the synaptic vesicle proteome.pdf

REVIEW The synaptic vesicle proteome

Jacqueline Burre and Walter Volknandt

Institute of Cell Biology and Neuroscience, Neurochemistry, JW Goethe University, Frankfurt, Germany

Abstract

Synaptic vesicles are key organelles in neurotransmission.

Vesicle integral or membrane-associated proteins mediate

the various functions the organelle fulfills during its life cycle.

These include organelle transport, interaction with the nerve

terminal cytoskeleton, uptake and storage of low molecular

weight constituents, and the regulated interaction with the

pre-synaptic plasma membrane during exo- and endocytosis.

Within the past two decades, converging work from several

laboratories resulted in the molecular and functional char-

acterization of the proteinaceous inventory of the synaptic

vesicle compartment. However, up until recently and due to

technical difficulties, it was impossible to screen the entire

organelle thoroughly. Recent advances in membrane protein

identification and mass spectrometry (MS) have dramatically

promoted this field. A comparison of different techniques for

elucidating the proteinaceous composition of synaptic vesi-

cles revealed numerous overlaps but also remarkable dif-

ferences in the protein constituents of the synaptic vesicle

compartment, indicating that several protein separation

techniques in combination with differing MS approaches are

required to identify and characterize the synaptic vesicle

proteome. This review highlights the power of various gel

separation techniques and MS analyses for the characteri-

zation of the proteome of highly purified synaptic vesicles.

Furthermore, the newly detected protein assignments to

synaptic vesicles, especially those proteins which are new to

the inventory of the synaptic vesicle proteome, are critically

discussed.

Keywords: electrophoresis, mass spectrometry, proteome,

proteomics, synaptic vesicle, synaptic vesicle protein.

J. Neurochem. (2007) 101, 14481462.

The synaptic vesicle compartment

Neurotransmission is based primarily on the regulatedrelease of neurotransmitters from synaptic vesicles. Synapticvesicles govern essential pre-synaptic tasks such as theuptake, storage and Ca2+-regulated release of neurotrans-mitters. Upon arrival of an action potential, synapticvesicles docked and primed at the active zone of the pre-synaptic plasma membrane fuse with the membrane andrelease neurotransmitters into the synaptic cleft. Neuro-transmitters diffuse to the post-synaptic membrane toactivate neurotransmitter receptors. Synaptic vesicles areretrieved by endocytosis to restore the primed vesicle pool(Littleton 2006; Ryan 2006).These essential tasks are controlled by a unique inventory

of integral membrane proteins and also of membrane-associated proteins that attach and detach during the vesiclecycle in a time-dependent manner (reviewed in Li and Chen2003; Sudhof 2004). The exploration of the proteinaceouscomposition of the synaptic vesicle is essential for anunderstanding of the molecular mechanisms of neurotrans-mitter release.

Proteomic strategies

One major advantage of proteomic approaches for the studyof tissue, cell and organelle proteomes is the sequenceinformation immediately provided by mass spectrometry(MS). Yet, many studies of the past few years indicate thatthe proteomic analysis of whole cells or tissues is often too

Received October 16, 2006; revised manuscript received December 1,

2006; accepted December 22, 2006.

Address correspondence and reprint requests to Jacqueline Burre,

Institute of Cell Biology and Neuroscience, Neurochemistry, JW Goethe

University, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany.

E-mail: [email protected]

Abbreviations used: 1D, one-dimensional; 2D, two-dimensional; BAC,

benzyldimethyl-n-hexadecyl ammonium chloride; CCV, clathrin-coated

vesicle; ER, endoplasmic reticulum; GAPDH, glyceraldehyde-3-phos-

phate dehydrogenase; LC, liquid chromatography; MAP, mitogen-acti-

vated protein; MS, mass spectrometry; NECAP, adaptin ear-binding

coat-associated protein; PAGE, polyacrylamide gel electrophoresis;

SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein

receptor; TGN, trans-Golgi network; VAMP, vesicle-associated mem-

brane protein.

Journal of Neurochemistry, 2007, 101, 14481462 doi:10.1111/j.1471-4159.2007.04453.x

1448 Journal Compilation 2007 International Society for Neurochemistry, J. Neurochem. (2007) 101, 14481462 2007 The Authors

complex for the available technology to obtain a highlyresoluted protein separation and is thus unsuitable for thestudy of proteins that are present in low copy numbers(Stasyk and Huber 2004). Proteins in low abundance areinevitably masked by highly abundant proteins (Ahmed andRice 2005; Righetti et al. 2006). Therefore, an appropriateproteomic strategy to address these limitations is to enrichparticular subcellular structures by subcellular fractionation.One advantage of subcellular proteomic approaches is thatthe identied proteins can be directly assigned to denedorganelles. Especially for novel proteins, this can providevaluable information for further investigation into theirfunctional role. For the evaluation of novel proteins andproteins newly assigned to a subcellular structure, correlationproling has been proposed (Andersen et al. 2003). Thismethod assumes that constituents of the same organellereveal the same degree of enrichment during the sequentialpurication steps. In addition, for morphological analysis ofall fractions obtained during organelle isolation, electronmicroscopy can be employed. Finally, the combination ofMS, bioinformatics, and high-resolution two-dimensional(2D) gel electrophoresis provides the basis for the large-scaleidentication of proteins in complex mixtures (Dhingra et al.2005; Domon and Aebersold 2006).Hydrophobic membrane proteins are a challenge for

proteomic approaches. Their amino acid sequences containonly a few tryptic cleavage sites, impairing their identica-tion using peptide mass ngerprinting (van Montfort et al.2002a; Tannu and Hemby 2006). In addition, these largetryptic peptides extract poorly from the gel matrix and escapedetection by MS. The combination of different proteases andchemical cleavage methods or shotgun approaches (vanMontfort et al. 2002a,b; Zhang et al. 2004) can only partiallyovercome these limitations.In proteomic studies using conventional 2D electrophoresis

(OFarrell 1975), membrane proteins are notoriously under-represented (Rabilloud et al. 1999; Santoni et al. 2000). Thedevelopment of techniques especially designed for theseparation of hydrophobic proteins is essential. On a globalproteomic level, these efforts comprise renements of theconventional 2D polyacrylamide gel electrophoresis (PAGE)(Kashino 2003; Luche et al. 2003) and partial or fullreplacement of gel electrophoretic separation steps (Simpsonet al. 2000; Schirle et al. 2003). Moreover, non-gel shotgunmethods (Gevaert et al. 2003; Wu and Yates 2003; Zhanget al. 2004) or in-liquid pre-fractionation techniques like freeow electrophoresis (Righetti et al. 2005) have been applied.Hartinger et al. (1996) optimized benzyldimethyl-n-hexa-decyl ammonium chloride (BAC)/sodium dodecyl sulphate(SDS)PAGE (Macfarlane 1986) for the separation ofsynaptic vesicle proteins. Other gel electrophoretic tech-niques especially designed for the separation of membraneproteins including double SDSPAGE technique (Rais et al.2004), blue native/SDSPAGE (Brookes et al. 2002) and

cetyl trimethyl ammonium bromide (CTAB)/SDSPAGE(Navarre et al. 2002) were successfully applied for theresolution of mitochondrial proteins and protein complexesand yeast plasma membrane proteins respectively.

Neuroproteomics

The Human Proteome Organization (HUPO) brain proteomeproject was launched to analyse human and mouse brainsamples (Hamacher et al. 2004). It became obvious that evenupon application of identical data processing guidelines, eachgroup identied, for the most part, different proteins (seeHamacher et al. 2006; Reidegeld et al. 2006 and the entireProteomics issue, Vol. 6, No. 18, Sept 2006). For example, of792 mouse brain proteins identied by seven groups, nearly80%were found in one particular study only. Differences arosefrom sample preparation protocols, separation techniques,mass spectrometers, bioinformatic tools and databases used,revealing that strict methods of quality control and assurancehave to be applied to increase the level of condence.Subjecting crude samples to proteomic analysis such as

whole-brain tissue from human (Dumont et al. 2006; Parket al. 2006), mouse (Gauss et al. 1999; Wang et al. 2006) orrat (Fountoulakis et al. 1999; Krapfenbauer et al. 2003)resulted in a very limited number of identied proteins for aspecic organelle such as synaptic vesicles. Similarly, work-ing with brain parts such as mouse cerebellum (Beranova-Giorgianni et al. 2002), human parietal cortex (Langen et al.1999) or human hippocampus (Edgar et al. 1999) lead to theidentication of only a few synaptic vesicle proteins. Analysisof subproteomes include the synaptic plasma membranefraction of rat forebrain that resulted in the identication of six(Prokai et al. 2005) and 17 (Stevens et al. 2003) synapticvesicle proteins, respectively, whereas the proteomic analysisof synaptic membranes and post-synaptic density isolatedfrom whole rat brain upon isotope-coded afnity tagging(ICAT) labelling (Li et al. 2005) lead to the identication ofsix synaptic vesicle proteins. A proteomic analysis ofsynaptosomes derived from rat cerebral cortex (Witzmannet al. 2005) identied 246 proteins, comprising 12 proteinsbeing part of the synaptic vesicle compartment. Similarly, in aproteomic analysis using synaptosomes from mouse brain(Schrimpf et al. 2005), peptides representing a total of 1131database entries were identied, including 15 proteinsinvolved in the exocytosis, and six proteins in the endocytosis,of synaptic vesicles. This strongly suggests that synapticvesicles should be puried before proteomic analysis.

Synaptic vesicle proteomics

Recent studies succeeded in the elucidation of the proteina-ceous inventory of several organelles (reviewed in Yateset al. 2005). In particular, proteomic analyses of brain tissuebecame an integral component of neuroscientic research

The synaptic vesicle proteome 1449

2007 The AuthorsJournal Compilation 2007 International Society for Neurochemistry, J. Neurochem. (2007) 101, 14481462

(reviewed in Abul-Husn and Devi 2006; Becker et al. 2006);however, comprehensive studies on the synaptic vesicleproteome are rare (Blondeau et al. 2004; Coughenour et al.2004; Morciano et al. 2005; Burre et al. 2006b; Takamoriet al. 2006) as synaptic vesicles have been difcult to purifyto homogeneity in large quantities and contain numeroushydrophobic proteins.

Isolation of synaptic vesicles

For the proteomic analysis of synaptic vesicles, differentisolation protocols have been applied. Importantly, synapticvesicles and clathrin-coated vesicles (CCV) were isolatedfrom puried synaptosomes by hypo-osmotic shock. Thisexcludes vesicular organelles of similar biophysical orbiochemical properties from non-terminal compartments ofthe neuron.Hartinger et al. (1996) further puried synaptic vesicles by

subcellular fractionation, according to Huttner et al. (1983),and replaced the controlled-pore glass bead column as thenal purication step with high-velocity glycerol gradientcentrifugation. Similarly, velocity gradient centrifugation wasthe nal step for the synaptic vesicle isolation protocoldescribed by Coughenour et al. (2004). Electron microscopyand immunoblot analyses of marker enzymes for mitochon-dria, plasma membrane, lysosomes, endoplasmic reticulum(ER) and cytosol were applied to screen for potentialcontamination. Low levels of ER were still detected in thepuried vesicle sample whereas no contamination of otherorganelles was observed. Contaminations with fragmentsderived from the ER were shown to be present also in otherstudies using controlled-pore glass beads (Hell et al. 1988).In addition, voltage-dependent anion channel 1, a knownmitochondrial outer membrane protein (Rostovtseva andColombini 1996; Hodge and Colombini 1997) was identiedin isolated vesicle fractions (Coughenour et al. 2004). Yet,voltage-dependent anion channel-like transport activity wasascribed to the plasma membrane of synaptosomes (Sharet al. 1998) and it is also present in phagosomes (Garin et al.2001) and Golgi-derived vesicles (Bell et al. 2001).Another protocol for the isolation of synaptic vesicles from

rat brain is based on continuous sucrose density gradientcentrifugation and subsequent immunoprecipitation (Morci-ano et al. 2005; Burre et al. 2006b). The purity of the isolatedorganelles was evaluated by electron microscopy, markerprotein analysis (Morciano et al. 2005) and correlationproling (Burre et al. 2006b). No immunosignal was detectedfor marker proteins of organelles such as mitochondria,lysosomes, peroxisomes, nuclei, Golgi stacks, ER or constit-uents of the pre- and post-synaptic membrane. However, MSanalysis led to the identication of proteins assigned tomitochondria, ER and the pre-synaptic membrane.According to a recent proteomic study of Takamori et al.

(2006) synaptic vesicles were isolated according to Huttner

et al. (1983) with controlled-pore glass bead chromatographyas the nal purication step. In addition, they subjected theorganelles to carbonate treatment to remove peripherallyassociated proteins. Electron microscopy and correlationproling were performed for evaluation of organelle purity.However, MS analysis resulted in the identication of severalplasma membrane proteins.In another study, CCVs were isolated according to Maycox

et al. (1992) with sucrose density gradient centrifugation asthe nal purication step (Blondeau et al. 2004). Correlationproling and electron microscopy for the evaluation oforganelle purity revealed a contribution of 73% of CCVs inthe preparation used for proteomic analysis. The residualuncoated structures were heterogeneous in shape and size andvaried from 13 to >200 nm in diameter. Analysis of CCVsrevealed a number of pre-synaptic plasma membrane-locatedproteins. In this context it is important to note that the pre-synaptic plasma membrane protein Na+/K+-ATPase isenriched on CCVs when compared with total brain homo-genate, suggesting that pre-synaptic plasma membrane pro-teins may be incorporated into CCVs (Blondeau et al. 2004).

Identification of synaptic vesicle proteins

The highest number of synaptic vesicles proteins wasidentied using one-dimensional (1D) SDSPAGE forprotein separation (Blondeau et al. 2004; Burre et al.2006b; Takamori et al. 2006). The limiting separating abilityof 1D gels was overcome using nanoscale liquid chroma-tography (LC) to resolve the extracted peptides. A proteomicanalysis of CCVs (Blondeau et al. 2004) succeeded in theidentication of 209 proteins with up to 17 proteins in onegel slice. Synaptic vesicle proteins identied includedsynaptic vesicle protein 2 (Buckley and Kelly 1985; Bajjaliehet al. 1994), synaptophysin (Calakos and Scheller 1994),synaptoporin (Brandstatter et al. 1996), synaptogyrins 1 and3 (Baumert et al. 1990; Sugita et al. 1999), synaptotagmins Iand II (Ullrich et al. 1994), secretory carrier-associatedmembrane proteins 1, 3 and 5 (Castle and Castle 2005), thesoluble N-ethylmaleimide-sensitive factor attachment proteinreceptors (SNAREs) vesicle-associated membrane protein(VAMP)-2 (Rossetto et al. 1996), soluble NSF attachmentprotein (SNAP)-25 (Hodel 1998) and syntaxin-1 (Lin andScheller 1997), the VAMP-interacting protein (Skehel et al.1995), subunits of the vATPase (Nelson and Harvey 1999), avesicular glutamate transporter (Bellocchio et al. 2000), thevesicular GABA transporter (Chaudhry et al. 1998), cysteinestring protein (Chamberlain and Burgoyne 1998), heterotri-meric G proteins (Holtje et al. 2000; Pahner et al. 2003) andrab3 (Zerial and McBride 2001) (Table 1). All these proteinswere also identied in a proteomic approach applying thesame techniques but using immunopuried synaptic vesicles(Burre et al. 2006b) and synaptic vesicle puried bycontrolled-pore glass bead chromatography (Takamori et al.

1450 J. Burre and W. Volknandt

Journal Compilation 2007 International Society for Neurochemistry, J. Neurochem. (2007) 101, 14481462 2007 The Authors

2006). However, some proteins such as synaptoporin, thevesicular amine transporter (Linial et al. 1989), the vesicularzinc transporter ZnT-3 (Cole et al. 1999), synaptotagmin V(Craxton and Goedert 1999), and two subunits of thevATPase were only identied by two 1D approaches. Inaddition, tomosyn (McEwen et al. 2006) and vesicletransport through interaction with t-SNAREs homolog 1A(vti1A) (Antonin et al. 2000) was identied solely by oneanalysis.

Two-dimensional gel-electrophoretic techniques are cap-able of resolving proteins that migrate at the same rate withina 1D gel. A direct comparison of BAC/SDSPAGE withisoelectric focussing/SDSPAGE for the separation of syn-aptic vesicle proteins was reported by Coughenour et al.(2004). Separated proteins were subjected to tryptic in-geldigestion, capillary LC and electrospray ionization quadru-pole time of ight MS. This led to the identication of themajor protein components of synaptic vesicles (compare

Table 1 Established synaptic vesicle proteins

Proteins Function Membrane anchorage Proteomics approach

SV2s Transporter-like, release 12 TMH 15

SVOP Transporter-like 12 TMH

Synaptophysin Exocytosis 4 TMH 15

Synaptoporin Exocytosis 4 TMH 2, 5

Synaptogyrin 1 Exocytosis 4 TMH 2, 4, 5

Synaptogyrin 3 Exocytosis 4 TMH 2, 4, 5

Synaptotagmin I Exocytosis/endocytosis 1 TMH 15

Synaptotagmin II Exocytosis 1 TMH 25

Synaptotagmin V Exocytosis 1 TMH 4, 5

SCAMP 1 Endocytosis 4 TMH 2, 4, 5

SCAMP 3 Endocytosis 4 TMH 2, 4, 5

SCAMP 5 ? 4 TMH 2, 4, 5

VAMP-2 Exocytosis 1 TMH 15

SNAP-25 Exocytosis Palmitoylation 1, 2, 4, 5

Syntaxin-1 Exocytosis 1 TMH 15

Tomosyn Exocytosis 5

vti1A Exocytosis 1 TMH 5

VAP Exocytosis 1 TMH 2, 4, 5

vATPase V0 a Proton pump 6 TMH 2, 4, 5

V0 c Proton pump 4 TMH 4, 5

V0 d Proton pump 1, 2, 4, 5

V1 A Proton pump 14

V1 B Proton pump 15

V1 C Proton pump 15

V1 D Proton pump 15

V1 E Proton pump 15

V1 F Proton pump 4, 5

V1 G Proton pump 25

V1 H Proton pump 1, 2, 4, 5

vAChT Neurotransmitter transport 10 TMH

vMaT2 Neurotransmitter transport 11 TMH

vGaT Neurotransmitter transport 9 TMH 2, 4, 5

vGluT Neurotransmitter transport 11 TMH 1, 2, 4, 5

VaT1 Neurotransmitter transport 4, 5

ZnT-3 Zinc transporter 6 TMH 1, 3, 4, 5

ClC-3 Chloride channel 11 TMH

CSP Chaperone Palmitoylation 1, 2, 4, 5

Heterotrimeric G proteins Signal transduction Palmitoylation 1, 2, 4, 5

rab3 Vesicle trafficking Prenylation 1, 2, 4, 5

TMH, transmembrane helices; SV2, synaptic vesicle protein 2; SCAMP, secretory carrier-associated membrane protein; VAMP, vesicle-associ-

ated membrane protein; vGaT, vesicular GABA transporter; vAChT, vesicular acetylcholine transporter; VAP, vesicle-associated membrane-

interacting protein; VaT1, vesicular amine transporter; ZnT3, vesicular zinc transporter; ClC, chloride channel; CSP, cysteine string protein; 1,

Coughenour et al. 2004; 2, Blondeau et al. 2004; 3, Morciano et al. 2005; 4, Burre et al. 2006b; 5, Takamori et al. 2006.



Fig. 1 and Table 1). Isoelectric focussing gels poorlyresolved proteins with very basic isoelectric points and onlytwo integral membrane proteins were identied. A combi-nation of 1D, BAC/SDS and double SDS for the separationof synaptic vesicle proteins resulted in the identication of185 proteins (Burre et al. 2006b). Interestingly, only 19% ofthe proteins were identied by all three techniques. Further-more, 23% of the proteins detected were identied by the 2Dtechniques and matrix-assisted laser desorption ionization(MALDI) time of ight MS only. In general, the 1Dapproach together with LC-tandem MS has proved to besuperior for integral membrane protein identication. Incontrast, the study of Takamori et al. (2006) lead to theidentication of 410 proteins with similar values for 1D(56%) and 2D (44%) electrophoresis using LC-tandem MS.However, a combination of several gel electrophoreticseparation techniques allows the most comprehensive analy-sis of the synaptic vesicle proteome.Several proteins that are assumed to be only transiently

associated with the synaptic vesicle membrane during thesynaptic vesicle life cycle have been identied in mostproteomic studies (Table 2, Fig. 2). Coughenour et al. (2004)described the presence of calcium/calmodulin-dependentprotein kinase II (CaMKII) (Grifth et al. 2003), synapsin I(De Camilli et al. 1983; Benfenati et al. 1989), the chaper-ones N-ethyl maleimide sensitive factor (NSF) (Sollner et al.1993) and Hsc70 (Chamberlain and Burgoyne 2000; Jianget al. 2000), adaptor protein complex 2 (AP-2) (Royle andLagnado 2003), actin (Dillon and Goda 2005), tubulin

(Honda et al. 2002) and glyceraldehyde-3-phosphate dehy-drogenase (GAPDH) (Schlafer et al. 1994; Ikemoto et al.2003) on the synaptic vesicle membrane. The presence of allthese proteins was conrmed in other investigations (Blond-eau et al. 2004; Burre et al. 2006b; Takamori et al. 2006). Inaddition, these studies reported the presence of synapsin II(Hosaka and Sudhof 1998), the small G proteins ADP-ribosylation factor (ARF) (Sewell and Kahn 1988) and rac(Didsbury et al. 1989), ral (Chardin and Tavitian 1986), rabGTPase-activating proteins (GAPs)/Guanine nucleotide ex-change factors (GEFs)/Guanine nucleotide dissociationinhibitors (GDIs) (Schimmoller et al. 1998), rabconnectin-3(Nagano et al. 2002), rabphilin-3A (Mollard et al. 1991),munc-18 (Ciufo et al. 2005), the chaperone Hsp90 (Sakisakaet al. 2002), AP-1 (reviewed in Boehm and Bonifacino2001), AP-180 (Bao et al. 2005), AP-2-associated kinase(Conner and Schmid 2002), clathrin (Kirchhausen andHarrison 1981), phosphatidylinositol binding clathrin assem-bly protein (PICALM) (Yao et al. 2005), dynamin (Severet al. 2000), synaptojanin (Haffner et al. 2000), alpha-internexin (Yuan et al. 2006), complex of actin relatedproteins 2 and 3 (ARP2/3) (Merrield 2004), capping protein(Hart et al. 1997), p25 (Tirian et al. 2003), microtubuleassociated protein 6 (Eastwood et al. 2006), elongation factor1 (EF-1) (Yang et al. 1993), myosin-Va (Doussau andAugustine 2000), dynein (Holleran et al. 2001a) and 3-phosphoglycerate kinase (Ikemoto et al. 2003). Proteins iden-tied in one or two investigation only were synapsin III (Fenget al. 2002), spectrin (Sikorski et al. 1991), neurolament

Fig. 1 Established synaptic vesicle pro-

teins.



proteins (Lariviere and Julien 2004), the actin-interactingprotein prolin (Schluter et al. 1997), and the microtubuleinteracting proteins MAP-1 (Halpain and Dehmelt 2006),kinesin (Bloom 2001) and dynactin 1 (Holleran et al. 2001b).Endocytosis is described as a fast event and the endocytic

proteins clathrin, dynamin, synaptojanin and AP-2 are

assumed to associate with the vesicular membrane onlytransiently (Sudhof 2004). However, the data obtained byproteomic analyses of synaptic vesicles and CCVs indicatethat the apparatus for endocytosis might remain associatedwith the vesicle membrane for a longer period of time thanpreviously anticipated. It has recently been demonstated that

Table 2 Proteins transiently associated with the synaptic vesicle membrane during the vesicle life cycle

Proteins Function Interaction Proteomics approach

CaMKII Reserve pool regulation Cytoskeleton, SV 15

Synapsin I Reserve pool regulation Cytoskeleton, SV, PK 1, 35

Synapsin II Reserve pool regulation Cytoskeleton, SV 35

Synapsin III Reserve pool regulation Cytoskeleton, SV 5

Small G protein ARF-like Vesicle trafficking GP-MP 2, 4, 5

Small G protein rac Exocytosis SV, PK 2, 4, 5

Small G protein ralA Vesicle pool regulation GP-MP, PL, exocyst 4, 5

Rab GAP/GEF/GDI Vesicle trafficking Rab 2, 4, 5

Rabconnectin-3 Vesicle trafficking Rab3 4, 5

Rabphilin-3A Vesicle trafficking Rab3, SV 35

Munc18 Docking/priming Syntaxin-1 25

Chaperone NSF SNARE complex disassembly SNARE complex 15

Chaperone Hsc70 Clathrin coat disassembly CSP, SNAREs, synaptotagmin I 1, 2, 4

Chaperone Hsp90 Rab recycling Rab a-GDI 2, 4

AP-1 Endocytosis Synaptophysin, microtubule 2, 5

AP-2 Endocytosis

AP-2, AP180, amphiphysin, clathrin,

dynamin, synaptojanin, phospholipids

15

AP-180 Endocytosis 2, 4, 5

AP-2-associated kinase Endocytosis 4, 5

Amphiphysin Endocytosis

Clathrin Endocytosis 24

PICALM Endocytosis 2, 5

Dynamin Endocytosis 35

Synaptojanin Endocytosis 4, 5

Actin Cytoskeleton Synapsin 15

Tubulin Cytoskeleton Synaptotagmin I 15

Spectrin Cytoskeleton Cytoskeleton, synapsin I, munc13 4

Neurofilament proteins Cytoskeleton Neurofilament proteins, dynein 4

Alpha-internexin Cytoskeleton 4, 5

ARP2/3 Cytoskeleton dynamics Actin, dynamin 4, 5

Capping protein Cytoskeleton dynamics Actin 4, 5

Profilin Cytoskeleton dynamics Actin, ARP2/3 2

p25 Cytoskeleton dynamics Microtubules 2, 5

MAP-1 Cytoskeleton dynamics Microtubules 5

MAP-6 (STOP) Cytoskeleton dynamics Microtubules 4, 5

EF1 Cytoskeleton dynamics,

protein translation

Cytoskeleton 4, 5

Myosin-Va Molecular motor Actin, VAMP-2/synaptophysin complex, syntaxin-1A 4, 5

Dynein Molecular motor Microtubules, dynactin 2, 4, 5

Kinesin Molecular motor Microtubules 5

Dynactin 1 Molecular motor Dynein, Arp1 5

GAPDH Glycolysis vATPase 15

3-PGK Glycolysis ? 2, 4

SV, synaptic vesicle membrane; 3-PGK, 3-phosphoglycerate kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PK, protein kinase;

GP-MP, G protein-modulating proteins; PL, phospholipase; CSP, cysteine string protein; SNARE, soluble N-ethylmaleimide-sensitive factor

attachment protein receptor; VAMP, vesicle-associated membrane protein; MAP, microtubule-associated protein. 1, Coughenour et al. 2004; 2,

Blondeau et al. 2004; 3, Morciano et al. 2005; 4, Burre et al. 2006b; 5, Takamori et al. 2006.

}



a-adaptin is enriched in synaptic vesicle preparations(Murshid et al. 2006).Moreover, numerous proteins involved in cytoskeletal

rearrangements such as prolin, capping protein, ARP2/3,EF1, STOP and p25 were identied. These proteins areinvolved in cytoskeletal organization and might also play animportant role in vesicular dynamics within the nerveterminal.

Synaptic vesicle proteins eluding identification by

proteomic analysis

Several established integral synaptic vesicle proteins havenot been identied by MS in any of the vesicle proteomicanalyses, including the vesicular transporter for acetylcholine(Gilmor et al. 1996), for monoamines (Nirenberg et al.1995), the putative transporter SV2 related protein (SVOP)(Janz et al. 1998) and the chloride channel CIC-3 (Salazaret al. 2004). Moreover, the vesicle-attached amphiphysin hasnot been identied in any studies using MS although itspresence on CCVs has been demonstrated in an immunoblotanalysis by Blondeau et al. (2004).

Novel candidates

Numerous proteins have been identied by the proteomicapproaches that have not yet been assigned to the synapticvesicle compartment, or their putative presence has beenindicated only recently (Table 3 and Fig. 3). The identica-tion of these proteins in several proteomic analyses suggeststhat it might be worthwhile to investigate their potential

involvement in the synaptic vesicle function in more detail.Following, a brief summary of known functions or cellularallocations of these proteins is provided.

Isoforms of established synaptic vesicle proteins

Additional SNARE protein isoforms were identied onboth CCVs and synaptic vesicles (Blondeau et al. 2004;Burre et al. 2006b; Takamori et al. 2006). Syntaxin-6localizes to the trans-Golgi network (TGN) and endosomesand, unlike other SNAREs, reveals a broad variety ofinteraction partners (Wendler and Tooze 2001). Syntaxin-7 isconcentrated on late endosomes and lysosomes and isrequired for the fusion of both organelles (Mullock et al.2000). Syntaxin-12/13 is also localized on endosomes andmight either function to receive vesicles from the TGN or theearly endosomes (Tang et al. 1998), or may be involved inhomotypic early endosome fusion (Brandhorst et al. 2006).Syntaxin-16b is concentrated on the TGN and involved inTGNendosome transport (Wang et al. 2005). In contrast toubiquitous VAMP-2, VAMP-1 reveals a less abundantexpression in particular brain areas, and co-localizes withsynaptophysin (Raptis et al. 2005). Cellubrevin is a ubiqui-tously expressed membrane protein that localizes to endo-somes and functions in constitutive exocytosis (Gerst 1999).SNAP-29 is present at synapses and inhibits synaptictransmission, probably by competing with a-SNAP forbinding to the SNAREs and consequently inhibiting disas-sembly of the SNARE complex (Pan et al. 2005). SNAP-47reveals a widespread distribution on intracellular membranesand is also enriched in synaptic vesicle fractions (Holt et al.2006). These results suggest that synaptic vesicles are

Fig. 2 Proteins transiently associated with

the synaptic vesicle membrane during the

vesicle life cycle.



involved also in endosomal fusion reactions. SynaptotagminXVII (B/K protein) has been found in the ER in rathippocampus (Jang et al. 2004) and revealed an abundantpresence in the brain, kidney and prostate (Chin et al. 2006).Synaptotagmin XII, unlike most other synaptotagmins, doesnot bind syntaxin-1 and SNAP-25 in a Ca2+-dependentmanner (Chapman et al. 1995; Schiavo et al. 1997). Up untilnow, the physiological role of these proteins on the synapticvesicle compartment remains unclear.

GTP-binding proteins

In addition to the synaptic vesicle-associated rab3 isoformsother members of the rab family were identied in the vesicle

proteome, although it has been demonstrated, for most rabproteins, that they reside on organelles other than synapticvesicles, e.g. early and late endosomes or TGN (Pfeffer 2001).Of these, endosome-localized rab5, in addition, was found toassociate with synaptic vesicles specically (Shimizu et al.2003). The GTP-binding protein septins 2, 5, 7 and 11 belongto a multigene family with multiple splice variants (Xue et al.2004). They associate with biological membranes through ahighly conserved polybasic region at the N-terminus of theGTP-binding domain (Casamayor and Snyder 2003; Spiliotisand Nelson 2006). Septin 2 is essential for the retention ofnormal actin levels and structures (Schmidt and Nichols2004). Septin 5 associates with membranes of GABAergic

Table 3 Putative novel synaptic vesicle proteins

Proteins Putative function Interaction Proteomics approach

Rabs Vesicle trafficking GP-MP 25

Small G protein rap Signal transduction Ras effectors 2, 4, 5

Small G protein K-ras Signal transduction PLC, Erk 2, 4, 5

VAMP-1 Exocytosis SNAREs 2, 4, 5

Cellubrevin Exocytosis SNAREs 5

SNAP-29 Endosomal fusion SNAREs 5

SNAP-47 Endosomal fusion SNAREs 5

Syntaxin 6 Endosomal fusion SNAREs 2, 5

Syntaxin 7 Endosomal fusion SNAREs 4, 5

Syntaxin 12 Endosomal fusion SNAREs 2, 4, 5

Syntaxin 13 Endosomal fusion SNAREs 5

Syntaxin 16b Golgi trafficking SNAREs 5

Thy-1 Release, signalling GPI anchor 2, 4, 5

Glucosephosphate Isomerase Glycolysis ? 2

PFK Glycolysis vATPase 25

FBP aldolase Glycolysis vATPase 25

Phosphoglycerate mutase Glycolysis ? 2

Enolase Glycolysis ? 2, 5

Pyruvate kinase Glycolysis ? 25

Lactate dehydrogenase Glycolysis ? 25

Septin 2

Cytoskeleton, plasticity, exocytosis

Actin, PIP, exocyst, septins 4

Septin 5 SNAREs, synaptophysin, PIP, septins 4

Septin 7 PIP, exocyst complex, septins 2, 4, 5

Septin 11 PIP, cytoskeleton 4

Reticulon 1

Membrane curvature

AP50, SNAREs 2, 4, 5

Reticulon 3 ? 2, 4

Reticulon 4 ? 4

NTT4 Neurotransmitter transport ? 2, 4, 5

VILIP 1 Neuronal Ca2+ sensor nACh-R, guanylate cyclase receptor 2, 4, 5

VILIP 3 Neuronal Ca2+ sensor ? 2, 4

Proteasomal proteins Protein degradation Munc18, syntaxin-1, synaptophysin 4, 5

Synaptotagmin XII ? ? 4, 5

Synaptotagmin XVII ? ? 5

Svap30 ? ? 4

DKFZp566N034 Cation transporter ? 4, 5

GP-MP, G protein-modulating proteins; VAMP, vesicle-associated membrane protein; NTT, neurotransmitter transporter; SNARE, soluble

N-ethylmaleimide-sensitive factor attachment protein receptor; Svap30, synaptic vesicle-associated protein of 30 kDa; VILIP, visinin-like proteins;

FBP, fructose bisphosphate; PFK, phosphofructokinase; PIP, phosphatidyl inositol phosphates. 1, Coughenour et al. 2004; 2, Blondeau et al.

2004; 3, Morciano et al. 2005; 4, Burre et al. 2006b; 5, Takamori et al. 2006.

}}



synaptic vesicles (Caltagarone et al. 1998), co-localizespartially with VAMP-2 (Peng et al. 2002) and interacts withsynaptophysin (Caltagarone et al. 1998). Moreover, it bindsto SNARE complexes via syntaxin-1 (Beites et al. 1999), andcompetes with NSF and SNAPs for its binding (Beites et al.2005). Septin 5 complexes with the septins 2 and 7 (Penget al. 2002). Septin 7 in concert with other septins associatewith the exocyst complex (Hsu et al. 1998b) and might beimportant for the maintenance of neuronal polarity andsynaptic plasticity (Hsu et al. 1998a). The small G protein,ras, communicates extracellular signals to the nucleus andthereby regulates a variety of intracellular processes (Marshall1995). Sustained ras activity revealed an increase in thepackage density of synaptic vesicles docked at active zones(Seeger et al. 2004). The increased size of the readilyreleasable vesicle pool potentially results in a modulation ofthe release probability at glutamatergic synapses. Moreover,ras has been shown to be a constitutive CCV component(Howe et al. 2001). Rap is predominantly localized post-synaptically and binds the G protein, raf, which is known toactivate the MAP kinase pathway (Ohtsuka et al. 1999). Therole of these two small G proteins with respect to theregulation of the synaptic vesicle cycle must be furtherelucidated.

Putative transporters

The orphan neurotransmitter transporter, NTT4, belongs tothe family of Na+/Cl)-dependent transporters of the plasmamembrane (Liu et al. 1993). The protein has 12 putativetransmembrane helices, is present on synaptic vesicles insubpopulations of GABAergic and glutamatergic neuronsand might possibly be involved in the vesicular transport ofATP, calcium, zinc or chloride (Masson et al. 1999).However, a general vesicular function can be ruled out since

the protein is only present in subpopulations of neurons. Itremains to be analysed whether this transporter activelytransports a substrate into the synaptic vesicle lumen, or if itrepresents a protein inserted into the plasma membrane ondemand. The putative novel synaptic vesicle proteinDKFZp566N034 (Burre et al. 2006b; Takamori et al.2006) with six predicted transmembrane domains revealshomology to bacterial cation transporters (Haney et al.2005). No functional data exists for the latter protein.

Glycolytic machinery for ATP production

The presence of almost all enzymes of the glycolyticmachinery has been reported both for CCVs and synapticvesicles (Blondeau et al. 2004; Burre et al. 2006b; Takamoriet al. 2006). In addition to GAPDH and 3-phosphoglyceratekinase, these include phosphofructokinase, fructose bisphos-phate (FBP) aldolase, enolase, pyruvate kinase and lactatedehydrogenase. Additional proteins identied on CCVsincluded glucose phosphate isomerase and phosphoglyceratemutase. ATP produced by glycolysis rather than mitochond-rial ATP has been demonstrated to drive the vATPase(Ikemoto et al. 2003). In support of the notion of anassociation of these proteins with the vesicular surface,interactions of the fructose bisphosphate aldolase andphosphofructokinase with subunits of the vATPase havebeen described (Lu et al. 2001; Su et al. 2003). Moreover,GAPDH has been found to be a part of the fusion complextogether with the vATPase and Ca2+/calmodulin (Peters et al.2001).

Membrane curvature

The reticulons 1, 3 and 4 were identied on CCVs andsynaptic vesicles. Reticulons are a family of integralmembrane proteins (GrandPre et al. 2000) that are predom-

Fig. 3 Recently assigned and putative

novel synaptic vesicle proteins.



inantly localized to the ER (Velde et al. 1994; Ortle andSchwab 2003). Their physiological function has yet to beelucidated. Reticulon 1A and 1B interact with AP50, acomponent of the AP-2 adaptor complex required forendocytosis (Iwahashi and Hamada 2003). Reticulon 1Cprecipitates with individual SNARE proteins but does notbind the SNARE complex (Steiner et al. 2004), possiblybeing involved in the biosynthesis or targeting of SNAREproteins. Reticulon 3 presumably plays a role in membranetrafcking of the early secretory pathway (Wakana et al.2005), and reticulon 4 (Nogo-A) is an important regulator ofregeneration and plasticity within the adult CNS (Simonenet al. 2003) and is present in Golgi and ER membranes ofneurons (Oertle et al. 2003). Recent results indicate thatreticulons play an important role in creating membranecurvature at the ER and might help to sustain these structures(Voeltz et al. 2006). Possibly, they also exert these functionson synaptic vesicles.

Visinin-like proteins and thy-1

The visinin-like proteins (VILIPs) are neuronal Ca2+ sensorproteins which attach to membranes by Ca2+-dependentmyristoylation (Ames et al. 1997). They are involved inCa2+-dependent signalling cascades, such as the metabolismof cyclic nucleotides, the release of neurotransmitters, themodulation of ion channels, the regulation of gene expres-sion and possibly also synaptic plasticity (Braunewell et al.2001; Burgoyne and Weiss 2001; Brackmann et al. 2005).Whereas VILIP-1 is primarily associated with neuronalplasma membranes, VILIP-3 is localized on intracellularmembranes where it exerts an effect on signal cascades viaMAP kinase (Spilker et al. 2002). VILIP-1 has beendemonstrated to be associated with CCVs and might beinvolved in endocytosis via interaction with AP-2 (Blondeauet al. 2004). The GPI-anchored membrane protein, thy-1,involved in cellcell and cellmatrix interaction such asinhibition of neurite outgrowth, apoptotic signalling, leuco-cyte and melanoma cell adhesion and migration, tumoursuppression and broblast proliferation (reviewed in Regeand Hagood 2006), was identied both on CCVs andsynaptic vesicles (Blondeau et al. 2004; Burre et al. 2006a;Takamori et al. 2006). Besides its localization at the plasmamembrane (Green and Kelly 1992) more than 50% of theprotein is localized in intracellular vesicles (Jeng et al. 1998).After fusion of vesicles with the plasma membrane, asignicant part remains at the membrane and does notrecycle. A functional implication in cellular signalling hasbeen proposed because antibodies against thy-1 reduce theamount of released neurotransmitter (Jeng et al. 1998).

Proteasomal degradation in the nerve terminal

Most cellular proteins are degraded by the ubiquitinproteasome system. Pre-synaptic proteins regulated by theubiquitinproteasome system include munc18 (Speese et al.

2003), syntaxin-1 (Chin et al. 2002) and synaptophysin(Wheeler et al. 2002). The physiological consequence ofdegradation of these proteins is not yet understood, but theubiquitinproteasome system may acutely determine thelocal concentration of key regulatory proteins at neuronalsynapses as a means for locally modulating synaptic efcacyand the strength of neurotransmission.

Proteins in search for a function

Eight of the numerous proteins identied in CCVs werehypothetical gene products that had not been detectedpreviously at the protein level (Blondeau et al. 2004).Enthoprotin has been demonstrated to be a novel clathrin-associated protein (Wasiak et al. 2002) that stimulatesclathrin assembly. Adaptin ear-binding coat-associated pro-tein (NECAP)-1 and NECAP-2 are proteins present on CCVsthat interact with AP-2 (Ritter et al. 2003). In clathrin heavychain depleted Hela cells NECAP-1 is approximatelythreefold less abundant compared with wild type (Borneret al. 2006). It has been demonstrated that NECAP-1 isenriched during synaptic vesicle preparation and might play arole in pre-synaptic endocytosis (Murshid et al. 2006).Similarly, 9 (Burre et al. 2006b) and 15 (Takamori et al.2006) novel proteins, where only the DNA sequence isknown, have been reported to reside on synaptic vesicles.Synaptic vesicle-associated protein of 30 kDa has recentlybeen identied in an approach where functional differencesbetween synaptic vesicle proteins under conditions of restand activation were observed (Burre et al. 2006a). Theprotein revealed an increase in abundance following stimu-lation.

Summary and outlook

The number of proteins identied by proteomic approachesin both synaptic vesicles and CCVs is unexpectedly high.Only about one quarter of these proteins are well-establishedplayers in the synaptic vesicle life cycle. An additionalsurprising outcome of these studies is the large number ofvesicle-associated proteins, many of which are expected toassociate only transiently with synaptic vesicles. In thiscontext, it is important to note that not all the proteins mayreside on the same vesicles. Some proteins, such astransporters, are contained only in subpopulations of synapticvesicles. Other proteins may be primarily localized toendosomes, or the plasma membrane, and may be capturedduring cycles of exo- and endocytosis. A proteomic analysisof subpopulations of synaptic vesicles might clarify thisissue. Similarly, proteins expressed in subpopulations ofneurons that to date eluded identication by proteomicapproaches might be detected more easily when subpopula-tions of synaptic vesicles are screened. Further investigationis required to elucidate whether vesicular transport systemsthat have previously been characterized in functional terms,



such as the transporters for nucleotides or calcium, can beattributed to the novel vesicle proteins.The functional characterization of the novel synaptic

vesicle proteins, and of proteins previously not assigned tothe synaptic vesicle compartment, will be an important issuefor future studies. It is expected that the precise functionalrole of individual synaptic vesicle proteins will be resolvedleading to a deeper understanding of the regulation of thesynaptic vesicle compartment regarding both neurotransmit-ter release and the plasticity of the nerve terminal compart-ment.

Acknowledgements

We thank the Deutsche Forschungsgemeinschaft for nancial

support (SFB628, P16). We are indebted to Herbert Zimmermann

for helpful discussions and critical reading of the manuscript. We

thank Jeremy Hack for critically editing the manuscript.

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