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Molecular determinants of presynaptic active zones
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Molecular determinants of presynaptic active zones Craig C Garner*, Stefan Kindler† and Eckart D Gundelfinger‡ The presynaptic cytoskeletal matrix (cytomatrix) assembled at active zones has been implicated in defining neurotransmitter release sites. Munc13, Rim, Bassoon and Piccolo/Aczonin are recently identified presynaptic cytomatrix proteins. These multidomain proteins are thought to organize the exocytotic and endocytotic machinery precisely at active zones. Addresses *Department of Neurobiology, University of Alabama at Birmingham, Birmingham, AL 35294-0021, USA; e-mail: [email protected] † Institute for Cellular Biochemistry and Clinical Neurobiology, University of Hamburg, D-20246 Hamburg, Germany; e-mail: [email protected] ‡ Leibniz Institute for Neurobiology, D-39118 Magdeburg, Germany; e-mail: [email protected] Current Opinion in Neurobiology 2000, 10:321–327 0959-4388/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations C1 domain C2 domain CaMKII CASK CAZ CNS DLG Doc2α EM GUK domain MAGUK Mint NSF PBH PDZ domain PRA1 PRS PSD-95 PSD Rim SAP SH3 domain SNAP SNARE SV Unc-13 VAMP2 ZO-1
PKC, conserved domain 1 (phorbol ester/ diacylglycerol binding) PKC, conserved domain 2 (Ca2+/phospholipid binding) Ca2+/calmodulin-dependent protein kinase II CaMK/SH3/guanylate kinase domain protein cytomatrix assembled at active zones central nervous system discs large double C2α electron microscopy guanylate kinase-like domain membrane-associated guanylate kinase Munc18/nSec1-interacting protein N-ethylmaleimide-sensitive factor Piccolo Bassoon homology domain PSD-95/SAP90/DLG/ZO1 domain prenylated Rab acceptor 1 proline-rich sequence postsynaptic density protein of 95 kDa postsynaptic density Rab3-interacting molecule synapse-associated protein src-homology 3 domain soluble NSF attachment protein SNAP receptor synaptic vesicle uncoordination mutant-13 vesicle-associated membrane protein 2 (synaptobrevin) zona occludens-1
Introduction Chemical synapses are highly specialized cellular junctions between neurons and their targets, designed for the rapid and efficient transmission of signaling information. Synapses are asymmetric junctions composed of a presynaptic terminal (bouton) filled with neurotransmitter-containing synaptic vesicles (SVs), a synaptic cleft, and a postsynaptic reception apparatus. The region of the presynaptic plasma membrane at which SVs dock, fuse with the membrane, and release neurotransmitters is called the active zone [1]. The
postsynaptic reception apparatus, juxtaposed to active zones, contains clusters of neurotransmitter receptors and ion channels and is referred to as the postsynaptic density (PSD). The molecular characterization of PSDs over the last 10 years has led to the identification of several classes of PDZ-containing proteins that are directly involved in the clustering of ion channels and the assembly of macromolecular signaling complexes (for reviews see [2–4]). This review focuses on more recent advances in the identification and characterization of several novel proteins that may structurally define the cortical cytomatrix assembled at active zones (CAZ).
Organization of presynaptic boutons Presynaptic boutons of conventional central nervous system (CNS) synapses are composed of distinct structural and functional compartments (Figure 1). These include the active zone where SVs dock, fuse and recycle, as well as a large reservoir of SVs that can be functionally divided into three pools. The reserve pool comprises SVs that are more than about 200 nm away from active zones and are thought to be held in place by microfilaments. SVs in the release-ready pool are docked to the presynaptic plasma membrane. Situated between these two pools is the proximal pool of SVs that appears to be clustered near the active zone via a matrix of fine filaments [5–7]. SVs present in the reserve pool are tethered to microfilaments by members of the synapsin family (Figure 1). Phosphorylation of synapsins by Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates this interaction. Disruption of the different synapsin genes in mice drastically reduces the number of SVs present in the reserve SV pool, but has no effect on the clustering of SVs in the proximal and release-ready pools (see [7,8]). The docking and fusion of SVs from the release-ready pool at active zones is mediated by, among other factors, components of the SNARE (SNAP receptor) complex and synaptotagmin [9,10]. Neurotransmitter release is triggered by the local entry of Ca2+ through N-, P/Q- and R-type Ca2+ channels that are specifically localized in the active zonal membrane [11–13]. Endocytosis occurs in a dynamin/clathrin-dependent process lateral to transmitter release sites [7,14,15]. While great progress has been made in the molecular characterization of these events, the cellular mechanisms that spatially restrict these processes and the clustering of proximal pool vesicles to the active zone are unresolved. Analogous to the PSD on the postsynaptic side, the CAZ is likely to play an organizational role in defining presynaptic sites of SV fusion and recycling. This concept was initially suggested by freeze-fracture deep-etch electron
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Cytoskeletal elements defining active zones Recent studies on the molecular composition of the active zone have led to the characterization of several classes of cytoskeletal proteins including members of the membrane-associated guanylate kinase (MAGUK) superfamily as well as Munc13 (the mouse homolog of Unc-13), Rim (Rab3-interacting molecule), Bassoon and Piccolo/Aczonin. All are multidomain proteins that are tightly associated with synaptic junctions. MAGUKs contain PDZ, SH3 (src-homology 3) and GUK (guanylate kinase-like) domains, each of which functions as a site of protein–protein interaction. They are thought to function as adaptor proteins involved in the localization and assembly of pre- and postsynaptic membrane-associated signaling complexes [3,4,17]. Three MAGUKs, SAP90/PSD-95 (synapse-associated protein 90/postsynaptic density protein 95), SAP97 (synapse-associated protein 97) and CASK (CaMK/SH3/guanylate kinase protein), are found in presynaptic boutons [18–20,21•]. Of these, CASK has been shown to interact with the cell-adhesion molecules syndecan 2 and β-neurexin, cytosolic proteins, as well as N- and
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microscopy (EM) showing that a matrix of fine filaments is present at the active zone of CNS synapses [1,5]. A similar cytomatrix has been observed in presynaptic boutons associated with neuromuscular junctions, as well as in retinal ribbon synapses [16]. In all instances, these fine filaments of the CAZ are observed to reach out and make contact with SVs, suggesting a role in the clustering of SVs in the proximal pool [5].
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Schematic diagram of a presynaptic bouton at an asymmetric type 1 glutamatergic synapse. Three distinct pools of SVs are shown. SVs in the large reserve pool (R) are tethered to microfilaments by short fine filaments composed of synapsin (syn; see inset). Binding of syn to SVs and microfilaments is regulated by phosphorylation via different kinases. A smaller proximal pool (P) of SVs is imbedded in a meshwork of fine filaments associated with the presynaptic plasma membrane at neurotransmitter release sites. This cytomatrix assembled at active zones is referred to as the CAZ and can extend several hundred nm into the bouton. Extraction of the CAZ components Piccolo/Aczonin, Bassoon, Rim and Munc13-1 requires harsh conditions, suggesting that they are tightly anchored to the cortical actin/spectrin cytoskeleton. SVs of the third pool are physically docked (D) with the active zone membrane in a fusion-ready state. They are closely associated with voltage-gated Ca2+ channels. After fusion (F), SV proteins are recycled via clathrin (T)-mediated endocytosis (E). The postsynaptic membrane is associated with a cortical cytoskeletal structure named the postsynaptic density (PSD).
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P/Q-type voltage-gated Ca2+ channels [21•,22,23•,24•]. These interactions may serve to cluster Ca2+ channels at active zones. CASK also binds the PDZ containing proteins Veli and Mint1 (a Munc18/nSec1[neuronal homolog of the yeast secretion mutant 1]-interacting protein) [23•]. The latter may help to couple Ca2+ channels to the SV fusion machinery [24•]. It should be noted that neither CASK nor SAP90/PSD-95 nor SAP97 are restricted to the active zone and also exert their clustering functions at other membrane specializations [3,4,17,21•].
Munc13-1 In contrast to the MAGUKs, Munc13-1, Rim, Bassoon and Piccolo/Aczonin are spatially restricted in nerve terminals to active zones [25,26,27•,28••,29••]. Initially, mutations in the unc-13 (uncoordination mutant 13) gene in C. elegans were found to cause severe uncoordinated movements in worms [30]. The protein product of a mammalian homologue, Munc13-1, contains three C2 (protein kinase C, conserved domain 2) domains that are involved in binding Ca2+ and phospholipids, as well as a C1 domain which, like that in protein kinase C, binds phorbol esters and diacylglycerol (Figure 2) [27•,31]. Experimental evidence suggests that phorbol ester binding to Munc13-1 is involved in regulating neurotransmitter release [27•]. Disruption of the C. elegans (unc-13), Drosophila (dunc-13) or mouse (munc13-1) genes dramatically reduces both the evoked and the spontaneous release of neurotransmitter ([32••,33••,34••]; see Brose et al., pp 303–311, this issue). Loss of the related proteins does not affect the morphological structure of synaptic junctions, nor does it
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Domain structure of CAZ proteins. Bassoon and Piccolo/Aczonin are structurally related CAZ proteins composed of 10 regions of clear homology called Piccolo Bassoon homology domains (PBHs, numbered 1–10) as well as two zinc fingers (Zn), three coiled-coil (CC) domains and several proline-rich sequences. In the carboxyl-terminus of Piccolo/Aczonin is a PDZ domain and two C2 domains. Rim contains a single amino-terminal zinc finger domain, a PDZ domain and two C2 domains. These domains share limited homology with the analogous domains in Piccolo/Aczonin. Oboe, identified on the basis of its
homology to the carboxyl-terminal tails of Rim and Piccolo, contains a PDZ domain and two C2 domains and appears to lack a zinc finger. It is unclear whether it is also a component of the CAZ. Other than the presence of several C2 domains, Munc13-1 appears mostly unrelated to Rim, Oboe, Bassoon and Piccolo/Aczonin. Munc13-1 also contains a C1 domain involved in binding phorbol esters. CASK is a member of the MAGUK superfamily and is composed of a CaMKII-like domain, a PDZ, an SH3 and a GUK domain.
disrupt the clustering of SVs near active zones. The number of SVs is unchanged in mice, and observed to be abnormally large in C. elegans and Drosophila mutants. Munc13-1 is thought to regulate the release of neurotransmitter through its interactions with the SV-associated
protein Doc2α (double c2α) [35•], a Ca2+/phospholipid binding protein, and syntaxin, a component of the SNARE complex. As shown in C. elegans, the regulated release of neurotransmitter by Munc13-1 may be achieved by displacing UNC18/Munc18/nSec1 from syntaxin,
Figure 3 Localization of Bassoon in the CAZ of various types of CNS synapses. (a) Mossy fiber terminal (mf) in the CA3 region of the rat hippocampus. Immunoreactivity is highly concentrated at active zones opposite to PSDs (arrowheads). Scale bar 500 nm. Reproduced with permission from [28••]. (b) Immunogold localization of Bassoon in an ultrathin cryosection of a CA1 synapse in the rat hippocampus (pre, presynaptic terminal; post, postsynapse). Scale bar 100 nm. (Courtesy of Karin Richter, Magdeburg; reprinted from the research report 1998/99 of the Leibniz Institute for Neurobiology.) (c) Immunogold localization of Bassoon at a rod photoreceptor ribbon synapse of the rabbit retina. H, postsynaptic processes of horizontal cells. The arrowhead marks the tip, the arrow the acriform density at the base of the ribbon. Scale bar 100 nm. Reproduced with permission from [42•].
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Molecular model of the structure and interactions of CAZ proteins. The membrane-associated cortical cytoskeleton is thought to provide a scaffold for different classes of proteins involved in SV exo- and endocytosis. CASK, a member of the superfamily of membraneassociated guanylate kinase homologs (MAGUK), contains CaMKII-like, SH3, PDZ and guanylate kinase (GK)-like domains. The first domain binds the Munc18-interacting protein Mint1, while the PDZ domain interacts with the cytoplasmic tails of β-neurexin and syndecan. CASK also associates with the Veli proteins, a group of proteins containing a single PDZ domain. More recently, the cytoplasmic tail of N-type Ca2+ channels was shown to interact with both the SH3 domain in CASK and the PDZ domain in Mint1. The assembly of voltage-gated Ca2+ channels and Munc18 (a negative regulator of SV fusion) into one complex suggests a role of this structure in neurotransmitter release. Several studies have directly implicated the CAZ protein Munc13 in regulating SV fusion with the plasma membrane. Munc13 is thought to antagonize
Munc18 and to promote the assembly of syntaxin into the SNARE fusion complex. It contains several domains that interact with β-spectrin, syntaxin, phorbol esters (C1) and Ca2+/phospholipids (C2). The CAZ protein Rim comprises a zinc finger, a PDZ domain and C2 domains. Its ability to bind Rab3A/C in a GTP-dependent manner, similar to rabphilin, suggests a function in the translocation of SVs from the proximal to the release-ready pool. The largest identified CAZ proteins, Bassoon and Piccolo/Aczonin, may extend several hundred nm in length. Both are structurally related proteins containing 10 PBH domains, a pair of zinc fingers and, in the case of Piccolo/Aczonin, a PDZ and two C2 domains. Piccolo zinc fingers interact with the prenylated Rab3 acceptor protein PRA1, suggesting a role in SV exocytosis. Proline-rich sequences (PRS) in both proteins may play a role in dynamically tethering components of the endocytotic machinery near active zones via SH3 domains. Relative distances of Rim, Bassoon and/or Piccolo/Aczonin from the membrane of the active zone are not known.
allowing syntaxin to join the exocytotic synaptic core complex [36]. This finding suggests a role for the CAZ protein Munc13-1 in the achievement of fusion competence of docked SVs. An association of Munc13-1 with a novel brain-specific isoform of β-spectrin may act to anchor this molecule to the CAZ [37].
zinc finger domain, a PDZ domain, and two C2 domains (Figure 2). The Rim zinc finger is about 42% identical to the zinc finger in rabphilin, a soluble Rab3 effector molecule. As with rabphilin, the Rim zinc finger binds specifically to Rab3 in a GTP-dependent manner, but does not bind to other Rabs such as Rab5, Rab7, Rab17, or Rab22 [26]. Rab3 is a SV-associated protein that may regulate exocytosis by limiting the extent of Ca2+-triggered membrane fusion. This limiting function is thought to occur during a late step in exocytosis, after the docking step (for a review see [38]). It has been proposed that the interaction of Rim with GTP-Rab3 may act to clamp the
Rim A second CAZ protein is Rim, a 180 kDa protein identified by its ability to interact with Rab3A/C [26]. Rim is enriched in the synaptic plasma membrane fraction but is absent from SV preparations. It contains an amino-terminal
Molecular determinants of presynaptic active zones Garner, Kindler and Gundelfinger
extent of SV fusion at the active zone [26]. This interpretation is not completely consistent with the subsynaptic localization of Rim at the retinal ribbon synapse [26]. This type of synapse is designed for the rapid and continuous release of transmitter. The ribbon is a highly specialized CAZ, which tethers and directs SVs towards the active zone at the base of the ribbon. At these synapses, Rim is found all along the surface of the ribbon. This distribution is compatible with a role of Rim in tethering SVs to the ribbon in a Rab3/GTP-dependent manner. In ribbon synapses, this would place the function of Rim upstream of the docking of SVs to the active-zonal plasma membrane.
Piccolo and Bassoon Piccolo and Bassoon are the largest members (530 and 420 kDa, respectively) of the ensemble of proteins orchestrating events at the presynaptic active zone [25,28••,39,40••]. Aczonin, a recently described CAZ protein [29••], is an ortholog of Piccolo. Spatially, Piccolo/Aczonin and Bassoon are restricted to the CAZ within the nerve terminals of excitatory and inhibitory synapses throughout the CNS (Figure 3). In primary hippocampal cultures, Piccolo and Bassoon have been co-localized at both glutamatergic and GABAergic synapses [28••,39,40••]. Temporally, their appearance at early stages of synaptogenesis corresponds to the clustering of SVs, and is prior to the recruitment of postsynaptic neurotransmitter receptors [41]; this suggests a role for these CAZ proteins during synapse assembly. Bassoon is also present at the photoreceptor ribbon synapse [42•]. In contrast to Rim, which is found along the surface of the ribbon, Bassoon appears to be concentrated close to the ribbon base (Figure 3), where SVs fuse with the plasma membrane. Neither Piccolo nor Bassoon are present at the cholinergic neuromuscular junction [40••], nor has Bassoon been found at cholinergic synapses in the retina [42•]. Piccolo/Aczonin and Bassoon are structurally related CAZ proteins present in a variety of vertebrate species [28••,29••,40••,43,44]. They contain 10 clear regions of homology called Piccolo Bassoon homology (PBH) domains (Figure 2) [40••]. Embedded in the first two PBH domains are two double zinc fingers that exhibit limited homology to the zinc fingers in rabphilin and Rim. The zinc fingers in Piccolo/Aczonin and Bassoon do not interact with Rab3 [29••,40••]. Instead, Piccolo zinc fingers have been found to bind the prenylated Rab acceptor PRA1 [40••]. PRA1 is a soluble protein that can interact with both the v-SNARE VAMP2/synaptobrevin II as well as Rab3 and Rab5, two proteins involved in exo- and endocytosis, respec tively [45]. Binding of Piccolo and Rab3 to PRA1 is competitive. These interactions may play a role in the maturation of SVs as they pass through the proximal pool to the release-ready pool (Figure 4). The PBH4, PBH6 and PBH8 domains contain coiled-coil regions. Situated in the carboxyl terminus of Piccolo/Aczonin (and absent in Bassoon) are a single PDZ and two C2 domains. Both proteins also contain proline-rich sequences (PRS).
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In vitro, profilin has been shown to bind a PRS in Aczonin [29••]. PRS may also act as SH3 binding sites for various proteins involved in endocytosis, such as endophilins (SH3p4/SH3p8/SH3p13) [46], amphiphysins I and II [47] and intersectin [48], which have also been localized to active zones [14,15,49,50]. These data suggest that Piccolo/Aczonin and Bassoon are multidomain CAZ scaffolding proteins that may serve to localize components of the exo- and endocytotic machinery. On the basis of structural considerations, Piccolo/Aczonin and Bassoon are members of a new gene family not found in C. elegans. While Rim shares some structural features with Piccolo such as the zinc finger, PDZ and two C2 domains, the absence of PBH domains indicates that Rim belongs to a different protein family. A second member of the Rim family is Oboe. Although Oboe has a domain structure similar to that of Rim [40••] (Figure 2), it is unclear whether Oboe is a component of the CAZ. Members of the Rim/Oboe family are present in C. elegans, suggesting a more ancient function for these proteins.
Conclusions The identification of CASK, Munc13-1, Rim, Piccolo/Aczonin and Bassoon probably represents only the tip of the iceberg with respect to the complexity of the protein constituents of the CAZ. In many respects, the CAZ appears to perform an organizational function at the active zone similar to the function of the PSD at the postsynapse. For example, the CAZ proteins described thus far are composed of multiple domains that may enable them to interact with a variety of proteins. Therefore, CAZ proteins may act as adaptors that interconnect cell adhesion molecules, ion channels/receptors, the cortical cytoskeleton and components of the neurotransmitter release machinery. Functionally, these interactions may hold the active zone in register with the postsynaptic reception apparatus, may regulate the recruitment of SVs to the release-ready pool, and may restrict endo- and exocytosis to active zones.
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18. Kistner U, Wenzel BM, Veh RW, Cases-Langhoff C, Garner AM, Appeltauer U, Voss B, Gundelfinger ED, Garner CC: SAP90, a rat presynaptic protein related to the product of the Drosophila tumor suppressor gene dlg-A. J Biol Chem 1993, 268:4580-4583. 19. Muller BM, Kistner U, Veh RW, Cases-Langhoff C, Becker B, Gundelfinger ED, Garner CC: Molecular characterization and spatial distribution of SAP97, a novel presynaptic protein homologous to SAP90 and the Drosophila discs-large tumor suppressor protein. J Neurosci 1995, 15:2354-2366. 20. Koulen P, Fletcher EL, Craven SE, Bredt DS, Wassle H: Immunocytochemical localization of the postsynaptic density protein PSD-95 in the mammalian retina. J Neurosci 1998, 18:10136-10149. 21. Hsueh YP, Yang FC, Kharazia V, Naisbitt S, Cohen AR, Weinberg RJ, • Sheng M: Direct interaction of CASK/LIN-2 and syndecan heparan sulfate proteoglycan and their overlapping distribution in neuronal synapses. J Cell Biol 1998, 142:139-151. This paper describes the interaction of the MAGUK CASK with the proteoglycan syndecan. The interaction between the PDZ domain in CASK and the carboxyl-terminus of syndecan may play a role in synaptic adhesion and/or signaling via receptor tyrosine kinases. This study also examines the spatial distribution of CASK by immunogold electron microscopy. Importantly, while CASK is found in presynaptic terminals, it is not restricted to this compartment but is also found in the postsynaptic compartment and in extrasynaptic compartments, indicating that its neuronal functions are not restricted to those within the active zone. 22. Hata Y, Butz S, Sudhof TC: CASK: a novel dlg/PSD95 homolog with an N-terminal calmodulin-dependent protein kinase domain identified by interaction with neurexins. J Neurosci 1996, 16:2488-2494. 23. Butz S, Okamoto M, Sudhof TC: A tripartite protein complex with • the potential to couple synaptic vesicle exocytosis to cell adhesion in brain. Cell 1998, 94:773-782. This paper describes a salt-resistant protein complex containing CASK, the small PDZ domain protein Veli/LIN-7, and the Munc18-interacting molecule Mint1, which may link the complex to presynaptic Ca2+ channels [24•]. This study, and others by this group, are providing clues as to how synaptic celladhesion molecules may promote the assembly of the CAZ and keep it in register with the postsynaptic reception apparatus. 24. Maximov A, Sudhof TC, Bezprozvanny I: Association of neuronal • calcium channels with modular adaptor proteins. J Biol Chem 1999, 274:24453-24456. These authors provide biochemical evidence for a tight interaction of presynaptic voltage-gated Ca2+ channels with the Munc18-interacting protein Mint1. Thus the presynaptic Ca2+ entry sites appear to be directly linked to the tripartite adapter complex [23•] and, in turn, to the exocytotic machinery and the β-neurexin/neuroligin cell adhesion complex linking pre- and postsynaptic compartments.
25. Cases-Langhoff C, Voss B, Garner AM, Appeltauer U, Takei K, Kindler S, Veh RW, De Camilli P, Gundelfinger ED, Garner CC: Piccolo, a novel 420 kDa protein associated with the presynaptic cytomatrix. Eur J Cell Biol 1996, 69:214-223. 26. Wang Y, Okamoto M, Schmitz F, Hofmann K, Sudhof TC: Rim is a putative Rab3 effector in regulating synaptic-vesicle fusion. Nature 1997, 388:593-598. 27. •
Betz A, Ashery U, Rickmann M, Augustin I, Neher E, Sudhof TC, Rettig J, Brose N: Munc13-1 is a presynaptic phorbol ester receptor that enhances neurotransmitter release. Neuron 1998, 21:123-136. The paper demonstrates that Munc13-1 is a direct receptor for phorbol esters and diacylglycerol. Munc13-1 can translocate to the membrane in a PKC-independent manner and can act as an enhancer of neurotransmitter release. Munc13-1 immunoreactivity is highly concentrated near the active zone. Genetic studies [32••–34••] confirm the significance of Munc13-1 in regulated exocytosis. 28. tom Dieck S, Sanmarti-Vila L, Langnaese K, Richter K, Kindler S, •• Soyke A, Wex H, Smalla KH, Kampf U, Franzer JT, Stumm M, Garner CC, Gundelfinger ED: Bassoon, a novel zinc-finger CAG/glutamine-repeat protein selectively localized at the active zone of presynaptic nerve terminals. J Cell Biol 1998, 142:499-509. This study identifies the 420 kDa protein Bassoon as a specific component of the presynaptic cytoskeleton, and the authors document its highly restricted localization near the active zone. Together with more recent studies showing that Bassoon is structurally related to Aczonin/Piccolo (see [29••,40••]), this study paves the way for a detailed molecular analysis of the protein scaffold of the CAZ. 29. Wang X, Kibschull M, Laue MM, Lichte B, Petrasch-Parwez E, •• Kilimann MW: Aczonin, a 550-kD putative scaffolding protein of presynaptic active zones, shares homology regions with Rim and Bassoon and binds profilin. J Cell Biol 1999, 147:151-162. Aczonin is orthologous to Piccolo [40••]. The study reveals the domain structure of Aczonin, and provides evidence that it is a major cytoskeletal element of the CAZ that can interact with the actin regulatory protein profilin. Together with the studies on Bassoon and Piccolo [28••,40••], this work allows a more detailed functional analysis of the CAZ. 30. Tokumaru H, Augustine GJ: UNC-13 and neurotransmitter release. Nat Neurosci 1999, 2:929-930. 31. Kazanietz MG, Lewin NE, Bruns JD, Blumberg PM: Characterization of the cysteine-rich region of the Caenorhabditis elegans protein Unc-13 as a high affinity phorbol ester receptor. Analysis of ligand-binding interactions, lipid cofactor requirements, and inhibitor sensitivity. J Biol Chem 1995, 270:10777-10783. 32. Richmond JE, Davis WS, Jorgensen EM: UNC-13 is required for •• synaptic vesicle fusion in C. elegans. Nat Neurosci 1999, 2:959-964. This is one of three studies genetically assessing the function of UNC13/Munc13-1 (see also [33••,34••]). Using an allelic series of unc-13 mutants, the authors demonstrate that the protein plays a key role in both regulated and spontaneous neurotransmitter release. Consistent with the other genetic studies, SVs appear to dock normally, but do not attain fusion competence. 33. Aravamudan B, Fergestad T, Davis WS, Rodesch CK, Broadie K: •• Drosophila UNC-13 is essential for synaptic transmission. Nat Neurosci 1999, 2:965-971. This study evaluates the function of UNC-13/Munc13-1 (see also [32••,34••]). It demonstrates the fundamental function of the protein for neurotransmitter release. Molecular physiological and ultra-structural studies suggest a role for UNC-13 that occurs between the docking and the fusion steps of exocytosis. 34. Augustin I, Rosenmund C, Sudhof TC, Brose N: Munc13-1 is •• essential for fusion competence of glutamatergic synaptic vesicles. Nature 1999, 400:457-461. In this study the function of UNC-13/Munc13-1 is genetically examined (see also [32••,33••]). Using Munc13-1-deletion mutant mice, the paper demonstrates the fundamental role of the protein in neurotransmitter release. Sophisticated electrophysiological and ultra-structural studies on mutant neurons in culture indicate a role for Munc13-1 in the maturation of docked SVs. 35. Duncan RR, Betz A, Shipston MJ, Brose N, Chow RH: Transient, • phorbol ester-induced DOC2-Munc13 interactions in vivo. J Biol Chem 1999, 274:27347-27350. This study shows that phorbol ester-stimulated enhancement of exocytosis is, in part, mediated by an association of Doc2 with Munc13-1. 36. Sassa T, Harada S, Ogawa H, Rand JB, Maruyama IN, Hosono R: Regulation of the UNC-18-Caenorhabditis elegans syntaxin complex by UNC-13. J Neurosci 1999, 19:4772-4777.
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37.
Sakaguchi G, Orita S, Naito A, Maeda M, Igarashi H, Sasaki T, Takai Y: A novel brain-specific isoform of beta spectrin: isolation and its interaction with Munc13. Biochem Biophys Res Commun 1998, 248:846-851.
38. Geppert M, Sudhof TC: RAB3 and synaptotagmin: the yin and yang of synaptic membrane fusion. Annu Rev Neurosci 1998, 21:75-95. 39. Richter K, Langnaese K, Kreutz MR, Olias G, Zhai R, Scheich H, Garner CC, Gundelfinger ED: Presynaptic cytomatrix protein bassoon is localized at both excitatory and inhibitory synapses of rat brain. J Comp Neurol 1999, 408:437-448. 40. Fenster SD, Chung WJ, Zhai R, Cases-Langhoff C, Voss B, •• Garner AM, Kaempf U, Kindler S, Gundelfinger ED, Garner CC: Piccolo, a presynaptic zinc finger protein structurally related to Bassoon. Neuron 2000, 25:203-214. The domain structure of the high-molecular-weight CAZ protein Piccolo and its interaction with the vesicle-associated protein PRA-1 is described. Together with the related protein Bassoon [28••], Piccolo (also described as Aczonin; [29••]) is the prime candidate for the formation of the proteinaceous scaffold of the CAZ. 41. Zhai R, Olias G, Chung WJ, Lester RAJ, tom Dieck S, Langnaese K, Kreutz MR, Kindler S, Gundelfinger ED, Garner CC: Temporal appearance of the presynaptic cytomatrix protein Bassoon during synaptogenesis. Mol Cell Neurosci 2000, in press. 42. Brandstatter JH, Fletcher EL, Garner CC, Gundelfinger ED, • Wassle H: Differential expression of the presynaptic cytomatrix protein bassoon among ribbon synapses in the mammalian retina. Eur J Neurosci 1999, 11:3683-3693. This study uses in situ immunogold EM to determine the subsynaptic localization of Bassoon at excitatory ribbon synapses and at conventional inhibitory synapses in the mammalian retina. The authors demonstrate the highly restricted localization of the protein at the active zone. Interestingly, Bassoon was found at photoreceptor, but not at bipolar cell, ribbon synapses.
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43. Hashida H, Goto J, Zhao N, Takahashi N, Hirai M, Kanazawa I, Sakaki Y: Cloning and mapping of ZNF231, a novel brainspecific gene encoding neuronal double zinc finger protein whose expression is enhanced in a neurodegenerative disorder, multiple system atrophy (MSA). Genomics 1998, 54:50-58. 44. Winter C, tom Dieck S, Boeckers TM, Bockmann J, Kampf U, Sanmarti-Vila L, Langnaese K, Altrock W, Stumm M, Soyke A et al.: The presynaptic cytomatrix protein Bassoon: sequence and chromosomal localization of the human BSN gene. Genomics 1999, 57:389-397. 45. Martincic I, Peralta ME, Ngsee JK: Isolation and characterization of a dual prenylated Rab and VAMP2 receptor. J Biol Chem 1997, 272:26991-26998. 46. Micheva KD, Kay BK, McPherson PS: Synaptojanin forms two separate complexes in the nerve terminal. Interactions with endophilin and amphiphysin. J Biol Chem 1997, 272:27239-27245. 47.
David C, McPherson PS, Mundigl O, De Camilli P: A role of amphiphysin in synaptic vesicle endocytosis suggested by its binding to dynamin in nerve terminals. Proc Natl Acad Sci USA 1996, 93:331-335.
48. Yamabhai M, Hoffman NG, Hardison NL, McPherson PS, Castagnoli L, Cesareni G, Kay BK: Intersectin, a novel adaptor protein with two eps15 homology and five src homology 3 domains. J Biol Chem 1998, 273:31401-31407. 49. Estes PS, Roos J, van der Bliek A, Kelly RB, Krishnan KS, Ramaswami M: Traffic of dynamin within individual Drosophila synaptic boutons relative to compartment specific markers. J Neurosci 1996, 16:5443-5456. 50. Haffner C, Takei K, Chen H, Ringstad N, Hudson A, Butler MH, Salcini AE, Di Fiore PP, De Camilli P: Synaptojanin 1: localization on coated endocytotic intermediates in nerve terminals and interaction of its 170 kDa isoform with Eps15. FEBS Lett 1997, 419:175-180.
Molecular determinants of presynaptic active zones Craig C Garner*, Stefan Kindler† and Eckart D Gundelfinger‡ The presynaptic cytoskeletal matrix (cytomatrix) assembled at active zones has been implicated in defining neurotransmitter release sites. Munc13, Rim, Bassoon and Piccolo/Aczonin are recently identified presynaptic cytomatrix proteins. These multidomain proteins are thought to organize the exocytotic and endocytotic machinery precisely at active zones. Addresses *Department of Neurobiology, University of Alabama at Birmingham, Birmingham, AL 35294-0021, USA; e-mail: [email protected] † Institute for Cellular Biochemistry and Clinical Neurobiology, University of Hamburg, D-20246 Hamburg, Germany; e-mail: [email protected] ‡ Leibniz Institute for Neurobiology, D-39118 Magdeburg, Germany; e-mail: [email protected] Current Opinion in Neurobiology 2000, 10:321–327 0959-4388/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations C1 domain C2 domain CaMKII CASK CAZ CNS DLG Doc2α EM GUK domain MAGUK Mint NSF PBH PDZ domain PRA1 PRS PSD-95 PSD Rim SAP SH3 domain SNAP SNARE SV Unc-13 VAMP2 ZO-1
PKC, conserved domain 1 (phorbol ester/ diacylglycerol binding) PKC, conserved domain 2 (Ca2+/phospholipid binding) Ca2+/calmodulin-dependent protein kinase II CaMK/SH3/guanylate kinase domain protein cytomatrix assembled at active zones central nervous system discs large double C2α electron microscopy guanylate kinase-like domain membrane-associated guanylate kinase Munc18/nSec1-interacting protein N-ethylmaleimide-sensitive factor Piccolo Bassoon homology domain PSD-95/SAP90/DLG/ZO1 domain prenylated Rab acceptor 1 proline-rich sequence postsynaptic density protein of 95 kDa postsynaptic density Rab3-interacting molecule synapse-associated protein src-homology 3 domain soluble NSF attachment protein SNAP receptor synaptic vesicle uncoordination mutant-13 vesicle-associated membrane protein 2 (synaptobrevin) zona occludens-1
Introduction Chemical synapses are highly specialized cellular junctions between neurons and their targets, designed for the rapid and efficient transmission of signaling information. Synapses are asymmetric junctions composed of a presynaptic terminal (bouton) filled with neurotransmitter-containing synaptic vesicles (SVs), a synaptic cleft, and a postsynaptic reception apparatus. The region of the presynaptic plasma membrane at which SVs dock, fuse with the membrane, and release neurotransmitters is called the active zone [1]. The
postsynaptic reception apparatus, juxtaposed to active zones, contains clusters of neurotransmitter receptors and ion channels and is referred to as the postsynaptic density (PSD). The molecular characterization of PSDs over the last 10 years has led to the identification of several classes of PDZ-containing proteins that are directly involved in the clustering of ion channels and the assembly of macromolecular signaling complexes (for reviews see [2–4]). This review focuses on more recent advances in the identification and characterization of several novel proteins that may structurally define the cortical cytomatrix assembled at active zones (CAZ).
Organization of presynaptic boutons Presynaptic boutons of conventional central nervous system (CNS) synapses are composed of distinct structural and functional compartments (Figure 1). These include the active zone where SVs dock, fuse and recycle, as well as a large reservoir of SVs that can be functionally divided into three pools. The reserve pool comprises SVs that are more than about 200 nm away from active zones and are thought to be held in place by microfilaments. SVs in the release-ready pool are docked to the presynaptic plasma membrane. Situated between these two pools is the proximal pool of SVs that appears to be clustered near the active zone via a matrix of fine filaments [5–7]. SVs present in the reserve pool are tethered to microfilaments by members of the synapsin family (Figure 1). Phosphorylation of synapsins by Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates this interaction. Disruption of the different synapsin genes in mice drastically reduces the number of SVs present in the reserve SV pool, but has no effect on the clustering of SVs in the proximal and release-ready pools (see [7,8]). The docking and fusion of SVs from the release-ready pool at active zones is mediated by, among other factors, components of the SNARE (SNAP receptor) complex and synaptotagmin [9,10]. Neurotransmitter release is triggered by the local entry of Ca2+ through N-, P/Q- and R-type Ca2+ channels that are specifically localized in the active zonal membrane [11–13]. Endocytosis occurs in a dynamin/clathrin-dependent process lateral to transmitter release sites [7,14,15]. While great progress has been made in the molecular characterization of these events, the cellular mechanisms that spatially restrict these processes and the clustering of proximal pool vesicles to the active zone are unresolved. Analogous to the PSD on the postsynaptic side, the CAZ is likely to play an organizational role in defining presynaptic sites of SV fusion and recycling. This concept was initially suggested by freeze-fracture deep-etch electron
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Cytoskeletal elements defining active zones Recent studies on the molecular composition of the active zone have led to the characterization of several classes of cytoskeletal proteins including members of the membrane-associated guanylate kinase (MAGUK) superfamily as well as Munc13 (the mouse homolog of Unc-13), Rim (Rab3-interacting molecule), Bassoon and Piccolo/Aczonin. All are multidomain proteins that are tightly associated with synaptic junctions. MAGUKs contain PDZ, SH3 (src-homology 3) and GUK (guanylate kinase-like) domains, each of which functions as a site of protein–protein interaction. They are thought to function as adaptor proteins involved in the localization and assembly of pre- and postsynaptic membrane-associated signaling complexes [3,4,17]. Three MAGUKs, SAP90/PSD-95 (synapse-associated protein 90/postsynaptic density protein 95), SAP97 (synapse-associated protein 97) and CASK (CaMK/SH3/guanylate kinase protein), are found in presynaptic boutons [18–20,21•]. Of these, CASK has been shown to interact with the cell-adhesion molecules syndecan 2 and β-neurexin, cytosolic proteins, as well as N- and
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microscopy (EM) showing that a matrix of fine filaments is present at the active zone of CNS synapses [1,5]. A similar cytomatrix has been observed in presynaptic boutons associated with neuromuscular junctions, as well as in retinal ribbon synapses [16]. In all instances, these fine filaments of the CAZ are observed to reach out and make contact with SVs, suggesting a role in the clustering of SVs in the proximal pool [5].
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Schematic diagram of a presynaptic bouton at an asymmetric type 1 glutamatergic synapse. Three distinct pools of SVs are shown. SVs in the large reserve pool (R) are tethered to microfilaments by short fine filaments composed of synapsin (syn; see inset). Binding of syn to SVs and microfilaments is regulated by phosphorylation via different kinases. A smaller proximal pool (P) of SVs is imbedded in a meshwork of fine filaments associated with the presynaptic plasma membrane at neurotransmitter release sites. This cytomatrix assembled at active zones is referred to as the CAZ and can extend several hundred nm into the bouton. Extraction of the CAZ components Piccolo/Aczonin, Bassoon, Rim and Munc13-1 requires harsh conditions, suggesting that they are tightly anchored to the cortical actin/spectrin cytoskeleton. SVs of the third pool are physically docked (D) with the active zone membrane in a fusion-ready state. They are closely associated with voltage-gated Ca2+ channels. After fusion (F), SV proteins are recycled via clathrin (T)-mediated endocytosis (E). The postsynaptic membrane is associated with a cortical cytoskeletal structure named the postsynaptic density (PSD).
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P/Q-type voltage-gated Ca2+ channels [21•,22,23•,24•]. These interactions may serve to cluster Ca2+ channels at active zones. CASK also binds the PDZ containing proteins Veli and Mint1 (a Munc18/nSec1[neuronal homolog of the yeast secretion mutant 1]-interacting protein) [23•]. The latter may help to couple Ca2+ channels to the SV fusion machinery [24•]. It should be noted that neither CASK nor SAP90/PSD-95 nor SAP97 are restricted to the active zone and also exert their clustering functions at other membrane specializations [3,4,17,21•].
Munc13-1 In contrast to the MAGUKs, Munc13-1, Rim, Bassoon and Piccolo/Aczonin are spatially restricted in nerve terminals to active zones [25,26,27•,28••,29••]. Initially, mutations in the unc-13 (uncoordination mutant 13) gene in C. elegans were found to cause severe uncoordinated movements in worms [30]. The protein product of a mammalian homologue, Munc13-1, contains three C2 (protein kinase C, conserved domain 2) domains that are involved in binding Ca2+ and phospholipids, as well as a C1 domain which, like that in protein kinase C, binds phorbol esters and diacylglycerol (Figure 2) [27•,31]. Experimental evidence suggests that phorbol ester binding to Munc13-1 is involved in regulating neurotransmitter release [27•]. Disruption of the C. elegans (unc-13), Drosophila (dunc-13) or mouse (munc13-1) genes dramatically reduces both the evoked and the spontaneous release of neurotransmitter ([32••,33••,34••]; see Brose et al., pp 303–311, this issue). Loss of the related proteins does not affect the morphological structure of synaptic junctions, nor does it
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Domain structure of CAZ proteins. Bassoon and Piccolo/Aczonin are structurally related CAZ proteins composed of 10 regions of clear homology called Piccolo Bassoon homology domains (PBHs, numbered 1–10) as well as two zinc fingers (Zn), three coiled-coil (CC) domains and several proline-rich sequences. In the carboxyl-terminus of Piccolo/Aczonin is a PDZ domain and two C2 domains. Rim contains a single amino-terminal zinc finger domain, a PDZ domain and two C2 domains. These domains share limited homology with the analogous domains in Piccolo/Aczonin. Oboe, identified on the basis of its
homology to the carboxyl-terminal tails of Rim and Piccolo, contains a PDZ domain and two C2 domains and appears to lack a zinc finger. It is unclear whether it is also a component of the CAZ. Other than the presence of several C2 domains, Munc13-1 appears mostly unrelated to Rim, Oboe, Bassoon and Piccolo/Aczonin. Munc13-1 also contains a C1 domain involved in binding phorbol esters. CASK is a member of the MAGUK superfamily and is composed of a CaMKII-like domain, a PDZ, an SH3 and a GUK domain.
disrupt the clustering of SVs near active zones. The number of SVs is unchanged in mice, and observed to be abnormally large in C. elegans and Drosophila mutants. Munc13-1 is thought to regulate the release of neurotransmitter through its interactions with the SV-associated
protein Doc2α (double c2α) [35•], a Ca2+/phospholipid binding protein, and syntaxin, a component of the SNARE complex. As shown in C. elegans, the regulated release of neurotransmitter by Munc13-1 may be achieved by displacing UNC18/Munc18/nSec1 from syntaxin,
Figure 3 Localization of Bassoon in the CAZ of various types of CNS synapses. (a) Mossy fiber terminal (mf) in the CA3 region of the rat hippocampus. Immunoreactivity is highly concentrated at active zones opposite to PSDs (arrowheads). Scale bar 500 nm. Reproduced with permission from [28••]. (b) Immunogold localization of Bassoon in an ultrathin cryosection of a CA1 synapse in the rat hippocampus (pre, presynaptic terminal; post, postsynapse). Scale bar 100 nm. (Courtesy of Karin Richter, Magdeburg; reprinted from the research report 1998/99 of the Leibniz Institute for Neurobiology.) (c) Immunogold localization of Bassoon at a rod photoreceptor ribbon synapse of the rabbit retina. H, postsynaptic processes of horizontal cells. The arrowhead marks the tip, the arrow the acriform density at the base of the ribbon. Scale bar 100 nm. Reproduced with permission from [42•].
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Molecular model of the structure and interactions of CAZ proteins. The membrane-associated cortical cytoskeleton is thought to provide a scaffold for different classes of proteins involved in SV exo- and endocytosis. CASK, a member of the superfamily of membraneassociated guanylate kinase homologs (MAGUK), contains CaMKII-like, SH3, PDZ and guanylate kinase (GK)-like domains. The first domain binds the Munc18-interacting protein Mint1, while the PDZ domain interacts with the cytoplasmic tails of β-neurexin and syndecan. CASK also associates with the Veli proteins, a group of proteins containing a single PDZ domain. More recently, the cytoplasmic tail of N-type Ca2+ channels was shown to interact with both the SH3 domain in CASK and the PDZ domain in Mint1. The assembly of voltage-gated Ca2+ channels and Munc18 (a negative regulator of SV fusion) into one complex suggests a role of this structure in neurotransmitter release. Several studies have directly implicated the CAZ protein Munc13 in regulating SV fusion with the plasma membrane. Munc13 is thought to antagonize
Munc18 and to promote the assembly of syntaxin into the SNARE fusion complex. It contains several domains that interact with β-spectrin, syntaxin, phorbol esters (C1) and Ca2+/phospholipids (C2). The CAZ protein Rim comprises a zinc finger, a PDZ domain and C2 domains. Its ability to bind Rab3A/C in a GTP-dependent manner, similar to rabphilin, suggests a function in the translocation of SVs from the proximal to the release-ready pool. The largest identified CAZ proteins, Bassoon and Piccolo/Aczonin, may extend several hundred nm in length. Both are structurally related proteins containing 10 PBH domains, a pair of zinc fingers and, in the case of Piccolo/Aczonin, a PDZ and two C2 domains. Piccolo zinc fingers interact with the prenylated Rab3 acceptor protein PRA1, suggesting a role in SV exocytosis. Proline-rich sequences (PRS) in both proteins may play a role in dynamically tethering components of the endocytotic machinery near active zones via SH3 domains. Relative distances of Rim, Bassoon and/or Piccolo/Aczonin from the membrane of the active zone are not known.
allowing syntaxin to join the exocytotic synaptic core complex [36]. This finding suggests a role for the CAZ protein Munc13-1 in the achievement of fusion competence of docked SVs. An association of Munc13-1 with a novel brain-specific isoform of β-spectrin may act to anchor this molecule to the CAZ [37].
zinc finger domain, a PDZ domain, and two C2 domains (Figure 2). The Rim zinc finger is about 42% identical to the zinc finger in rabphilin, a soluble Rab3 effector molecule. As with rabphilin, the Rim zinc finger binds specifically to Rab3 in a GTP-dependent manner, but does not bind to other Rabs such as Rab5, Rab7, Rab17, or Rab22 [26]. Rab3 is a SV-associated protein that may regulate exocytosis by limiting the extent of Ca2+-triggered membrane fusion. This limiting function is thought to occur during a late step in exocytosis, after the docking step (for a review see [38]). It has been proposed that the interaction of Rim with GTP-Rab3 may act to clamp the
Rim A second CAZ protein is Rim, a 180 kDa protein identified by its ability to interact with Rab3A/C [26]. Rim is enriched in the synaptic plasma membrane fraction but is absent from SV preparations. It contains an amino-terminal
Molecular determinants of presynaptic active zones Garner, Kindler and Gundelfinger
extent of SV fusion at the active zone [26]. This interpretation is not completely consistent with the subsynaptic localization of Rim at the retinal ribbon synapse [26]. This type of synapse is designed for the rapid and continuous release of transmitter. The ribbon is a highly specialized CAZ, which tethers and directs SVs towards the active zone at the base of the ribbon. At these synapses, Rim is found all along the surface of the ribbon. This distribution is compatible with a role of Rim in tethering SVs to the ribbon in a Rab3/GTP-dependent manner. In ribbon synapses, this would place the function of Rim upstream of the docking of SVs to the active-zonal plasma membrane.
Piccolo and Bassoon Piccolo and Bassoon are the largest members (530 and 420 kDa, respectively) of the ensemble of proteins orchestrating events at the presynaptic active zone [25,28••,39,40••]. Aczonin, a recently described CAZ protein [29••], is an ortholog of Piccolo. Spatially, Piccolo/Aczonin and Bassoon are restricted to the CAZ within the nerve terminals of excitatory and inhibitory synapses throughout the CNS (Figure 3). In primary hippocampal cultures, Piccolo and Bassoon have been co-localized at both glutamatergic and GABAergic synapses [28••,39,40••]. Temporally, their appearance at early stages of synaptogenesis corresponds to the clustering of SVs, and is prior to the recruitment of postsynaptic neurotransmitter receptors [41]; this suggests a role for these CAZ proteins during synapse assembly. Bassoon is also present at the photoreceptor ribbon synapse [42•]. In contrast to Rim, which is found along the surface of the ribbon, Bassoon appears to be concentrated close to the ribbon base (Figure 3), where SVs fuse with the plasma membrane. Neither Piccolo nor Bassoon are present at the cholinergic neuromuscular junction [40••], nor has Bassoon been found at cholinergic synapses in the retina [42•]. Piccolo/Aczonin and Bassoon are structurally related CAZ proteins present in a variety of vertebrate species [28••,29••,40••,43,44]. They contain 10 clear regions of homology called Piccolo Bassoon homology (PBH) domains (Figure 2) [40••]. Embedded in the first two PBH domains are two double zinc fingers that exhibit limited homology to the zinc fingers in rabphilin and Rim. The zinc fingers in Piccolo/Aczonin and Bassoon do not interact with Rab3 [29••,40••]. Instead, Piccolo zinc fingers have been found to bind the prenylated Rab acceptor PRA1 [40••]. PRA1 is a soluble protein that can interact with both the v-SNARE VAMP2/synaptobrevin II as well as Rab3 and Rab5, two proteins involved in exo- and endocytosis, respec tively [45]. Binding of Piccolo and Rab3 to PRA1 is competitive. These interactions may play a role in the maturation of SVs as they pass through the proximal pool to the release-ready pool (Figure 4). The PBH4, PBH6 and PBH8 domains contain coiled-coil regions. Situated in the carboxyl terminus of Piccolo/Aczonin (and absent in Bassoon) are a single PDZ and two C2 domains. Both proteins also contain proline-rich sequences (PRS).
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In vitro, profilin has been shown to bind a PRS in Aczonin [29••]. PRS may also act as SH3 binding sites for various proteins involved in endocytosis, such as endophilins (SH3p4/SH3p8/SH3p13) [46], amphiphysins I and II [47] and intersectin [48], which have also been localized to active zones [14,15,49,50]. These data suggest that Piccolo/Aczonin and Bassoon are multidomain CAZ scaffolding proteins that may serve to localize components of the exo- and endocytotic machinery. On the basis of structural considerations, Piccolo/Aczonin and Bassoon are members of a new gene family not found in C. elegans. While Rim shares some structural features with Piccolo such as the zinc finger, PDZ and two C2 domains, the absence of PBH domains indicates that Rim belongs to a different protein family. A second member of the Rim family is Oboe. Although Oboe has a domain structure similar to that of Rim [40••] (Figure 2), it is unclear whether Oboe is a component of the CAZ. Members of the Rim/Oboe family are present in C. elegans, suggesting a more ancient function for these proteins.
Conclusions The identification of CASK, Munc13-1, Rim, Piccolo/Aczonin and Bassoon probably represents only the tip of the iceberg with respect to the complexity of the protein constituents of the CAZ. In many respects, the CAZ appears to perform an organizational function at the active zone similar to the function of the PSD at the postsynapse. For example, the CAZ proteins described thus far are composed of multiple domains that may enable them to interact with a variety of proteins. Therefore, CAZ proteins may act as adaptors that interconnect cell adhesion molecules, ion channels/receptors, the cortical cytoskeleton and components of the neurotransmitter release machinery. Functionally, these interactions may hold the active zone in register with the postsynaptic reception apparatus, may regulate the recruitment of SVs to the release-ready pool, and may restrict endo- and exocytosis to active zones.
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