synaptic vesicle exocytosis

Synaptic Vesicle Exocytosis

Thomas C. Sudhof1 and Josep Rizo2

1Department of Molecular and Cellular Physiology, and Howard Hughes Medical Institute, StanfordUniversity Medical School, Stanford, California 94305

2Departments of Biochemistry and Pharmacology, University of Texas Southwestern Medical Center,Dallas, Texas 75390

Correspondence: [email protected]

Presynaptic nerve terminals release neurotransmitters by synaptic vesicle exocytosis.Membrane fusion mediating synaptic exocytosis and other intracellular membrane trafficis affected by a universal machinery that includes SNARE (for “soluble NSF-attachmentprotein receptor”) and SM (for “Sec1/Munc18-like”) proteins. During fusion, vesicularand target SNARE proteins assemble into an a-helical trans-SNARE complex that forcesthe two membranes tightly together, and SM proteins likely wrap around assembling trans-SNARE complexes to catalyze membrane fusion. After fusion, SNARE complexes aredissociated by the ATPase NSF (for “N-ethylmaleimide sensitive factor”). Fusion-competentconformations of SNARE proteins are maintained by chaperone complexes composedof CSPa, Hsc70, and SGT, and by nonenzymatically acting synuclein chaperones; dysfunc-tion of these chaperones results in neurodegeneration. The synaptic membrane-fusionmachinery is controlled by synaptotagmin, and additionally regulated by a presynapticprotein matrix (the “active zone”) that includes Munc13 and RIM proteins as centralcomponents.

Synaptic vesicles are uniform organelles of�40 nm diameter that constitute the central

organelle for neurotransmitter release. Eachpresynaptic nerve terminal contains hundredsof synaptic vesicles that are filled with neu-rotransmitters. When an action potential de-polarizes the presynaptic plasma membrane,Ca2þ-channels open, and Ca2þ flows into thenerve terminal to trigger the exocytosis of syn-aptic vesicles, thereby releasing their neuro-transmitters into the synaptic cleft (Fig. 1). Ca2þ

triggers exocytosis by binding to synaptotag-min; after exocytosis, vesicles are re-endocytosed,

recycled, and refilled with neurotransmitters.Recycling can occur by multiple parallel path-ways, either by fast recycling via local reuse ofvesicles (“kiss-and-run” and “kiss-and-stay”),or by slower recycling via an endosomal inter-mediate (Fig. 1).

Due to their small size, synaptic vesiclescontain a limited complement of proteins thathave been described in detail (Sudhof 2004;Takamori et al. 2006). Although the functionsof several vesicle components remain to beidentified, most vesicle components participatein one of three processes: neurotransmitter

Editors: Morgan Sheng, Bernardo Sabatini, and Thomas C. Sudhof

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uptake and storage, vesicle exocytosis, and ves-icle endocytosis and recycling. In addition, itis likely that at least some vesicle proteins areinvolved in the biogenesis of synaptic vesiclesand the maintenance of their exquisite uni-formity and stability, but little is known abouthow vesicles are made, and what determinestheir size.

SNARE PROTEINS MEDIATE SYNAPTICVESICLE FUSION

Membrane fusion is a universal process ineukaryotic cells—the formation and fusion ofmembranous organelles forms the basis of allof life’s processes, from the compartmentalorganization of the most archaic cells to the

Neurotransmitteruptake

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Figure 1. The synaptic vesicle cycle. A presynaptic nerve terminal is depicted schematically as it contacts a post-synaptic neuron. The synaptic vesicle cycle consists of exocytosis (red arrows) followed by endocytosis and recy-cling (yellow arrows). Synaptic vesicles (green circles) are filled with neurotransmitters (NT; red dots) by activetransport (neurotransmitter uptake) fueled by an electrochemical gradient established by a proton pump thatacidifies the vesicle interior (vesicle acidification; green background). In preparation to synaptic exocytosis, syn-aptic vesicles are docked at the active zone, and primed by an ATP-dependent process that renders the vesiclescompetent to respond to a Ca2þ-signal. When an action potential depolarizes the presynaptic membrane, Ca2þ-channels open, causing a local increase in intracellular Ca2þ at the active zone that triggers completion of thefusion reaction. Released neurotransmitters then bind to receptors associated with the postsynaptic density(PSD). After fusion pore opening, synaptic vesicles probably recycle via three alternative pathways: local refillingwith neurotransmitters without undocking (“kiss-and-stay”), local recycling with undocking (“kiss-and-run”),and full recycling of vesicles with passage through an endosomal intermediate. (Adapted from Sudhof 2004.)

T.C. Sudhof and J. Rizo

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sophistication of events such as synaptic trans-mission, fertilization, and immunity. The firstclue to which molecules mediate membranefusion was obtained at the synapse whenSNARE proteins were identified as targets ofclostridial botulinum and tetanus toxins. Thesepowerful neurotoxins enter a presynaptic termi-nal and act as highly specific proteases thatselectively block presynaptic membrane fusionwithout altering the morphological structureof the terminal. In 1992, the vesicular SNARE-protein synaptobrevin/VAMP (for “vesicle-associated membrane protein”)—whose desig-nation as a “SNARE protein” occurred onlylater—was identified as the first essential fusionprotein when it was shown that tetanus toxinand some botulinum toxin subtypes specificallycleave it in half (Link et al. 1992; Schiavo et al.1992). Rapidly afterward, SNAP-25 and syn-taxin-1 were shown to be substrates for differentbotulinum toxin subtypes, with several toxinsoften cleaving the same protein, but at differentpositions (Blasi et al. 1993a,b). These resultsprovided the first evidence that these three pro-teins are essential components of the membranefusion machinery, and suggested an immediateexplanation for the finding that Caenorhabditiselegans homologs of these proteins were essen-tial for nervous system function (Brenner1974), and that yeast homologs of these proteinswere required for membrane traffic in the secre-tory pathway (Novick et al. 1980).

Prior to the work on clostridial neurotoxins,in vitro fusion assays in a cell-free transport sys-tem had isolated NSF (for N-ethylmaleimidesensitive factor) as an essential factor for in vitromembrane traffic, and had shown that NSFacts via adaptor proteins called “SNAPs” (for“soluble NSF-attachment proteins,” no relationto SNAP-25 and its homologs; Wilson et al.1989). Immediately after the neurotoxindata identified synaptobrevin, SNAP-25, andsyntaxin as essential for fusion, Rothman andcolleagues discovered that these three proteinsform a complex with each other that is dissoci-ated by NSF, which acts as an ATPase (Sollneret al. 1993), leading to the “SNARE” (for “solu-ble NSF-attachment protein receptor”) desig-nation. These findings led to the hypothesis

that trans-SNARE complexes bridging thevesicle and plasma membranes mediate vesicledocking and targeting specificity, and that byanalogy other SNAREs discovered by geneticscreens in yeast and C. elegans as essential formembrane traffic might function similarly.Subsequently, SNARE complexes were foundto be resistant to SDS, revealing their highstability (Hayashi et al. 1994), and to involve aparallel arrangement of helical regions of synap-tobrevin and syntaxin-1 adjacent to their trans-membrane regions (Hanson et al. 1997). At thesame time, NSF was shown to act upstream offusion and not in fusion as originally thought(Mayer et al. 1996). Together, these findingsled to the notion that SNARE-complex assem-bly releases the energy that fuels membranefusion by forcing membranes together. Consis-tent with this notion, the three-dimensionalstructure of the synaptic SNARE complexrevealed that it forms a parallel four-helix bun-dle (Poirier et al. 1998; Sutton et al. 1998). Aftermuch additional work (reviewed in Jahn et al.2003), all SNARE complexes are now believedto assemble similarly, and to mediate intracellu-lar fusion by the same overall mechanism,although without their SM protein partners,SNARE-proteins are unable to effect fusionunder physiological conditions (see below).The function of SNARE-proteins in fusion hasbeen referred to as the “SNARE hypothesis,” aterm that has been applied with very differentmeanings over the years, and may be somewhatoutdated because at least in our view the roleof SNARE proteins in fusion is no longer ahypothesis.

SNARE proteins function in fusion by acycle of assembly into complexes that fuelfusion, and disassembly of the complexes byNSF and SNAPs that makes SNARE proteinsavailable again for another round of fusion(Figs. 2 and 3). The SNARE cycle starts withthe amino- to carboxy-terminal zippering oftrans-SNARE complexes that bridge the gapbetween the membranes destined to fuse. Fullzippering of trans-SNARE complexes likelyproduces fusion-pore opening, although it ispossible that the full zippering only stressesthe membranes and that fusion-pore opening


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occurs subsequently mediated by the SM pro-tein (see below). After fusion pore opening,the two membranes completely merge, andtrans-SNARE complexes are converted intocis-SNARE complexes (i.e., SNARE complexesare on a single membrane), which are dissoci-ated into monomers by NSF and SNAPs(Fig. 2). It is likely that NSF also acts on trans-SNARE complexes; moreover, SNARE proteinsalso associate in vitro into other complexesthat are composed of different stoichiometries

of only two proteins (e.g., syntaxin-SNAP-25heterodimers) and may also be substrates forNSF. Independent of these other actions ofNSF, however, fusion is driven overall by a cycleof SNARE association and dissociation. In thiscycle, NSF “loads” SNARE proteins with energyby the ATP-dependent dissociation of SNAREcomplexes into reactive monomeric SNAREproteins; the energy of reactive SNARE proteinsis then transformed into fusion via SNAREcomplex assembly.

1. SNARE-complexassembly

(chaperones: CSPα/β/γ +α/β/γ-synucleins)

4. SNARE-complex disassemblyand vesicle recycling

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Figure 2. The SNARE/SM protein cycle. The diagram on top depicts SNARE and SM proteins prior to fusion,when they are localized to the membranes either as natively unfolded proteins (e.g., the R-SNARE synaptobre-vin/VAMP) or as proteins folded in complexes distinct from canonical SNARE complexes (e.g., binding of theSM protein Munc18-1 to the closed conformation of syntaxin-1 as shown here, or as syntaxin-1/SNAP-25heterodimeric complexes). During priming, SNARE proteins partially zipper up into trans-complexes, andthe SM protein associates with the trans-complexes by binding to the syntaxin amino terminus (left diagram).Full SNARE-complex assembly then pulls the membranes apart, opening the fusion pore (bottom diagram),which expands such that the vesicle membrane collapses into the target membrane, and the trans-SNARE com-plexes are converted into cis-SNARE complexes (right diagram). Afterward, cis-SNARE complexes are dissoci-ated by the ATPase NSF acting in conjunction with its adaptors a/b/g-SNAPs (no relation to SNAP-25 and itshomologs—an unfortunate coincidence of acronyms), and vesicles recycle to start another round of the cycle.





SPECIFICITY OF SNARE–PROTEINCOMPLEX ASSEMBLY

SNARE-proteins contain a characteristic se-quence, referred to as the SNARE motif (Fig.3A), that comprises 60–70 residues composed

of eight heptad repeats. Most SNARE proteinscontain one SNARE motif, whereas SNAP-25and its homologs (SNAP-23, -29, and -47)include two SNARE motifs. The four SNAREmotifs of synaptic SNARE proteins assembleinto the four-helix coiled coil structure of the

SM proteins

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Figure 3. Structures of synaptic SNARE and SM proteins. (A) Schematic diagram of the domain structures ofsyntaxin, SNAP-25, and synaptobrevin/VAMP (Habc, Habc-domain; TM, transmembrane region). (B,C) Car-toon of the two modes of interaction of the SM protein Munc18 with SNARE proteins during synaptic exocy-tosis: Binding of Munc18 to the closed conformation of syntaxin-1 that occludes the SNARE motif (B), andbinding of Munc18 to assembling SNARE trans-complexes that depends on the syntaxin-1 amino terminus(C). Note that the precise mode of Munc18 binding to assembling SNARE complexes is unknown, apartfrom the fact that it is anchored by interaction of the syntaxin amino terminus (indicated by an N) with theN-lobe of Munc18; the arrow indicates the uncertain atomic nature of this binding, which may involve wrappingof Munc18 around the SNARE helical bundle analogous to the binding of Munc18 to the closed conformation ofsyntaxin (Sudhof and Rothman 2009). (i) Atomic structures of the fully assembled SNARE complex, thesynaxin-1A Habc domain, and Munc18 containing a bound syntaxin amino-terminal peptide (blue), drawnto scale. (Data for structures are from Sutton et al. 1998, Fernandez et al. 1998, and Hu et al. 2011, respectively.)Arrow indicates uncertainty in how precisely Munc18 binds to SNARE complexes apart from the interaction ofthe syntaxin amino terminus with the Munc18 N-lobe.





SNARE complex (Fig. 3C,D) (Poirier et al.1998; Sutton et al. 1998), with one SNAREmotif each from synaptobrevin and syntaxin-1,and two SNARE motifs from SNAP-25. Amongsynaptic SNARE proteins, synaptobrevin isthe simplest as it is only composed of a shortamino-terminal sequence, a SNARE motif,and a carboxy-terminal membrane anchor(Fig. 3A). SNAP-25 is unique in containingtwo SNARE motifs but otherwise includesonly a linker sequence that serves for mem-brane-anchoring via palmitoylation. Syntaxin-1and its homologs, in contrast, are rather com-plex molecules that contain a conserved non-structured amino-terminal sequence followedby an autonomously folded amino-terminalHabc-domain (Fernandez et al. 1998), a SNAREmotif, and a transmembrane anchor (Fig. 2A).Of these sequences, the SNARE motif andtransmembrane region only account for a thirdof the entire protein, which is thus largelycomposed of “non-SNARE” sequences. As wewill see below, the interesting architecture ofsyntaxin-1 reflects a richer function in fusion,illustrating the fact that the function of SNAREproteins is not limited to assembly into SNAREcomplexes.

The four-helix bundle of a SNARE com-plex is formed primarily by hydrophobic inter-actions. All SNARE complexes, however, con-tain a conserved central hydrophilic layer ofinteracting amino acids that is composed ofthree glutamine and one arginine residue (Fas-shauer et al. 1998). Sequence analyses suggestedthat SNARE motifs can be classified into fourgroups that are referred to as Qa-, Qb-, Qc-,and R-SNAREs based on this central layer resi-due. All physiological SNARE complexes arecomposed of one of each of the four SNAREgroups, but otherwise SNARE-complex assem-bly is rather promiscuous (Fasshauer et al. 1999;Yang et al. 1999). Often, an R-SNARE is local-ized to a transport vesicle that then fuses withan acceptor membrane containing all threeQ-SNAREs, in which case the R-SNARE isalso called a v-SNARE (for “vesicular SNARE,”although not all v-SNAREs act on vesicles)and the three Q-SNAREs are referred to ast-SNAREs (for “target-membrane SNARE”).

During fusion, SNARE proteins initiallyassemble into trans-complexes in an amino- tocarboxy-terminal direction (Fig. 3). How assem-bly is nucleated remains unclear, although itappears likely that SM proteins contribute tothe assembly initiation. SNARE-complex assem-bly is a highly exergonic process. Current evi-dence suggests that SNARE-complex formationpromotes membrane fusion by simple mechani-cal force. The energy released during SNARE-complex assembly likely fuels membrane fusion,i.e., provides the power that overcomes the energybarriers to membrane fusion—in that sense,SNARE proteins are truly the fusion motors.

Although much is understood about mem-brane fusion at the synapse and elsewhere, manymajor issues remain unresolved. For example,how does the specificity of SNARE-complexassembly arise, given the fact that such assemblyis promiscuous in vitro, and that many organ-elles, including synaptic vesicles, contain multi-ple SNARE proteins that participate in selectivebut distinct fusion reactions of the same organ-elle (e.g., exocytosis vs. endosome fusion ofsynaptic vesicles)? Why is there a topologicalrestriction in fusion in that the R-SNAREsynaptobrevin on synaptic vesicles acts in con-junction with plasma membrane Q-SNAREsduring synaptic exocytosis, even though theQ-SNARES are actually present on synapticvesicles as well (Otto et al. 1997)?

SM PROTEINS

SM proteins are essential partners for SNAREproteins in fusion—without one or the other,no fusion occurs physiologically. SM proteinsare evolutionarily conserved cytosolic proteinsof approximately 600 residues that are com-posed of three lobes folding into an arch-shaped“clasp” structure (Fig. 3D). Despite the fact thatan essential role for SM proteins in fusion wassuggested in initial studies of Munc18-1 in syn-aptic exocytosis immediately following the dis-covery of the function of SNARE proteins (Hataet al. 1993), the importance of SM proteins wasquestioned for more than a decade.

Two circumstances contributed to the un-certainty of SM protein function. First, SNARE





proteins can induce fusion of liposomes with-out SM proteins (Weber et al. 1998; van denBogaart et al. 2010), and second, the biochem-istry of the SNARE/SM protein interactionwas initially difficult to fit into a simple model(Dulubova et al. 1999). The first circumstancewas more intuitive than rational, becausemany proteins including bovine-serum albu-min are known to promote liposome fusion;hence, in liposome fusion studies it is criticalto show that fusion shows similar requirementsto those observed physiologically before func-tional conclusions can be drawn.

The second circumstance, however, posed agenuine conceptual challenge. Initial studiessuggested that SM proteins interact withSNARE proteins not by a uniform mechanism,but in diverse and occasionally incompatibleways. Specifically, Munc18 was shown to bindto the closed conformation of syntaxin-1(Fig. 3B) (Dulubova et al. 1999). This bindingappeared to prevent syntaxin-1 from engaginginto SNARE complexes, and led to the sugges-tion that Munc18 is an inhibitor of fusioninstead of a component of the fusion machine(Wu et al. 2001), despite the fact that deletionof Munc18 caused a loss of fusion instead ofincreased fusion (Verhage et al. 2000). More-over, the Munc18 homolog in yeast, Sec1p,was shown to bind only to assembled SNAREcomplexes (Carr et al. 1999), creating an addi-tional interpretational dilemma.

This puzzle was only resolved, at least asregards mammalian fusion, when it was shownthat a conserved sequence at the amino termi-nus of syntaxins binds SM proteins by a novelmechanism that is compatible with full SNARE-complex assembly (Fig. 3C). Initially describedfor SNARE and SM proteins involved in endo-plasmic reticulum, Golgi, and endosome traffic(Dulubova et al. 2002; Yamaguchi et al. 2002),this mechanism was extended to the corre-sponding proteins involved in exocytosis(Dulubova et al. 2007; Khvotchev et al. 2007;Shen et al. 2007). As a result, a unified view ofSM/SNARE protein interactions emerged thatappears to apply to most intracellular fusionreactions, whereby SM proteins are attached toassembling SNARE complexes via short peptide

sequences, usually localized to syntaxin, whichallows the SM proteins to hang onto the assem-bling SNARE complexes independent of theconformation of the SNARE motif (Fig. 2).The amino-terminal peptide on syntaxin-1that tethers the SM protein Munc18 is abso-lutely essential for fusion in vivo, whereas theclosed syntaxin conformation may be dispens-able, at least for fusion as such (Khvotchev et al.2007; Gerber et al. 2008; Rathore et al. 2010).

Despite these more recent results, the initialfindings of Munc18-binding to the closed con-formation of syntaxin did not turn out to beirrelevant. Knock-in mice in which syntaxin-1carries a point mutation that renders it predom-inantly open, thereby decreasing Munc18-bind-ing to the closed monomeric conformation ofsyntaxin-1 without altering Munc18-bindingto syntaxin-1 containing SNARE complexes,show a dramatic synaptic transmission pheno-type (Gerber et al. 2008). This phenotypeincluded an increased release probabilitycombined with a decreased number of synapticvesicles available for release, suggesting that theMunc18 interaction with the Habc domain ofsyntaxin-1 in its closed conformation mayregulate release, as opposed to the Munc18interaction with the amino-terminal syntaxin-1peptide that is essential for fusion as such. Thus,at present, the role of the syntaxin Habc domainand its binding of Munc18 are only incom-pletely understood.

COOPERATION OF SNARE AND SMPROTEINS IN SYNAPTIC EXOCYTOSIS

Why do SNARE proteins require SM proteinsfor fusion? Although no definitive answer tothis question is available, the structure of SMproteins suggests that they may wrap aroundassembling SNARE complexes (Fig. 3D). Thisinteraction may spatially organize SNARE com-plexes, keep them from sliding into the spacebetween the fusing membranes and therebyblock fusion, and promote SNARE complexassembly (Dulubova et al. 2007). Moreover,SM proteins may directly catalyze phospholipidmixing during fusion by interacting with thephospholipid membranes close to SNARE





complexes, perhaps altering the membranecurvature (Carr and Rizo 2010). Some otherhypotheses, however, appear to be ratherunlikely. SM proteins probably do not conferspecificity to fusion reactions because thesame SM protein is often used in differentfusion reactions, as are less frequently thesame SNARE proteins. Specificity likely residesprimarily in adaptor proteins, including Rabproteins and their effectors, which act onSNARE and SM proteins. Similarly, althoughSM proteins continue to be referred to as regu-lators, their primary function does not appearto be regulatory but executive because deletionsof SM proteins block fusion, and because SMproteins are not widely regulated by classicalsignaling mechanisms, such as calcium, phos-phorylation, or other second messengers. Test-ing these and additional hypotheses will bedifficult until a better understanding of thebiophysical chemistry of membrane fusion isavailable.

Conceptually, we differentiate in synapticexocytosis between synaptic vesicle dockingand fusion, with the latter probably startingduring the priming of vesicles, and finishingduring the Ca2þ-triggering of fusion-poreopening by synaptotagmin (Figs. 1 and 2).Although SNARE and SM proteins are notessential for synaptic vesicle docking, definedas the attachment of vesicles to the active zoneat the synapse, binding of Munc18 to the closedconformation of syntaxin-1 is essential forvesicle docking in chromaffin cell exocytosis(Gerber et al. 2008). Thus, SNARE and SM pro-tein complexes may stabilize the attachment ofvesicles to the target membrane and therebyparticipate in docking, consistent with thenotion that SNARE and SM protein complexesassemble at least partly prior to exocytosis dur-ing vesicle priming.

CSPa AND SYNUCLEINS—SNARECHAPERONES PREVENTINGNEURODEGENERATION

At a synapse, every release event is associatedwith assembly of SNARE complexes and theirdisassembly by NSF and SNAPs. At any given

time, a presynaptic terminal likely containshundreds of unfolded or partly folded SNAREprotein molecules, carrying exposed reactiveSNARE motifs that are prone to nonspecificinteractions and/or misfolding. It is thus notsurprising that neurons in particular, and allcells in general, developed at least two chaper-one systems to maintain functional SNAREconformations and promote SNARE-complexassembly (Fig. 4). Both of these chaperones—a complex composed of CSPa (for “cysteinestring protein a”), Hsc70, and SGT (for“glutamate- and threonine-rich protein”; Toba-ben et al. 2001) and the synucleins (Burreet al. 2010)—are linked to neurodegeneration,suggesting that abnormal exposure of neuronsto misfolded SNAREs and/or abnormalSNARE-complex assembly may impair neuro-nal survival.

CSPa is an evolutionarily conserved synap-tic vesicle protein containing a DNA-J domaintypical for cochaperones. When complexedwith the DNA-K-domain protein Hsc70 andthe tetrotricopeptide repeat protein SGT(Fig. 4), CSPa mediates the ATP-dependentrefolding of denatured luciferase as a modelsubstrate (Tobaben et al. 2001) and prevents inan ATP-dependent manner the aggregation ofSNAP-25 (Sharma et al. 2011). Deletion ofCSPa in mice has no immediate effects on neu-rotransmitter release, but leads to increasedubiquitination and degradation of SNAP-25and to decreased SNARE-complex assembly,resulting in fulminant neurodegeneration thatkills the mice after 2–3 mo (Fernandez-Chaconet al. 2004; Sharma et al. 2011).

Synucleins comprise a family of three pro-teins (a-, b-, and g-synuclein) that attractedattention because a-synuclein mutations oroverexpression lead to Parkinson’s disease inhuman patients, and because many neurodege-nerative disorders feature inclusions calledLewy bodies that contain a-synuclein (Galvinet al. 2001). Synucleins are small proteins thatbind to negatively charged phospholipids asa-helices. Different from CSPa, synucleins areonly observed in vertebrates. A role for synu-cleins in SNARE-complex assembly emergedfrom the surprising finding that moderate





overexpression of a-synuclein completely res-cues the lethal neurodegeneration producedby the knockout of CSPa (Chandra et al.2005). Biochemically, a-synuclein rescued theSNARE-complex assembly deficit in CSPaknockout mice, but not their loss of SNAP-25.Subsequent studies uncovered a direct enhance-ment of SNARE-complex assembly by a-synuclein in a purified reconstituted systemcontaining only recombinant SNARE poteinsand a-synuclein (Burre et al. 2010). Moreover,deletion of all synuclein isoforms, althoughnot lethal, leads to destabilization of SNAREcomplexes in vivo, and to an age-dependentneurological phenotype that increasingly inca-pacitates mice after 1 year of age (Burre et al.2010).

Because a-synuclein normally functions topromote SNARE-complex assembly, how does

overexpression of a-synuclein in patientscarrying duplication or triplication of the a-synuclein gene cause neurodegeneration (Erik-sen et al. 2005)? At present, two alternativehypotheses are plausible. First, an increase inSNARE chaperoning may be detrimental, pos-sibly by upsetting the balance of fusion innerve terminals; second, misfolding of a-synu-clein—which is a reactive protein due to itsfunction—may produce toxic conformers thatcause neuronal damage. The second hypothesisappears more likely because only a-synucleinbut not b- or g-synuclein mutations havebeen identified in Parkinson’s disease. If thenormal function of a-synuclein was involvedin its pathogenic action, one would expect theother synucleins, which likely have similar func-tions, to be also pathogenic. However, until themechanism of synuclein toxicity is uncovered,

Vesicle recycling

Synuclein

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Figure 4. Two types of chaperones support SNARE protein function. Synaptic SNARE proteins are subject tocontinuous folding and unfolding reactions, creating the potential for misfolding that is counteracted by at leasttwo SNARE chaperones implicated in neurodegeneration: the classical chaperone complex containing CSPa(for cysteine-string protein a), Hsc70, and SGT (red shapes), and the nonclassical chaperones a/b/g-synucleins (yellow shapes). CSPa and synucleins are vesicle proteins that act by distinct mechanisms.CSPa forms a complex with Hsc70 and SGT on the vesicle that binds to SNAP-25 on the target membraneand supports the functional competence of SNAP-25 to engage in SNARE complexes, whereas synucleins arebound to phospholipids and synaptobrevin (Syb)/VAMP on the vesicles, and bind to assembling SNARE com-plexes to support their folding.





both hypotheses, and probably others as well,are conceivable.

EMBEDDING THE SNARE/SM PROTEINFUSION MACHINERY IN THE ACTIVE ZONE

During synaptic exocytosis, synaptic vesiclesdock and fuse at the presynaptic active zone,an electron-dense proteinaceous structure.Active zones contain at least four key proteincomponents (Fig. 5): Munc13’s (no relationto Munc18’s; Brose et al. 1995), RIMs (forRab3-interacting molecules; Wang et al. 1997),RIM-BPs (for RIM-binding proteins; Wanget al. 2000), and a-liprins (Zhen and Jin1999). In addition, piccolo/bassoon (a familyof large cytomatrix proteins; piccolo is alsocalled aczonin; tom Dieck et al. 1998; Wang

et al. 1999; Fenster et al. 2000) and ELKS (alsoknown as Rab6-interacting protein, CAST, orERC; Ohtsuka et al. 2002; Wang et al. 2002)are genuine active zone proteins. Finally, adap-tor proteins such as CASK, Velis, and Mints aswell as endocytic scaffolding proteins such asintersectin, syndapin, amphiphysin, and othersare probably peripherally associated with theactive zone. It is likely that additional obligatoryactive zone proteins exist that have not yet beenidentified; for example, a scaffolding proteincalled Syd-1 is an essential presynaptic proteinin C. elegans (Hallam et al. 2002), but no truevertebrate homolog has been described. Thesestandard active zone proteins are present in alltypes of synapses, not only classical excitatoryand inhibitory central synapses but also in neu-romuscular junctions and ribbon synapses. The

Munc13inactive

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Munc13active

X

Figure 5. Model of active zone protein function. A complex composed of four out of the six canonical compo-nents of active zones (Munc13, a-liprins, RIMs, and RIM-BPs) is shown; the remaining two canonical activezone proteins (ELKS and Piccolo/Bassoon) bind peripherally to the complex, and are not shown. The activezone complex is formed by an interaction of the amino-terminal zinc-finger domain of RIM with the amino-terminal C2A-domain of Munc13; by an interaction of a proline-rich sequence in RIM with a RIM-BPSH3-domain, and by a binding of a-liprins to the RIM C2B-domain. The complex is anchored on synapticvesicles via RIM binding to Rab3 and possibly also via RIM binding synaptotagmin (not shown), and on theplasma membrane via direct binding of RIM and of RIM-BP to N- and P/Q-type Ca2þ-channels. Note thatthere are likely additional interactions among the various active zone proteins, and between these proteinsand the presynaptic plasma membrane. Ca2þ-ions are shown as dots; most of the active zone proteins containC2-domains similar to synaptotagmin, but only a subset of the C2-domains bind Ca2þ.





latter, however, contain an additional specialprotein component called Ribeye that is evolu-tionarily derived from the fusion of thetranscription factor CtBP2 with a novel amino-terminal Ribeye-specific domain (Schmitz et al.2000).

Active zones mediate at least three broad,overlapping functions of presynaptic terminals:(1) They physically link synaptic vesicles, cal-cium channels, and other presynaptic compo-nents into a single complex, thereby ensuringefficient colocalization of the elements thatonly enables fast synaptic transmission; (2)They organize presynaptic receptors to allowpresynaptic modulation of neurotransmitterrelease, for example by endocannabinoids orby autoreceptors; and (3) They mediate variousforms of short- and long-term presynaptic plas-ticity. All of these three functions are crucial forsynaptic transmission.

Munc13 and RIM proteins, currently thebest understood active zone proteins, are multi-domain proteins that are expressed in severalisoforms transcribed from multiple promotersin multiple genes. The predominant Munc13isoforms contain an amino-terminal C2A-domain that does not bind Ca2þ, but forms aconstitutive homodimer (Fig. 5). Munc13 alsocontains a central regulatory cassette composedof a calmodulin-binding sequence (Junge et al.2004), a C1-domain that is regulated by dia-cylglycerol (Rhee et al. 2002), and a centralC2B-domain that binds Ca2þ in a PIP2-dependent manner (Shin et al. 2010). Thecarboxy-terminal half of Munc13 proteins iscomposed of its executive “MUN” domainand a carboxy-terminal C2C-domain that alsodoes not bind Ca2þ. Functionally, Munc13 isessential for synaptic vesicle priming (Augustinet al. 1999); its function is tightly regulated byCa2þ (via the C2B-domain, diacylglycerol syn-thesis, and calmodulin) and RIM (see below).In priming, Munc13 probably acts via a director indirect interaction of the MUN-domainwith SNARE and/or SM proteins (Ma et al.2011).

The domain structure of RIM proteins iseven more complex than that of Munc13’s(Wang et al. 1997, 2000). a-RIM proteins, the

predominant isoforms, contain an amino-ter-minal sequence that includes two nested do-mains: two a-helices that interact with thevesicle GTP-binding protein Rab3 (hence thename “RIM”), and that are separated by a zinc-finger that binds to the Munc13 C2A-domain(Fig. 5) (Lu et al. 2006). Interestingly, RIMzinc-finger binding to the Munc13 C2A-domain competes with the homodimerizationof the C2A-domain; because the former showsa higher affinity than the latter, the presence ofthe RIM zinc-finger converts the Munc13 C2A-domain homodimer into a RIM/Munc13 heter-odimer (Lu et al. 2006). RIMs contain a centralPDZ-domain that binds to ELKS proteins andto N- and P/Q-type Ca2þ-channels (Wanget al. 2000; Ohtsuka et al. 2002; Kaeser et al.2011), and two carboxy-terminal C2-domainsthat do not bind Ca2þ. The sequence betweenthe two C2-domains includes a short proline-rich sequence that binds to RIM-BPs (RIM-binding proteins; Wang et al. 2000), whereasthe second RIM C2-domain (the C2B-domain)binds to the active zone protein a-liprin (Schochet al. 2002). RIM proteins are thus central ele-ments of active zones that interact with mostother active zone proteins.

Functionally, RIM proteins perform at leasttwo essential functions. First, RIM proteins reg-ulate the priming activity of Munc13 (Denget al. 2011). Deletions of RIM proteins producea severe priming defect (Koushika et al. 2001;Schoch et al. 2002); strikingly, this defect canbe partly reversed by expression of the RIMzinc-finger alone (Deng et al. 2011). The pri-ming defect of RIM-deficient synapses is alsocompensated by expression of mutant Munc13(but not a wild-type Munc13) either lackingthe homodimerizing C2A-domain, or con-taining a point mutation in the C2A-domainthat blocks homodimerization (Deng et al.2011). These data indicate that Munc13 homo-dimerization inhibits the priming functionof Munc13, and that RIM activates Munc13by converting the homodimer into a RIM/Munc13 heterodimer.

Second, RIM proteins tether Ca2þ-channelsto the active zone. It was known for many yearsthat Ca2þ-channels are enriched at the site of





neurotransmitter release, thereby allowing tightcoupling of Ca2þ-influx to triggering of fusion(Simon and Llinas 1985). However, only veryrecently the molecular mechanism recruitingCa2þ-channels to active zones was shown todepend on RIM proteins (Kaeser et al. 2011).Specifically, RIM proteins bind directly to thecarboxyl terminus of N- and P/Q-type Ca2þ-channels via their PDZ-domain, and indirectlyto the cytoplasmic tails of the same Ca2þ-channels via RIM-BP (Fig. 5). Deletion ofRIM proteins from synapses leads to a selectiveloss of Ca2þ-channels from presynaptic special-izations, and a decrease in action-potentialinduced Ca2þ-influx; this phenotype can onlybe rescued by RIM fragments containing boththe PDZ-domain and the RIM-BP bindingsequence (Kaeser et al. 2011). Thus, RIM pro-teins tether Ca2þ-channels to active zones, con-necting the channels to synaptic vesicles and toother active zone proteins via their variousbinding activities.

CONCLUDING REMARKS

As we described in this review, synaptic exocyto-sis is mediated by a hierarchically organizedmachinery that contains SNARE and SM pro-teins at its core, is maintained by NSF andSNAPs and by specific SNARE chaperones dedi-cated to its continuous operation, and is regu-lated by active zone proteins. In addition, theSNARE and SM fusion machine is controlledby Ca2þ via synaptotagmin and complexin.

ACKNOWLEDGMENTS

We are indebted to the NIH and the HowardHughes Medical Institute for continuous sup-port (grants supporting the work describedhere: MH086403, NS053862, and MH089054to T.C.S., and NS37200 and NS40944 to J.R.).

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October 25, 20112011; doi: 10.1101/cshperspect.a005637 originally published onlineCold Spring Harb Perspect Biol

Thomas C. Südhof and Josep Rizo Synaptic Vesicle Exocytosis

Subject Collection The Synapse

SpinesStudying Signal Transduction in Single Dendritic

Ryohei Yasuda

Synaptic Vesicle EndocytosisYasunori Saheki and Pietro De Camilli

Synaptic Vesicle Pools and DynamicsAbdulRasheed A. Alabi and Richard W. Tsien

Short-Term Presynaptic PlasticityWade G. Regehr

Synapses and Memory Storage

KandelMark Mayford, Steven A. Siegelbaum and Eric R.

(LTP/LTD)Potentiation and Long-Term Depression NMDA Receptor-Dependent Long-Term

Christian Lüscher and Robert C. MalenkaSynapses and Alzheimer's Disease

C. SüdhofMorgan Sheng, Bernardo L. Sabatini and Thomas Brain

Ultrastructure of Synapses in the Mammalian

Kristen M. Harris and Richard J. WeinbergSynaptic Cell Adhesion

BiedererMarkus Missler, Thomas C. Südhof and Thomas

Calcium Signaling in Dendritic SpinesMichael J. Higley and Bernardo L. Sabatini

DisabilitiesIntellectualDisorders Associated with Autism and

Synaptic Dysfunction in Neurodevelopmental

Huda Y. Zoghbi and Mark F. Bear

Synaptic Neurotransmitter-Gated ReceptorsTrevor G. Smart and Pierre Paoletti

The Postsynaptic Organization of SynapsesMorgan Sheng and Eunjoon Kim

Synaptic Vesicle ExocytosisThomas C. Südhof and Josep Rizo

Inhibitory SynapsesPresynaptic LTP and LTD of Excitatory and

Pablo E. CastilloNeurotransmittersVesicular and Plasma Membrane Transporters for

Randy D. Blakely and Robert H. Edwards

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