ras opposite (rop) gene is an essential component of secretion processes in Drosophila. The ROP gene product is homologous to the Caenorhabditis elegans UNC-18 and the rat munc-18/n-Sec1/rbSec1 proteins (implicated in the final steps of neurotransmitter exocytosis in nerve terminals) and the bovine mSec1 protein (implicated in the secretion of catecholamines in chromaffin cells). The mammalian brain protein has been shown to exert its activity in the presynaptic membrane through transient interaction with syntaxin, an integral component of this membrane. rop is highly expressed in the Drosophila nervous system, where it acts as both a positive and negative modulator of neurotransmitter release. It is also expressed in specialized tissues in which intensive exocytic/endocytic cycles take place, including the garland cells, a small group of nephrocytes that take up waste materials from the hemolymph by endocytosis. rop is regulated by a bidirectional promoter shared with Ras2, a member of the R-ras/TC21 branch of the ras supergene family (see Ras85). Ras2 is also highly expressed in the garland cells. These cells are characterized by their labyrinthine channels, long invaginations extending from the cell membrane into the cell, and a richly varied population of vesicles. In this study, the ultrastructural localization of the Rop and Ras2 proteins were analyzed in the garland cell. Rop is detected in the outer membranes of the labyrinthine channels, and in the outer membranes of many vesicles located nearby the labyrinthine channels, but not in vesicles located in inner parts of the cell. Using glutathione-S-transferase-syntaxin fusion, it has been shown that Rop is firmly bound to syntaxin (Halachmi, 1995).

The Sec1 family of proteins is thought to function in both non-neuronal and neuronal secretion, although the precise role of this protein family has not yet been defined. The function of ROP, the Drosophila Sec1 homolog, has been studied in neurotransmitter release. Electrophysiological analyses of transgenic lines overexpressing ROP and syntaxin, a presynaptic membrane protein, indicate that ROP interacts with syntaxin in vivo. Characterization of four point mutations in ROP shows that they fall into two phenotypic classes. Two mutations cause a dramatic reduction in both evoked and spontaneous neurotransmitter release. In contrast, the other two mutations reveal an increase in evoked neurotransmission. These data further show that neurotransmission is highly sensitive to the levels of ROP function. Studies on heterozygote animals indicate that half the amount of wild-type ROP results in a dramatic decrease in evoked and spontaneous exocytosis. Taken together, these results suggest that ROP interacts with syntaxin in vivo and is a rate-limiting regulator of exocytosis that performs both positive and inhibitory functions in neurotransmission. Since mutations in ROP can cause multiple effects, it is suggested that ROP may interact with multiple effector proteins in vivo (Wu, 1998).

In addition to syntaxin, studies of yeast mutants have suggested that the Sec1 family may interact with Rab family members. The vertebrate homolog of ROP, Munc-18, has been shown to biochemically interact with DOC2, a novel C2 domain-containing protein. Two potential mechanisms for ROP regulation are suggested. Munc-18 can be phosphorylated by protein kinase C and this phosphorylation inhibits binding to syntaxin. Nitric oxide has been shown to increase formation of the core complex (syntaxin, synaptobrevin and SNAP-25) and inhibit binding of Munc-18. Thus, two intracellular effectors (PKC and NO) that probably contribute to synaptic plasticity have been shown to regulate the syntaxin-ROP interaction (Wu, 1998).

The Drosophila ras2 promoter region exhibits bidirectional activity. Drosophila ras2 provides the only example to date in which the flanking gene (rop) and its product have been isolated. A linking mechanism of control suggests a mutual interaction between the two gene products. The Drosophila ras2 promoter region shares with the human c-Ha-ras1 promoter a CACCC box and an AP-1-like sequence. A 14 bp promoter fragment which holds a CACCC element is demonstrated to interact with a specific transcription factor (factor B). This CACCC promoter element represents a stretch of imperfect palindrome. This factor can form a complex with another specific DNA-binding protein (factor A). The binding sites (A + B) for these protein factors are essential for 95% expression of both genes flanking the promoter (ras2 and rop). Region A consists of four overlapping consensus sequences: a TATA-like element, a DSE-like motif (the core sequence of the serum response element), a DRE octamer, which has been shown to play a role in cell proliferation, and a 5 bp direct repeat representing the GATA consensus sequence. Factor A has a very weak affinity for the full promoter region, but when complexed with factor B binding efficiency is enhanced. Alterations of DNA-protein binding specificities can be achieved by supplementing the growth media with different sera (Lightfoot, 1994).

SNAP-24 and SNAP-25, t-SNAREs

The neuron-specific proteins SNAP-25 (synaptosome-associated protein 25 kDa), synaptobrevin and syntaxin, are localized to presynaptic terminals in mammals and have been found to associate with proteins involved in vesicle docking and membrane fusion. This study describes SNAP-25 cDNA clones from Drosophila and the ray Torpedo marmorata. In situ hybridization shows that SNAP-25 mRNA is exclusively found in brain and ganglia in Drosophila with a pattern suggesting expression in most neurons. The Drosophila and Torpedo proteins show 61 and 81% amino acid identity to mouse SNAP-25, a degree of conservation similar to that previously reported for synaptobrevin. None of the SNAP-25 sequences has a membrane-spanning region, but all contain a cluster of cysteine residues that can be palmitoylated for membrane attachment. SNAP-25 displays sequence similarity to syntaxin A and B. These data show that SNAP-25 and synaptobrevin, which are both implicated in vesicle docking and/or membrane fusion, have both been highly conserved during evolution. This supports the existence of a basic molecular machinery for synaptic vesicle docking in vertebrate and invertebrate synapses (Risinger, 1993).

The evolutionarily conserved protein SNAP-25 [synaptosome-associated protein 25 kDa (kilodaltons)] is a component of the protein complex involved in the docking and/or fusion of synaptic vesicles in nerve terminals. The SNAP-25 gene (Snap) in Drosophila has a complex organization with eight exons spanning more than 120 kb (kilobases). The exon boundaries coincide with those of the chicken SNAP-25 gene. Only a single exon 5 has been found in Drosophila, whereas human, rat, chicken, zebrafish and goldfish have two alternatively spliced versions of this exon. In situ hybridization and immunocytochemistry to whole mount embryos show that SNAP-25 mRNA and protein are detected in stage 14 and later developmental stages, and are mainly localized to the ventral nerve cord. Thus, Snap has an evolutionarily conserved and complex gene organization, and its onset of expression in Drosophila melanogaster correlates with a time in neuronal development when synapses begin to be formed and when other synapse-specific genes are switched on (Risinger, 1997).

Fusion of vesicles with target membranes is dependent on the interaction of target (t) and vesicle (v) SNARE proteins located on opposing membranes. For fusion at the plasma membrane, the t-SNARE SNAP-25 is essential. In Drosophila, the only known SNAP-25 isoform is specific to neuronal axons and synapses and additional t-SNAREs must exist that mediate both non-synaptic fusion in neurons and constitutive and regulated fusion in other cells. The identification and characterization is reported of SNAP-24, a closely related Drosophila SNAP-25 homolog, that is expressed throughout development. The spatial distribution of SNAP-24 in the nervous system is punctate and, unlike SNAP-25, is not concentrated in synaptic regions. In vitro studies, however, show that SNAP-24 can form core complexes with syntaxin and both synaptic and non-synaptic v-SNAREs. High levels of SNAP-24 are found in larval salivary glands, where SNAP-24 localizes mainly to granule membranes rather than the plasma membrane. During glue secretion, the massive exocytotic event of these glands, SNAP-24 containing granules fuse with one another and the apical membrane, suggesting that glue secretion utilizes compound exocytosis and that SNAP-24 mediates secretion (Niemeyer, 2000).

The synaptic protein SNAP-25 is an important component of the neurotransmitter release machinery, although its precise function is still unknown. Genetic analysis of other synaptic proteins has yielded valuable information on their role(s) in synaptic transmission. In this study, a mutagenesis screen was performed to identify new SNAP-25 alleles that fail to complement SNAP-25ts, a previously isolated recessive temperature-sensitive allele of SNAP-25. In a screen of 100,000 flies, 26 F1 progeny failed to complement SNAP-25ts and 21 of these were found to be null alleles of SNAP-25. These null alleles die at the pharate adult stage and electroretinogram recordings of these animals reveal that synaptic transmission is blocked. At the third instar larval stage, SNAP-25 nulls exhibit nearly normal neurotransmitter release at the neuromuscular junction. This is surprising since SNAP-25ts larvae exhibit a much stronger synaptic phenotype. A related protein, SNAP-24, can substitute for SNAP-25 at the larval stage in SNAP-25 nulls. However, if a wild-type or mutant form of SNAP-25 is present, then SNAP-24 does not appear to take part in neurotransmitter release at the larval NMJ. These results suggest that the apparent redundancy between SNAP-25 and SNAP-24 is due to inappropriate genetic substitution. In this situation, two related protein isoforms perform similar functions in distinct biochemical pathways. Normally, an individual isoform does not participate in the other pathway, and so hypomorphic mutations disrupting each protein show a distinct phenotype. However, a null allele that completely abolishes the expression of one protein could allow an isoform to step in and compensate for it. In this way, a null allele of a gene may paradoxically show a phenotype that is much weaker than that of a hypomorphic allele of that gene (Vilinsky, 2002).

What could account for the unexpectedly mild effects of abolishing SNAP-25 at the larval stage? Since SNAP-25 is thought to be a crucial component of the exocytosis machinery and since these mutants die just prior to eclosion, it was reasoned that another related protein, SNAP-24, may be substituting for SNAP-25 function in the larvae. The distributions of SNAP-24 and SNAP-25 in mutant and control animals was examined using an antibody raised against a peptide sequence from exon 4 of SNAP-25 that is identical in the SNAP-25 and SNAP-24 proteins. The Exon 4 antibody recognizes both proteins equally when used on a Western blot containing lanes of equal amounts of SNAP-25 and SNAP-24. Western blot analysis of different tissues in larvae shows that, while SNAP-25 is specifically neuronal, SNAP-24 is found in all tissues examined except the larval gut. In fact, SNAP-24 is found at relatively high levels within the CNS of both control and mutant animals. In the larvae, the level of SNAP-24 in the nervous system is at least as high as that of SNAP-25. This ratio shifts during metamorphosis, so that pharate adult heads express more SNAP-25 than SNAP-24. No clear evidence was found that levels of SNAP-24 in mutant animals are upregulated to compensate for the lack of SNAP-25 (Vilinsky, 2002).

The larval CNS and neuromuscular junctions were probed for immunoreactivity to the Exon 4 antibody. Staining in SNAP-25 null larvae revealed that SNAP-24 is indeed found within synaptic boutons at the neuromuscular junction and in synaptic regions of the CNS. The pattern of staining in SNAP-25 nulls is similar to that of controls, but control larvae show greater staining intensity due to the presence of both SNAP-24 and SNAP-25. Due to the fact that SNAP-24 is also present in larval muscle, detection of SNAP-24 in synaptic boutons required confocal microscopy to separate out the signal in muscle from that in boutons (Vilinsky, 2002).

If SNAP-24 can substitute for the function of SNAP-25, how well does SNAP-24 interact with other SNARE proteins? SNAP-24 can form the characteristic 73-kD SNARE complex with syntaxin and neuronal-synaptobrevin. The ability of SNAP-24, SNAP-25, and the temperature-sensitive form of SNAP-25, SNAP-25ts, to form SNARE complexes was compared using an in vitro assay. While SNAP-24 forms less 73-kD complex than does SNAP-25, it forms more complex than does SNAP-25ts. Interestingly, SNAP-24 is much better than either SNAP-25 or SNAP-25ts at forming a higher-order SNARE complex, indicating that the biochemical functions of SNAP-24 and SNAP-25 may have differences more subtle than those apparent in electrophysiological assays. In addition, SNARE complexes containing SNAP-24 migrate faster on SDS-PAGE gels than do those containing SNAP-25. The apparent size difference of SNARE complexes containing SNAP-24 cannot be accounted for by the small difference in molecular weight between SNAP-25 and SNAP-24. Thus, the size differences likely represent structural differences between complexes formed by these two proteins (Vilinsky, 2002).

While SNAP-25 is critical for adult Drosophila, the data suggest that SNAP-24 is sufficient for the animals during the larval stage. This division of function between developmental stages is not unprecedented for proteins involved in synaptic transmission and exocytosis. In mammals, SNAP-25 is alternatively spliced into two forms, SNAP-25a and SNAP-25b, which differ in the composition of the palmitoylated cysteine-rich domain thought to be responsible for association of the protein with membranes. SNAP-25a is the dominant form throughout embryonic development, while levels of SNAP-25b rise and exceed SNAP-25a postnatally. There is evidence that the developmentally significant SNAP-25a form is expressed in adult neurons that retain morphological plasticity or undergo regrowth. In Drosophila, SNAP-24 also differs from SNAP-25 in the cysteine-rich domain, where it contains three instead of four cysteine residues that could potentially affect its membrane association dynamics. SNAP-24 is found at high levels relative to SNAP-25 during the larval stage; during metamorphosis into adulthood SNAP-25 expression rises significantly relative to SNAP-24. Therefore, the roles of SNAP-24 and SNAP-25 in Drosophila may be in some ways analogous to the roles of SNAP-25a and SNAP-25b in mammals (Vilinsky, 2002).

Another parallel may be drawn between SNAP-24 and mammalian SNAP-23, a protein that is expressed widely throughout the body and is not restricted to the nervous system. While SNAP-23 is not highly expressed in the nervous system, it has been demonstrated that SNAP-23 can functionally substitute for SNAP-25 in at least some exocytosis processes, and it is found in regions of the hippocampus and cortex where SNAP-25 is absent. In flies, SNAP-24 is concentrated in mushroom body neuropil in adult brains, a region where SNAP-25 levels are very low. This segregation of expression between specific neuropils may point to more subtle differences in functional requirements for vesicle fusion. Thus, the role of SNAP-24 in the adult CNS may have parallels with the role of SNAP-23 in the mammalian brain (Vilinsky, 2002 and references therein).

In Drosophila other synaptic proteins have developmentally specific isoforms that roughly parallel those of SNAP-25 and SNAP-24. One example is N-ethylmaleimide-sensitive factor (NSF), a chaperone that uses ATP to dissociate SNARE complexes and is thought to be important for recycling SNARE proteins after a round of vesicle exocytosis. NSF has two isoforms in Drosophila, dNSF1 and dNSF2. dNSF1 and dNSF2 are expressed in the larval and adult CNS, with dNSF-2 also being expressed more widely in nonneuronal tissues. Like SNAP-25 nulls, dNSF1 loss-of-function mutants die at the pharate adult stage and are rescued by neuronal expression of a wild-type transgene. However, unlike the SNAP-25ts allele, temperature-sensitive paralytic alleles of dNSF1 have no synaptic phenotype at the third instar larval NMJ, presumably due to the presence of dNSF2 in the larval nervous system. Future studies may resolve how different isoforms of NSF may interact with different members of the SNAP-25 gene family (Vilinsky, 2002).

Overexpression of Cysteine-string proteins in Drosophila reveals interactions with Syntaxin

Cysteine-string proteins (CSPs) are associated with secretory vesicles and critical for regulated neurotransmitter release and peptide exocytosis. At nerve terminals, CSPs have been implicated in the mediation of neurotransmitter exocytosis by modulating presynaptic calcium channels; however, studies of CSPs in peptidergic secretion suggest a direct role in exocytosis independent of calcium transmembrane fluxes. The individual expression of various CSP isoforms in Drosophila similarly rescues the loss of evoked neurotransmitter release at csp null mutant motor nerve terminals, suggesting widely overlapping functions for each isoform. Thus, the structural difference of CSP variants may not explain the opposing putative functions of CSP in neurotransmitter and peptide exocytosis. Consistently, the individual overexpression of each CSP isoform in wild-type Drosophila shows similar effects such as impaired viability and interference with wing and eye development. The dominant effects caused by the overexpression of CSP are suppressed by the simultaneous overexpression of Syntaxin-1A but not by the coexpression of SNAP-25. Although overexpression of CSP itself has no apparent effect on the synaptic physiology of larval motor nerve terminals, it fully suppresses the decrease of evoked release induced by the overexpression of Syntaxin-1A. A direct protein-protein interaction of CSP with Syntaxin is further supported by coimmunoprecipitations of Syntaxin with CSP and by protein binding assays using recombinant fusion proteins. Together, the genetic and biochemical interactions of CSP and Syntaxin-1A suggest that CSP may chaperone or modulate protein-protein interactions of Syntaxin-1A with either calcium channels or other components of the regulatory machinery mediating depolarization-dependent neurotransmitter exocytosis (Nie, 1999).

Two different modes of CSP function have been suggested. In the first, for peptidergic secretion, CSP has been shown to mediate a direct step of stimulated exocytosis independent of calcium transmembrane fluxes. An increase or decrease of CSP levels in PC12 and insulin-secreting cells severely reduces exocytosis without affecting transmembrane calcium fluxes. Because these effects persist in permeabilized cells, any regulation via soluble second messengers can be excluded, suggesting that CSP may function directly in exocytosis. In contrast, for the second, fast neurotransmitter release, CSP has been suggested to link synaptic vesicles with calcium channels and to modulate channel activity because CSP is associated with synaptic vesicle membranes and modulated N-type calcium channel currents when ectopically coexpressed in frog oocytes. This idea has been further supported by the in vitro binding of CSP to P/Q-type calcium channels and by the consistent defects observed in csp mutant Drosophila like the reduction of evoked but not spontaneous neurotransmitter release and reduced presynaptic cytosolic calcium levels after repetitive stimulation (Nie, 1999 and references therein).

It has been further speculated that CSP may coordinate sequential protein-protein interactions between calcium channels and their associated synaptic proteins because CSP is presumably a vesicular, membrane-bound cofactor of the molecular chaperone Hsc70 and because CSP binds to the same cytoplasmic loopII-III of calcium channels that contains the synprint site mediating channel interactions with SNAP-25, synaptotagmin, and syntaxin. Interestingly, disruption of these interactions by the presynaptic injection of the synprint binding peptide in rat neurons causes strikingly similar defects of evoked neurotransmitter release as the deletion of the Drosophila csp gene: both reduce synchronous release but increase asynchronous release and paired pulse facilitation. Because the syntaxin/channel interaction reduces channel activity by prolonging an inactivated state, it has been speculated that CSP could promote the dissociation of a syntaxin/channel complex, if CSP promotes calcium channel activity as originally suggested (Nie, 1999 and references therein).

This study provides experimental evidence for this speculation showing that CSP interacts with syntaxin in vitro and in vivo. This interaction may modulate the dissociation of syntaxin from calcium channels. Alternatively, CSP may modulate protein interactions with other syntaxin-interacting proteins to mediate a calcium-dependent step of exocytosis as implied by the direct role of CSP in peptidergic exocytosis. An obvious candidate for this interaction is synaptobrevin, which has been shown to bind CSP in vitro. Both speculations are consistent with the current results and with the recent characterization of a mutation in Drosophila syntaxin-1A deleting a multiple protein binding domain that reduces CSP, synaptotagmin, and calcium channel binding. A third possible role for the CSP/syntaxin interaction is that CSP may simply chaperone protein folding or protein transport of syntaxin. Such a function should cause reduced levels of syntaxin in csp null mutants and proportionally reduce evoked and spontaneous release as observed in Drosophila syntaxin mutants. However, this possibility is unlikely because only evoked release but not spontaneous release is abolished in csp null mutants at restrictive temperatures. To finally determine whether the CSP/syntaxin interaction is required for a modulation of presynaptic calcium channel activity or for a step of evoked exocytosis independent of calcium transmembrane fluxes, or both, will require the analysis of mutations in CSP that specifically interrupt the interaction with syntaxin (Nie, 1999).

Tilting the balance between facilitatory and inhibitory functions of mammalian and Drosophila Complexins orchestrates synaptic vesicle exocytosis

SNARE-mediated synaptic exocytosis is orchestrated by facilitatory and inhibitory mechanisms. Genetic ablations of Complexins (see Drosophila complexin), a family of SNARE-complex-binding proteins, in mice and Drosophila cause apparently opposite effects on neurotransmitter release, leading to contradictory hypotheses of Complexin function. Reconstitution experiments with different fusion assays and Complexins also yield conflicting results. Cross-species rescue experiments were therefore performed to compare the functions of murine and Drosophila Complexins in both mouse and fly synapses. It was found that murine and Drosophila Complexins employ conserved mechanisms to regulate exocytosis despite their strikingly different overall effects on neurotransmitter release. Both mouse and fly Complexins contain distinct domains that facilitate or inhibit synaptic vesicle fusion, and the strength of each facilitatory or inhibitory function differs significantly between them. These results show that a relative shift in the balance of facilitatory and inhibitory functions results in differential regulation of neurotransmitter release by murine and Drosophila Complexins in vivo, reconciling previous incompatible findings (Xue, 2009).

SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor)-mediated synaptic vesicle exocytosis is tightly controlled by a large number of regulatory proteins to ensure the exquisite temporal and spatial precision of neurotransmitter release at synapses. Complexins constitute a family of small and highly charged proteins that bind to the assembled SNARE complex. They generally contain a central α helix and an accessory α helix in the middle portion of the protein, and the N- and C-terminal sequences that are probably largely unstructured. Complexins bind to the SNARE complex with high affinity. The central α helix of Complexins interacts with the SNARE motifs of Syntaxin-1 and Synaptobrevin-2 within the SNARE complex in an antiparallel fashion. Complexins can also bind to the target-SNAREs' (Syntaxin-1 and SNAP-25) heterodimer with a lower affinity (Xue, 2009 and references therein).

Biophysical and physiological studies have indicated diverse functions for Complexins in vesicle fusion, some of which are incompatible. Complexins have been shown to inhibit SNARE-mediated cell fusion and proteoliposome fusion in bulk ensemble assay, and this inhibition is released by the Ca2+ sensor Synaptotagmin-1 and Ca2+. Biochemically, Synaptotagmin-1 competes with Complexins for the SNARE complex binding and displaces Complexins from the SNARE complex in a Ca2+-dependent manner. These studies suggest a fusion clamp model for Complexin function, in which Complexins inhibit the transfer of the force generated by the SNARE complex assembly onto the fusing membranes and arrest synaptic vesicle fusion before Ca2+ influx. Upon Ca2+ binding, Synaptotagmin-1 displaces Complexins from the SNARE complex to release this inhibition and triggers exocytosis. However, Complexins have also been shown to stimulate proteoliposome fusion in both single-vesicle fusion assay and bulk ensemble assay, indicating a facilitatory role. These in vitro results are further confounded by in vivo genetic studies. Genetic knockout of Complexins in mice leads to a reduction in both evoked and spontaneous release at multiple glutamatergic and GABAergic synapses in cultures and in acute brain slicesand a decrease in Ca2+-triggered exocytosis in adrenal chromaffin cells, supporting a stimulatory function for Complexins. In contrast, genetic deletion of Complexin in fruit fly Drosophila melanogaster greatly enhances spontaneous release but decreases Ca2+-evoked release, favoring the fusion clamp model. Moreover, knockdown of Complexins by RNA interference in mass-cultured mouse cortical neurons decreases evoked release and increases spontaneous release at glutamatergic synapses. To explain the discrepancy between this result and those obtained previously from Complexin knockout mice, the authors (Maximov, 2009) suggest that this is due to the different preparations used (autaptic cultures for knockout studies versus mass cultures for knockdown study, disavowing the fact that the knockout studies also employed mass cultures and acute brain slices, and similar results were found to those obtained from autaptic cultures. Hence, many studies seem at odds with each other and the precise in vivo role of Complexins in exocytosis is still unclear (Xue, 2009 and references therein).

An in vivo structure-function analysis of murine Complexin I (CplxI) in Complexin I/II double knockout mouse neurons indicates that the SNARE complex binding is essential for CplxI function, and that the N terminus of CplxI facilitates release, whereas an accessory α helix between the N terminus and the central α helix inhibits release. A biophysical study reveals that CplxI inhibits SNARE complex formation, but strongly stimulates membrane fusion after the assembly of the SNARE complex in vitro. These studies indicate that Complexins play both facilitatory and inhibitory roles in exocytosis, but they still do not explain why genetic deletions of Complexins in two model organisms, mouse and fly, have such different effects on neurotransmitter release. Furthermore, the amino acid sequence homology is low between murine and Drosophila Complexins except for the central α helix that is essential for the binding to the SNARE complex, and part of the N terminus. Thus, the dramatic difference in loss-of-function phenotypes of Complexin-deficient mice and flies leads to the conclusion that Complexin function must differ between mice and flies (Xue, 2009).

To test whether the functions of murine and Drosophila Complexins are conserved in synaptic vesicle exocytosis, and to gain insight into their functional and structural differences, it is essential to compare murine and Drosophila Complexins in the same experimental in vivo systems. A detailed structure-function analysis is also necessary because a complete removal of Complexins is unlikely to reveal all aspects of their function. Therefore a systematic cross-species rescue approach was undertaken to compare the functions of murine and Drosophila Complexins at both mouse and fly synapses. It was found that both murine and Drosophila Complexins contain distinct functional domains and play dual roles in neurotransmitter release. They facilitate and inhibit release via similar domains, but the facilitatory or inhibitory strength of a given domain varies between murine and Drosophila Complexins. Thus, both murine and Drosophila Complexins utilize conserved mechanisms in release process, but the integration of facilitation and inhibition differs substantially between them, leading to an apparently opposite overall effect on exocytosis. These results reveal conserved functions of Complexins between species and indicate that the interplay of dual functions orchestrates neurotransmitter release (Xue, 2009).

Synaptic exocytosis is exquisitely controlled by a set of facilitatory and inhibitory mechanisms, some of which are often executed by the very same protein. As a key regulator of the release machinery, Complexins play both facilitatory and inhibitory roles in vesicle fusion through distinct mechanisms. However, the remarkable phenotypic difference between mouse and fly Complexin null animals remained unexplained. This work compared the functions of murine and Drosophila Complexins in cross-species rescue experiments. The data establish that murine and Drosophila Complexins share a set of conserved mechanisms in synaptic vesicle fusion (Xue, 2009).

First, the SNARE complex binding mediated by the central α helix (residues 48-70 for CplxI and 54-76 for dmCplx) is essential for Complexin function. Mutations that diminish the interaction between the central α helix and the SNARE complex abolish the functions of both CplxI and dmCplx, indicating that the actions of other domains all depend on this high-affinity interaction. The binding of the central α helix not only can stabilize the assembled SNARE complex, but perhaps more importantly, can strategically position the accessory α helix and the N terminus for their actions (Xue, 2009 and references therein).

Second, the accessory α helix (approximately residues 29-47 for CplxI and 33-53 for dmCplx) located between the N terminus and the central α helix inhibits vesicle fusion. It was proposed that the inhibitory action of the accessory α helix might arise from its interference with the binding of Synaptobrevin-2 to Syntaxin-1 and SNAP-25 heterodimer, which would consequently prevent the complete zippering of the SNARE complex. This model has recently been supported by the findings that Complexins can bind to Syntaxin-1 and SNAP-25 heterodimer in vitro and may form an alternative four-helix bundle with target-SNAREs to inhibit fusion in a reconstituted fusion system (Xue, 2009 and references therein).

Third, the N termini (residues 1-16) of both CplxI and dmCplx promote release. It has been speculated that the CplxI N terminus may interact with lipid membranes, but so far, there are no supporting biochemical data. Instead, this facilitatory effect is likely mediated by a direct interaction of the Complexin N terminus with the SNARE complex C terminus. Mutations of methionine 5 and lysine 6 of CplxI disrupt the binding of the CplxI N terminus to the SNARE complex C terminus and abolish the facilitatory activity of the N terminus. Interestingly, methionine 5 is not conserved in dmCplx and an alanine residue is at position 6 (corresponding to residue 5 of CplxI). It is possible that a methionine is not absolutely required for dmCplx and other residues may compensate for the interaction with the SNARE complex C terminus (Xue, 2009).

Furthermore, at fly neuromuscular junctions, both murine and Drosophila Complexins promote Ca2+-triggered release and suppress spontaneous release, but to very different degrees. Neuronal expression of murine or Drosophila Complexins rescues the lethality and sterility of Complexin null mutant flies, showing again that murine and Drosophila Complexins share conserved functions (Xue, 2009).

Therefore, these cross-species rescue experiments show that murine and Drosophila Complexins have both facilitatory and inhibitory functions associated with similar protein domains in synaptic vesicle exocytosis. It is proposed that the Complexin central α helix binds to the middle portion of the SNARE complex, stabilizing the SNARE complex and positioning the accessory α helix and the N terminus. The accessory α helix replaces the C terminus of the Synaptobrevin-2 SNARE motif in the four-helix bundle, preventing the full assembly of the SNARE complex to suppress fusion. The N terminus directly interacts with the C-terminal portion of the SNARE complex, likely stabilizing this unstable region of the SNARE complex to promote membrane fusion. However, the relative strengths of these functions are remarkably different between murine and Drosophila Complexins. It is proposed that the integration of facilitation and inhibition, which are associated with distinct domains, determines the overall effect of murine and Drosophila Complexins on neurotransmitter release in a given synapse. The overall action of murine and Drosophila Complexins is unlikely to be a linearly additive effect of all facilitatory and inhibitory actions. However, it is clear that the facilitatory function is preponderant in murine Complexins, whereas the inhibitory functions of the accessory α helix and the C terminus predominate in Drosophila Complexin. Thus, a relative shift in the balance of facilitatory and inhibitory functions results in differential roles of murine and Drosophila Complexins in neurotransmitter release, and leads to apparently very different loss-of-function phenotypes in flies and mice. These results emphasize the functional similarities and differences between murine and Drosophila Complexins, and reconcile previous contradictory hypotheses of Complexin in vivo function. Moreover, the data illustrate the complexity of Complexin function and strongly support the notion that Complexins play dual roles in vesicle fusion (Xue, 2009).

This model is clearly different from the previous models of Complexins based on the fusion clamp hypothesis. These models propose that Complexins arrest primed synaptic vesicles at a hemifused and metastable state, which provides the substrate for Ca2+-bound Synaptotagmin-1 to release the clamping function of Complexins, allowing the fast and synchronous fusion. The lack of Complexins and therefore the lack of metastable vesicles for Synaptotagmin-1 action causes excessive spontaneous release and deficient Ca2+-triggered fast release. However, the current in vivo results speak against this model because murine Complexins do not completely clamp the excessive spontaneous release in Drosophila Complexin null mutants, yet they actually enhance Ca2+-evoked fast release even better than Drosophila Complexin. This observation indicates that the decreased Ca2+-evoked fast release in Drosophila Complexin null mutants is not functionally coupled to the increased spontaneous release frequency. Could it be that the reduced evoked release in Drosophila Complexin null mutants is due to a partial depletion of readily releasable vesicles by the high-frequency spontaneous release? This is unlikely because the vesicle recruitment rate is usually at least 100-fold higher than the spontaneous release rate at resting intracellular Ca2+ level, and therefore a 20- to 30-fold increase in spontaneous release rate should not significantly change the vesicle pool size in Drosophila Complexin null mutants. In addition, murine-Complexin-rescued Drosophila Complexin null synapses still exhibit strongly increased spontaneous release, yet the evoked release is even larger than that of WT synapses, arguing that high-frequency spontaneous release in null mutants is unlikely to exhaust vesicles, causing a decreased evoked release (Xue, 2009).

A recent fusion clamp model proposes that Complexins control the force transfer from the SNARE complex to the membranes and assist the SNAREs in exerting force on the membranes (Maximov, 2009). This model assumes that Complexins are released from the SNARE complex by Synaptotagmin-1 and Ca2+, but it is physically unclear how Complexins can help SNAREs exert force on the membranes if they are dissociated upon Ca2+ influx. In contrast, the current model requires Complexins to remain bound to the SNARE complex upon Ca2+ influx and is consistent with the notion that Complexins could function independently from Synaptotagmin-1 (Xue, 2009).

Drosophila Complexin in Cplx-TKO neurons abolishes both evoked and spontaneous release without altering the number of fusion-competent vesicles measured by hypertonic sucrose solution. This effect is intriguing, because very few molecular manipulations specifically block the synaptic vesicle cycle at the final fusion step. Drosophila Complexin does not change the number of primed vesicles, indicating that the initial formation of the SNARE complex is not affected by Drosophila Complexin. The inhibitory effect of Drosophila Complexin requires its binding to the SNARE complex. Hence, it is hypothesized that when the Drosophila Complexin central α helix binds to the partially assembled SNARE complex, the accessory α helix together with the C terminus prevents the further assembly of the SNARE complex C terminus, thereby arresting vesicles at the primed state. It is currently unknown how, mechanistically, the C terminus of Drosophila Complexin inhibits release. One possibility is that the C terminus may fold back toward the N-terminal direction and cooperate with the accessory α helix to inhibit vesicle fusion (Xue, 2009).

The phenotypic differences between fly and mouse knockouts seem dramatic, but it is worth noting that an increase of just 1.4 kcal/mol in the strength of a protein-protein interaction, which can arise simply from the formation of one hydrogen bond or salt bridge, leads to a 10-fold increase in affinity according to the Boltzmann equation. Hence, subtle changes in the molecular interactions of murine and Drosophila Complexins can suffice to tip the balance between facilitatory and inhibitory strengths. For example, protein sequence alignments show that the lengths and the amino acid compositions of the accessory α helices differ among different Complexins, which may cause different interactions of the accessory α helix with Syntaxin-1 and SNAP-25 heterodimer, thus changing its inhibitory strength (Xue, 2009).

The effects of murine Complexins in murine and fly synapses are not identical, as murine Complexins promote evoked release and inhibit spontaneous release in fly neuromuscular junctions, and promotes both types of release in mouse central synapses. Likewise, the effects of Drosophila Complexin in murine and fly synapses are not identical either, as it strongly inhibits spontaneous release and mildly promotes evoked release in fly neuromuscular junctions, and strongly inhibits both types of release in mouse synapses. These observations indicate that in addition to the Complexin-intrinsic properties, the molecular differences between species or synapses could differentially affect the facilitatory and inhibitory functions of murine and Drosophila Complexins, thereby tilting the facilitation and inhibition balance and contributing to the phenotypic differences (Xue, 2009).

Complexins represent a family of proteins that maintain a highly conserved core of sequences and at the same time display great diversity across paralogs and orthologs. This is likely reflected in their functions, namely conserved facilitatory and inhibitory mechanisms with varying strengths in neurotransmitter release. It will be interesting to test Complexin function in some other model organisms along the phylogenetic tree, such as worm and fish, to determine if and how the balance between facilitatory and inhibitory functions of Complexins has changed during evolution. At different synapses, the strengths of facilitation and inhibition of Complexins may be differentially regulated in a paralog- and ortholog-dependent fashion, thereby regulating release in a synapse-specific manner, and contributing to synaptic diversity and specificity. Furthermore, the ability of Drosophila Complexin to inhibit neurotransmitter release in mammalian neurons potentially provides a powerful tool to manipulate synaptic function to study neural circuits, as one should be able to express Drosophila Complexin to inhibit or even abolish synaptic transmission in a spatially and temporally specific manner (Xue, 2009).

back to Syntaxin Protein Interactions part 1/3 | part 2/3

Syntaxin 1A: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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