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Invertebrate Neuroscience, 1, 3-13 (1995) Review Presynaptic proteins involved in exocytosis in melanogaster: a genetic analysis Drosophila j. TROY LITTLETON and H U G O J. BELLEN Howard Hughes Medical Institute, Department of Molecular and Human Genetics, Division of Neuroscience, Baylor College of Medicine, Houston, TX 77030, USA ABSTRACT Neuronal communication involves the fusion of neurotransmitter filled synaptic vesicles with the presynaptic terminal. This exocytotic event depends upon proteins present in three separate compartments: the synaptic vesicle, the synaptic cytosol, and the presynaptic membrane. Recent data indicate that the basic components of exocytotic pathways, including those used for neurotransmitter release, are conserved from yeast to human. Genetic dissection of the secretory pathway in yeast, identification of the target proteins cleaved by the clostridial neurotoxins and biochemical characterization of the interactions of synaptic proteins from vertebrates have converged to provide the SNARE (soluble NSF attachment protein receptor) hypothesis for vesicle trafficking. This model proposes that proteins present in the vesicle (v-SNAREs) interact with membrane receptors (t-SNAREs) to provide a molecular scaffold for cytosolic proteins involved in fusion. The hypothesis that these mechanisms function at the synapse relies largely upon in vitro evidence. Recently, genetic approaches in mice, C. elegans and the fruitf[y, Drosophila melanogaster, have been used to dissect the in vivo function of numerous proteins involved in synaptic transmission. This review covers recent progress and insights provided by a genetic dissection of neurotransmitter release in Drosophila. In addition, we will provide evidence that the mechanisms for synaptic communication are highly conserved from invertebrates to vertebrates, making Drosophila an ideal model system to further unravel the intricacies of synaptic transmission. KEY WORDS: exocytosis; synaptic vesicles; neurotransmitter release; Drosophila Neurotransmitter release and the general exocytotic pathway The fundamental aspects of neurotransmission have tong been studied at the electrophysiological level and were expounded upon by Katz (1969), who introduced the 'calcium hypothesis' of neurotransmitter release. This model is now widely accepted and can be summarized as follows. Following the integration of numerous synaptic signals at the soma and dendrites of a neuron, an all-or-none action potential is initiated and propagated down the axon. This electrical signal is carried primarily via the activation of voltage dependent sodium channels and the resultant depolarization due to influx of positively charged sodium ions. The depolarization is propagated into presynaptic terminals with the consequent activation of voltage dependent calcium channels. The resulting calcium influx triggers the fusion of docked synaptic vesicles with the presynaptic membrane, releasing their neurotransmitter contents into the synaptic cleft. Neurotransmitters then diffuse across the synaptic cleft to bind to postsynaptic receptors and initiate diverse processes within the postsynaptic cell. It is likely that proteins associated with synaptic vesicles, as well as those located in the presynaptic membrane and cytosol, play a role in mediating the temporal dynamics and spatial localization of this Ca2+-mediated fusion event. Fig. 1 shows a diagram of many of the proteins t h o u g h t to be involved in synaptic vesicle trafficking and fusion. Recent studies have shown that synaptic vesicle targeting, docking, and fusion has many similarities with vesicular transport in most cells, including yeast. Table 1 lists the proteins that are thought to play a role in neurotransmitter release and also have homologues in yeast. Based upon sequence homology, the similar subcellular distribution of these proteins in yeast and vertebrates, and the phenotype of mutant yeast, it is now generally accepted that a significant portion of the molecular pathway of neurotransmitter release relies on proteins that are evolutionarily conserved in all secretory systems (for review, see Bennett and Scheller, 1993). To date, the conserved proteins identified by this approach include s y n a p t o b r e v i n / S N C l p - S N C 2 p (Protopopov et al., 1993); rab3a/SEC4p (Salminen and Novick, 1987); syntaxin/SS01-SS02 (Aalto et al., 1993); 0t-SNAP/SEC17p (Griff et al., 1992); NSF/ SEC18p (Wilson et al., 1992); SNAP-25/SEC9p (Brennwald et aI., 1994); and rop/SEClp (Aalto et at., 1992). Corresponding author: Dr Hugo J. Belten 4 Littletonand Bellen Synaptic cleft neurotransmitter synaptotagmin neurotransmitters H+ pump + neurexins s y n a p t , ~ - synaptic vesicle ++ brevin ~ ~ "-~ NSF Channel synapsin ATP transporter Fig. 1. Proteins involved in neurotransmitter release. A schematic representation of proteins involved in neurotransmitter release is shown. See text for descriptions of the function of the various proteins. A large number of these proteins have now been cloned in Drosophila, and a substantial fraction have been targeted for mutagenesis. The function of several of these proteins in neurotransmission is highlighted by the finding that several clostridial neurotoxins (botulinum and tetanus) cleave synaptobrevin, SNAP-25, and syntaxin, leading to a reduction or block of neurotransmitter release (Schiavo et al., 1992; Blasi et al., 1993a, b). In addition to these ubiquitously used trafficking components, a number of proteins present in invertebrate and vertebrate synapses have not been identified in yeast. At least some of these proteins are thought to be specific to synaptic transmission and probably provide some of the key properties required to ensure the accuracy and speed of synaptic vesicle exocytosis. Proteins that may fall in this category are calcium channels, synaptotagmin (Littleton et al., 1993a; DiAntonio et al., 1993b), cysteine string proteins (Zinsmaier et al., 1994), neurexins (Ushkaryov et al., 1992), frequenin (Pongs et al., 1993) and rabphilin-3A (Shirataki et al., 1993). The SNARE hypothesis for neurotransmitter release Recent in vitro biochemical work with many of the proteins shown in Fig. 1 has provided a model for neurotransmitter release. The presynaptic membrane proteins SNAP-25 and syntaxin, termed t-SNAREs, have been shown to form a complex with the synaptic vesicle proteins, synaptobrevin (v-SNARE) and synaptotagmin (S611ner et al., 1993a, b). An additional protein complex can also be purified which contains synaptobrevin, syntaxin, and SNAP-25, along with the cytosolic proteins NSF and mSNAP (S611ner et al., 1993a). NSF is known to be an ATPase consisting of a homotetramer of 76 kDa subunits that is essential for intra-Golgi trafficking. The soluble NSF attachment proteins (SNAPs), consisting of cx, [3, and y SNAPs, are essential for NSF attachment to membranes, with a mechanism suggestive of SNAP association with integral membrane receptors (SNAREs) and subsequent binding of NSF (Clary et al., 1990). Following ATP hydrolysis by NSF, the complex dissociates (S611ner et al., 1993b). The cytoplasmic protein rop (also known as nSecl, Muncl8, and Uncl8) has also been demonstrated to bind to syntaxin and could also play a role in SNARE function (Hata et al., 1993; Garcia et al., 1994; Pevnser et al., 1994a). The biochemical characterization of these protein-protein interactions prompted the SNARE (soluble NSF attachment protein receptor) hypothesis of vesicle trafficking (S61lner et al., 1993a) in which a v-SNARE (vesicle SNAP receptors like synapto- Presynaptic function in exocytosis Synaptic Protein Synaptobrevin Rab3A Syntaxin 0t-SNAP NSF SNAP25 MUNC18/ n-Secl/Rop Yeast Homologues Transport Step S N C l p and SNC2p YPT1 SEC4p SED5 PEP12 SSO1 and SSO2 Golgi to plasma membrane ER to Golgi to plasma membrane SEC17p SEC18p SEC9p SEClp ER to Golgi Golgi to vacuole Golgi to plasma membrane ER to Golgi ER to Golgi Golgi to plasma membrane Golgi to plasma membrane 5 References Protopopov et al., 1993 Segev et al., 1988 Salminenetal., 1987 Hardwick and Pelham, 1992 Bennett et al., 1993 Aalto et al., 1993 Griff et al., 1992 Wilson et al., 1992 Brennwald et al., 1994 Aalto et al., 1992 Table 1. Yeast and synaptic homologues brevin) interacts with a specific t-SNARE (target membrane S N A P receptors like SNAP-25 and syntaxin) to form a fusion complex with NSF and SNAPs. This hypothesis provides a convenient system for specific vesicle targeting, since a v-SNARE would only interact with a specific t-SNARE (Pevsner et al., 1994b). However, the finding that docking of vesicles at the active zone appears normal in terminals treated with clostridial neurotoxins or with anti-synaptobrevin probes (Hunt et al., 1994) suggests that additional mechanisms may function in vesicle docking. Following docking, vesicle fusion could also be envisioned as being driven by ATP hydrolysis by NSF to overcome an energy barrier for membrane fusion. Other proteins, such as the recently identified cysteine string protein (csp), rop and the GTP-binding protein rab3, might play a regulatory role in the function of the fusion complex. Calcium binding proteins such as rabphilin, synaptotagmin and frequenin could be envisioned to confer calcium dependency to the constitutive components mediating the fusion event. The presence of additional yeast secretory mutants with no identified synaptic counterparts suggests that there are likely additional unidentified proteins participating in the release process. Dissection of neurotransmitter release in Drosophila The study of functional aspects of the nervous system of the fruitfly, Drosophila melanogaster, has been an area of active investigation for the last decade. Many of the presynaptic proteins identified in Drosophila are listed in Table 2. The cytological mapping position of the corresponding genes and the availability of mutants is also indicated. A number of these proteins have been implicated in membrane excitability (for review, see Wu and Ganetsky, 1992), and will not be discussed further. We will focus on what is known about proteins thought to be directly involved in neurotransmitter release. As shown in Table 3, all the major components of the SNARE complex (synaptobrevin, syntaxin, SNAP-25, rop, NSF, s-SNAP, and synaptotagmin) have been identified in Drosophila. In addition, rab3a and csp have also been cloned in Drosophila. The identity of the Drosophila proteins with their vertebrate homologues ranges from 57% to 78% over the entire length of the proteins. In addition, the molecular weight and subcellular localization of the majority of these proteins is identical between invertebrates and vertebrates (Schulze et al., 1995). Such dramatic similarities between proteins involved in the release process indicate that synaptic transmission is a highly evolutionarily conserved process. This is further supported by the observation that the electrophysiological characteristics of synaptic transmission in Drosophila and vertebrates share many properties (Jan and Jan, 1976). Given the genetic accessibility of Drosophila, and the now well characterized electrophysiological approaches in the fruitfly (Jan and Jan, 1976; Broadie and Bate, 1993; Broadie et al., 1994), dissection of the pathways for synaptic transmission has been an exciting and expanding area of study. Phenotypic 6 Littleton and Bellen Mutations Reference 23 B1-2 62A 47B 79E1-2 ? Ye s No No Yes No Perin et al., 1991 DiAntonio et al., 1993a Johnston et al., 1991 Zinsmaier et al., 1990 Heimbeck et al., 1991 Ye s Yes 95E1-2 ? Yes No Schulze et al., 1995 Risinger et al., 1993 Yes Ye s 35E3-F3 32D-E Yes No Zheng et al., 1995 Kousky et al., 1994 Yes Ye s 60E 14C7-8 No Yes Salkoff et al., 1987 Loughney et al., 1989 Yes 16F Yes Yes 12F6-13A4 Yes Yes Yes Yes Yes 63A 76B 24B-C 96F No No No Yes Kamb et al., 1987 Papazian et aI., 1987 Warmke et al., 1991 Bruggemann et al., 1993 Butler et al., 1989 Butler et al., 1989 Butler et al., 1989 Atkinson et al., 1991 Cloned Genetic Location A. Synaptic Vesicle Proteins Synaptotagmin Synaptobrevin Rab3 Cysteine String Protein Synapsin Yes Ye s Yes Yes Ye s B. Presynaptic Membrane Proteins Syntaxin SNAP25 Calcium Channels ~l-subunit 13-subunit Sodium Channels DSC1 para Potassium Channels Shaker Ether-a-go-go Shab Shal Shaw Slowpoke C. Presynaptic Cytostolic Proteins Rop NSF 0t-SNAP Dynamin ( shibire ) Ye s Yes Yes Y es 64B 11D9-E4 77B1-4 !3F-14A Ye s No No Yes Salzberg et al., 1993 Ordway et al., 1994 Ordway et al., 1994 van der Bliek and Meyerowitz, 1991 Pongs et al., 1993 Frequenin Ye s 16F5-8 Yes Acetyl cholinesterase Choline acetyltransferase Calmodulin Dunce (cAMP PDE) Ye s Yes Yes Yes 87E3 91C7-D2 49A 3Cll-D4 Ye s Yes Ye s Yes rutabaga (adenylate cyclase) Inebriated Hyperkinetic Yes 12F5-13A1 Yes Fournier et al., 1989 Itoh et al., 1986 Beckingham et al., 1987 Davis and Davidson, 1984 Levin et al., 1992 No No 24EF 9AC Yes Ye s Stem et al., 1992 Stem et al., 1989 D. Other Synaptic Proteins Table 2. Presynaptic proteins in Drosophila melanogaster Presynaptic function in exocytosis analyses of mutations in rop (Harrison et al., 1994; Schulze et al., 1994), synaptotagmin (Littleton et al., 1993, 1994; DiAntonio et al., 1993, 1994; Broadie et al., 1994), syntaxin (Schulze et al., 1995), frequenin (Pongs et al., 1993) and csp (Zinsmaier et al., 1994; Umbach et al., 1994) have been reported. In addition, synaptobrevin has been genetically targeted in Drosophila with a tetanus toxin construct (Sweeney et al., 1995). However, mutations in NSF, 0t-SNAP, synaptobrevin, SNAP25, and rab3a remain to be isolated. In the remainder of this review, we will discuss the results obtained in Drosophila that provide insights into the in vivo function of these proteins. We will first discuss the proteins of the constitutive pathway, and then cover what is known about the proteins that modulate this process. The core SNARE complex: synaptobrevin, syntaxin, SNAP-25, a - S N A P and NSF If the SNARE model of synaptic vesicle docking and fusion is correct, then disruptions in the docking Mammalian Synaptic Protein and Drosophila Homolog (% identity) Synaptotagmin Synaptotagmin (57%) Synaptobrevin/VAMP n-Synaptobrevin (70%) Rab3A dRab3 (78%) MW (kDa) 65 70 18 7 25 26 Subcellar Location proteins syntaxin, SNAP-25, and synaptobrevin, and the fusion proteins NSF and 0t-SNAP, would be expected to result in a complete block of synaptic transmission, assuming there are no redundant proteins. The genes corresponding to these proteins have now been cloned in Drosophila and localized to the nervous system (See Table 3). Mutations in NSF, c~-SNAP, SNAP-25 and synaptobrevin have not yet been described. However, syntaxin mutations have been isolated and characterized (Schulze et al., 1995). Synaptobrevin/VAMP, an integral membrane protein of synaptic vesicles (Trimble eta[., 1988), has recently been implicated in vesicle fusion, as a number of tetanus and clostridial neurotoxins have been shown to cleave the protein and disrupt neurotransmission (Schiavo et al., 1992). Two synaptobrevin genes have been identified in Drosophila, a neuronal specific synaptobrevin (n-syb) (DiAntonio et al., 1993a) and a ubiquitously expressed synaptobrevin (Siidhof et al., 1989; Chin et al., 1993). The neuronal specific synaptobrevin has been shown to be expressed Putative Functions synaptic vesicle fusion clamp, synaptic vesicle Ca 2+ sensor synaptic vesicle v-SNARE ? docking, fusion synaptic vesicle facilitation of synaptic vesicle docking Cysteine String Protein 32-34 dcsp (70%) 32-36 synaptic vesicle binds Ca 2+ synaptic vesicle channels, ? Munc-18/nSec-1 67 Rop (68%) SNAP-25 67 25 SNAP-25 (61%) 24 Syntaxin- 1A 35 Syx.lA (76%) 35 NSF dNSF (62%) 76 presynaptic negative membrane & regulator of cytosol same docking/fusion presynaptic t-SNARE, membrane I docking, axonal presynaptic outgrowth membrane presynaptic t-SNARE, membrane docking, fusion presynaptic membrane cytosol fusion cc/ 13 SNAP 33-34 ? cytosol ? dSNAP (62%) 7 fusion References Perin et al., 1990 Perin et al., 1991 Trimble et al., 1988DiAntonio et aI., 1993b Fischer yon Mallard et al., 1990 Johnston et al., 1991 Gundersen and Umbach, 1992 Zinsmaier et al., 1994 Hata et al., 1993 Schulze et al., 1994 Oyler et al., 1989 Risinger et al., 1993 Bennett et al., 1992 Schulze et al., 1995 Block et al., 1988 Ordway et al., 1994 Clary et al., 1990 Ordway et al., 1994 Table 3. Drosophila Homologues of Vertebrate Proteins Implicated in Neurotransmitter Release 8 Littleton and Bellen in the CNS and PNS, and its protein product has been localized to synapses (DiAntonio et al., 1993a). Sweeney et al. (1995) have expressed the tetanus toxin light chain specifically in the nervous system of transgenic Drosophila and have demonstrated embryonic cleavage of n-syb. This completely abolished the coordinated bodywall contractions typically present in late stage embryos. Expression of the toxin did not, however, alter the morphology of the embryo or development of the nervous system, as axonogenesis, synapse formation and postsynaptic muscle differentiation were unaffected. However, electrophysiological analysis revealed a complete block in evoked neuromuscular transmission. In addition, there was a 50% reduction in spontaneous vesicle fusions. Thus, synaptobrevin is essential for evoked neurotransmission, but some spontaneous vesicle fusion persists. The precise role of synaptobrevin in synaptic transmission, however, remains to be determined. It is possible that synaptobrevin functions in vesicle docking, or is a key component mediating vesicle fusion. The finding that spontaneous vesicle fusions can still be detected in this system suggests the possibility that there are two populations of vesicles undergoing spontaneous release: those that make use of the core components of the SNARE complex and likely represent fusion of docked vesicles; and a second population of vesicles that fuse with the membrane through a mechanism independent of the SNARE components. Syntaxins are a family of integral membrane proteins that were originally identified by their ability to bind synaptotagmin and N-type calcium channels (Bennett et al., 1992). Subsequent work has shown that these proteins are the target of the clostridial neurotoxin botulinum C1 (Blasi et al., 1993) and that members of this family are located in the presynaptic membrane (Bennett et al., 1993), consistent with a role in exocytosis. Syntaxin has been recently cloned in Drosophila and shown to be expressed in the CNS, PNS, garland cells, and epidermis (Schulze et al., 1995). Syntaxin protein is present along axonal tracts and at synaptic boutons (Schulze et al., 1995). Analysis of the subcellular distribution of the protein suggests that it may be present in synaptic vesicles, as well as at the presynaptic membrane. Null mutations in syntaxin result in a variety of phenotypes, including a failure to secrete cuticular structures and a lack of coordinated muscle contractions. Electrophysiological analysis of embryos having partial loss of function syntaxin mutations reveals a complete absence of endogenous neuromuscular transmission. In addition, evoked responses are severely reduced in amplitude and have a shift in the response histogram toward fewer synaptic vesicle fusion events. This profile is suggestive of a reduction in the number of vesicles available for fusion, consistent with a decreased number of docked or fusionready vesicles. Complete absence of syntaxin completely abolishes evoked neurotransmitter release, although spontaneous vesicles fusions can still be observed at a reduced frequency. These results imply that syntaxin is likely to be involved in secretion events in a variety of tissues, including neurons. The data currently available in Drosophila on the in vivo function of the core components of the SNARE complex support the hypothesis that these proteins are essential to synaptic vesicle fusion. However, it is still not understood exactly how these components function in vesicle fusion. Electron microscopic analysis of null mutations in these proteins may be very informative, and could reveal if this complex indeed functions to dock synaptic vesicles at the active zone, or might instead function to subserve the actual fusion event between the synaptic vesicle and presynaptic membrane. The presence of some spontaneous vesicle fusions in the absence of functional synaptobrevin or syntaxin indicates that at least a residual component of the fusion machinery is still intact. S N A R E m o d u l a t o r y p r o t e i n s : rop, rab3 a n d c y s t e i n e string p r o t e i n The presynaptic protein rop is a cytosolic protein that has been identified in several organisms including mammals (Hata et al., 1993), Drosophila (Salzberg et al., 1993) and C. elegans (Gengyo-Ando et al., 1993). The mammalian homologue of top, Muncl8/n-Secl, has been demonstrated to interact in vitro with syntaxin (Garcia et al., 1994; Hata et al., 1993; Pevsner et aI., 1994a), suggesting that top may participate in some aspect of vesicle trafficking. Interestingly, in vitro incubation of rop with syntaxin inhibits the association of syntaxin with synaptobrevin and SNAP-25 (Pevsner et al., 1994b), suggesting that rop may play a negative role in neurotransmitter release. Rop is expressed abundantly in the nervous system and many other tissues (Salzberg et al., 1993; Schulze et al., 1994; Harrison et al., 1994). Interestingly, even though rop lacks a transmembrane domain, a substantial amount of the protein is present in membrane fractions of nerve terminals (Schulze et al., 1994). This could be due to the previously described rop-syntaxin interaction. Mutations in the rop protein have been isolated and they result m a disruption of secretion in several tissues (Harrison et al., 1994), leading to embryonic lethality. Rop mutations cause defects in the gut, cuticle secretion, uric acid secretion from Malphigian tubules, and clearing of tracheal fluids. These results imply that top Presynaptic function in exocytosis plays a positive role in some aspect of secretion and endocytosis. Rop mutations also alter the on--off electrical transients in the eye, suggesting that rop functions in synaptic transmission. Another test of the role of rop in neurotransmission was performed by Schulze et al. (1994). They determined the electrophysiological consequences of rop overproduction on synaptic physiology. Rop overexpression results in decreased evoked responses, decreased miniature excitatory junctional potentials, and aberrant synaptic facilitation. However, the calcium dependence of neurotransmitter release and the postsynaptic responsiveness of the muscle fiber were unaltered. These results are in agreement with a role for rop in controlling the docking or activation of fusion-competent synaptic vesicles. The binding of rop to syntaxin may reduce the number of vesicles available for docking or fusion, and supports the hypothesis that the presynaptic membrane tSNARE complex is essential for vesicle docking and/or fusion. Rop overproduction not only decreases the response amplitude of an initial stimulus, but further decreases the amplitude of evoked responses during a train of stimuli. A similar phenotype has been reported in mice that lack the small GTP-binding protein rab3a (Geppert et al., 1994). Rab3a is a small G-protein localized to synaptic vesicles via isoprenylation (Johnston et al., 1991). Rab3a cycles between bound and free states during vesicle fusion (Fisher von Mollard et al., 1990). Mice that lack rab3a are viable, suggesting that the protein is not essential for neurotransmitter release. The only reported defects in these mice are a reduction in evoked responses during a train of stimuli, consistent with a role in the efficient recruiting of synaptic vesicles to docked sites (Geppert et al., 1994). No mutations in the Drosophila homologue of rab3a have yet been obtained, possibly due to its also being non-essential in synaptic transmission in Drosophila. However, the similar phenotype in the mouse loss of function rab3a mutation and the Drosophila rop gain of function mutations suggest that rab3a might modulate a rop-syntaxin interaction. Thus, loss of rab3a activity may unmask the negative role of a rop-syntaxin complex on vesicle docking, while rop overproduction masks rab3a's ability to modulate this interaction. In conclusion, it is possible that rab3a and rop's function is to ultimately control the number of docked synaptic vesicles present at the synapse by modulating the function of the SNARE complex discussed above. A newly identified synaptic vesicle protein shown to be important in release is the cysteine string protein (csp) (Zinsmaier et al., 1994). This protein has been shown to copurify with synaptic vesicles and csp muta- 9 tions have been isolated in Drosophila (Zinsmaier et al., 1994). Most embryos lacking csp fail to hatch, but flies carrying null mutations in csp can sometimes survive to adulthood, suggesting the persistence of the basic synaptic transmission pathway (Zinsmaier et al., 1994). However, these flies are paralyzed by increased temperature, implying that loss of csp unmasks a temperature sensitivity of synaptic transmission not found in the protein's presence. Larvae that lack csp exhibit a reduction in evoked response amplitude by approximately 50% at room temperature, and a complete absence of neurotransmitter release at elevated temperatures (Umbach et al., 1994). Spontaneous vesicle fusions persist at both temperatures, indicating that the mechanisms for evoked neurotransmission and spontaneous vesicle release are distinct. Cysteine string proteins have also been shown to be necessary for N-type channel activity (Gundersen and Umbach, 1992). This has led to speculation that the protein may allow docked synaptic vesicles to regulate presynaptic calcium channels and neurotransmitter release (Mastrogiacomo et al., 1994). Given the extensive homology of csp with dnaJ proteins, which have been shown to regulate assembly of multimeric protein complexes, it is possible that csp functions in synaptic transmission to stabilize the assembly and function of the SNARE protein complex. C a l c i u m r e g u l a t i o n of S N A R E function: synaptotagmin, rabphilin and frequenin The influx of calcium into the presynaptic terminal is known to trigger neurotransmitter release. Given that the constitutive pathway used in all cells functions in the absence of calcium, it is unlikely that components common to both the regulated and constitutive pathway would subserve this function. Therefore, the majority of interest into proteins that may function as calcium sensors has been directed towards the synapse specific proteins synaptotagmin, rabphilin and frequenin. All three of these proteins have been shown to directly bind calcium (Perin et al., 1990; Pongs et al., 1993; Shiritaki et al., 1993) and therefore are potential candidates to regulate the effect of calcium on neurotransmitter release. The most extensively studied mutations to date are in the synaptic vesicle protein synaptotagmin. The sequence of synaptotagmin in Drosophila (Perin et al., 1991) predicts a protein that spans the vesicle membrane once and has a short amino-terminal intravesicular domain and a larger cytoplasmic carboxy-terminal region (see Fig. 1). Following the transmembrane spanning region there are two 10 Littleton and Bellen cytoplasmic repeats that have homology to the regulatory domain (C2 domain) of protein kinase C (PKC) (Nishizuka, 1989). This domain has been implicated in CaZ+-dependent membrane interactions (Kaibuchi et al., 1989; Perin et al., 1990), suggesting that synaptotagmin may function as a calcium sensor in exocytosis. Synaptotagmin has also been shown to interact with the presynaptic membrane proteins syntaxin (Bennett et al., 1992) and neurexin (Petrenko et al., 1991), suggesting that synaptotagmin may play a role in synaptic vesicle docking. Interestingly, one of these interactions (syt-neurexin) has also been reported in Drosophila (Perin, 1994). Synaptotagmin is specifically expressed in the Drosophila CNS and PNS, and its protein product has been localized to synaptic contact sites (Littleton et al., 1993a). Mutations in synaptotagmin (syt) have been obtained through the use of chemical (DiAntonio et al., 1993b; Littleton et al., 1993b, 1994; Littleton and Bellen, 1994) and transposon mutagenesis (Littleton et al., 1993b). Null mutations in syt have been classified as embryonic (Littleton et al., 1993b, 1994) and first instar (DiAntonio et al., 1993b) lethal. The absence of synaptotagmin does not affect axonogenesis, synapse formation or synaptic vesicle clustering to synaptic contact sites (Littleton et al., 1995). However, syt deficient embryos show a severe reduction in coordinated muscle activity. Patch clamp analysis of synapses in syt embryonic null alleles reveals a dramatic defect in evoked neurotransmitter release (Broadie et al., 1994). For example, at 1.0 mM external calcium, synaptotagmin deficient boutons synapsing on muscle fiber 6 release on average 3 vesicles, compared to approximately 60 vesicles in wild type. In addition, 65% to 70% of the responses in syt null mutations at 1.0 mM Ca z§ result in a failure of synaptic transmission. Given the presence of residual calcium dependent exocytosis in syt null alleles, it is likely that there are additional calcium sensitive proteins participating in the release process. Data consistent with a role for synaptotagmin as a calcium sensor have been obtained in syt partial loss of function mutations. Approximately 20 alleles of syt have now been isolated (Littleton et al., 1993b, 1994; Littleton and Bellen, 1994; DiAntonio et al., 1993b). Many of these alleles show intragenic complementation, suggesting that synaptotagmin functions as a multimeric protein with independent functional domains (Littleton et al., 1994). Several of these mutant multimeric complexes contain truncated synaptotagmin proteins which lack the second C2 domain. These heteroallelic complexes cause a reduction in the order of the calcium dependence of neurotransmitter release by approximately half (Littleton et al., 1994). These data strongly support the hypothesis that synaptotagmin functions as a calcium sensor to activate synaptic vesicle fusion in vivo. In addition to synaptotagmin's role in promoting secretion, an additional finding from these studies is that the frequency of spontaneous vesicle fusions is increased in syt mutants (Littleton et al., 1993b, 1994; Broadie et al., 1994; DiAntonio et al., 1994). These data indicate that synaptotagmin might also play a role in preventing vesicle fusions in the absence of calcium. Consistent with a role for synaptotagmin as a negative regulator of neurotransmitter release, biochemical interactions of synaptotagmin with components of the SNARE complex have been identified that might allow synaptotagmin to inhibit spontaneous fusion (S611ner et al., 1993b). In conclusion, genetic dissection of the role of synaptotagmin suggests that the protein functions as a calcium sensitive activator of neurotransmitter release and an inhibitor of spontaneous vesicle fusion. Other roles for synaptotagmin have also been suggested, including a role in synaptic vesicle docking (DiAntonio et al., 1994; Perin, 1994) and endocytosis (Nonet et al., 1993; Zhang et al., 1994). Further experimental manipulations will be required before a more detailed understanding of synaptotagmin's function is achieved. Given that some residual calcium sensitive release persists in the complete absence of synaptotagmin, it is likely that other calcium sensors also modulate the release process (Broadie et al., 1994). One protein that has been proposed to play a role in calcium sensing is rabphilin-3a. (Shirataki et al., 1993). Rabphilin has been shown to bind the small GTP-binding protein rab3a and thus may function as a rab3a effector protein. Rabphilin also contains two C2 domains similar to synaptotagmin, and has been demonstrated to bind calcium in the presence of phosopholipids (Shirataki et al., 1993). To date, rabphilin has not been identified in Drosophila. Interestingly, mice lacking the rab3a gene show a depletion of rabphilin at synapses that lack the rab3a protein (Geppert et al., 1994). Synaptic transmission at these rabphilin depleted synapses shows normal calcium sensitivity and magnitude of evoked responses, except during repetitive stimulations. Thus, rabphilin may not be essential for calcium dependent evoked release, although it may play a modulatory role. Another calcium binding protein suggested to be important in calcium dependent modulation of synaptic transmission is frequenin (Pongs et al., 1993). Frequenin has been shown to bind calcium and to function as a calcium-sensitive guanylyl cyclase activator. Frequenin has been cloned in Drosophila (Pongs et al., 1993). Overexpression of frequenin either Presynaptic function in exocytosis through h e a t - s h o c k or mutagenesis causes a dramatic e n h a n c e m e n t of frequency d e p e n d e n t facilitation of n e u r o m u s c u l a r t r a n s m i s s i o n ( P o n g s et al., 1993; R i v o s e c c h i et al., 1994). T h e e x a c t m e c h a n i s m by which frequenin functions is unknown, but it has been s u g g e s t e d t h a t it m a y m o d u l a t e t h e a c t i v i t y o f a N a + - C a 2§ e x c h a n g e r , a n d t h u s l e a d to e n h a n c e d calcium levels and facilitated responses (Rivosecchi et al., 1 9 9 4 ) . If t h i s i n t e r p r e t a t i o n is c o r r e c t , t h e n frequenin may m o d u l a t e calcium sensitivity through an indirect mechanism that would ultimately impinge o n the actual calcium effectors of neurotransmission. Loss of function mutations in frequenin will have to be o b t a i n e d to d e t e r m i n e how frequenin modulates the release process. I n c o n c l u s i o n , t h e b e s t c a n d i d a t e to d a t e for calcium sensitive activation of the S N A R E complex is the synaptic vesicle protein synaptotagmin. A d d i t i o n a l calcium sensitivity may be conferred upon this complex by proteins such as rabphilin and frequenin, although their role as calcium effect0rs is unlikely to be as essential as t h a t of synaptotagmin. A d d i t i o n a l calcium sensitive proteins such as calmodulin may also play a role in controlling synaptic vesicle fusion, but their p o t e n t i a l roles await genetic dissection in vivo before any conclusions can be drawn. Concluding remarks In conclusion, the genetic dissection of proteins i n v o l v e d in s y n a p t i c t r a n s m i s s i o n in Drosophila has provided support for the S N A R E hypothesis of neurotransmitter release. In addition this approach has identified novel functions of many of the accessory proteins modulating the release pathway. 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