Molecular Anatomy of a Trafficking OrganelleIntroductionResultsProtein and Lipid Composition of SVsQuantitative Analysis of Physical ParametersConstruction of a Molecular ModelDiscussionSV Proteome and Compositional HeterogeneityImplications for Membrane StructureImplications for Membrane TraffickingImplications for Neurotransmitter Uptake andnbspStorageConclusionExperimental ProceduresPurification of SVs from Rat BrainProtein and Lipid AnalysisElectron MicroscopyMolecular Dynamics Simulations and Model ConstructionMiscellaneous ProceduresSupplemental DataAcknowledgmentsReferencesMolecular Anatomyof a Trafficking OrganelleShigeo Takamori,1,13Matthew Holt,1Katinka Stenius,7,14Edward A. Lemke,2Mads Grønborg,1,4Dietmar Riedel,3Henning Urlaub,4Stephan Schenck,1,13Britta Bru¨ gger,8Philippe Ringler,9Shirley A. Mu¨ ller,9Burkhard Rammner,10Frauke Gra¨ter,5Jochen S. Hub,5Bert L. De Groot,5Gottfried Mieskes,1Yoshinori Moriyama,11Ju¨ rgen Klingauf,2Helmut Grubmu¨ ller,6John Heuser,12Felix Wieland,8and Reinhard Jahn1,*1Department of Neurobiology2Department of Membrane Biophysics3Laboratory of Electron Microscopy4Laboratory of Bioanalytical Mass Spectrometry5Laboratory of Biomolecular Dynamics6Department of Theoretical and Computational BiophysicsMax-Planck Institute for Biophysical Chemistry, 37077 Go¨ttingen, Germany7Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06510, USA8Department of Biochemistry, University of Heidelberg, 69120 Heidelberg, Germany9M.E. Mu¨ller Institute, Biozentrum, University of Basel, CH-4056 Basel, Switzerland10Scimotion, Harkortstrasse 121, 22765 Hamburg, Germany11Laboratory of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry and PharmaceuticalScience, Okayama 700-8530, Japan12Department of Cell Biology, Washington University, St. Louis, MO 63110, USA13Present address: 21st Century COE Program, Department of Neurology and Neurological Science, Graduate School of Medicine,Tokyo Medical and Dental University, Tokyo 113-8519, Japan.14Present address: 31 Surry Road, Arlington, MA 02476, USA.*Contact: [email protected] 10.1016/j.cell.2006.10.030SUMMARYMembrane traffic in eukaryotic cells involvestransport of vesicles that bud from a donorcompartment and fuse with an acceptor com-partment. Common principles of budding andfusion have emerged, and many of the proteinsinvolved in these events are now known. How-ever, a detailed picture of an entire traffickingorganelle is not yet available. Using synapticvesicles as a model, we have now determinedthe protein and lipid composition; measuredvesicle size, density, and mass; calculated theaverage protein and lipid mass per vesicle;and determined the copy number of more thana dozen major constituents. A model has beenconstructed that integrates all quantitativedata and includes structural models of abun-dant proteins. Synaptic vesicles are dominatedby proteins, possess a surprising diversity oftrafficking proteins, and, with the exception ofthe V-ATPase that is present in only one totwo copies, contain numerous copies of pro-teins essential for membrane traffic and neuro-transmitter uptake.INTRODUCTIONEukaryotic cells are compartmentalized into membrane-bound organelles that communicate with each other bymembrane trafficking. While many organelles readily un-dergo fusion or fission, transport between different organ-elles usually involves specialized trafficking vesicles.These vesicles bud from the precursor compartmentand are transported to the target compartment, wherethey dock and fuse. Trafficking vesicles deliver both mem-brane constituents and soluble content material from thedonor to the acceptor compartment or to the extracellularspace (Bonifacino and Glick, 2004).Small trafficking vesicles, with diameters ranging be-tween 40 and 80 nm, can be considered as the basic min-imal units of membrane traffic. Vesicle transport, targetrecognition, docking, and fusion each involve the orderedand sequential recruitment of protein complexes from thecytoplasm. The membrane constituents of the organelleare ultimately responsible for orchestrating association ofthe complex, task execution, and complex disassembly.In recent years, many of the proteins involved in these re-actions have been identified (Bonifacino and Glick, 2004).However, little is known about overall membrane structure,including the concentration of integral membrane andmembrane-associated proteins, or about the surfacedensity of trafficking proteins such as SNAREs and Rabs.Cell 127, 831–846, November 17, 2006 ª2006 Elsevier Inc. 831In the present study, we have attempted to arrive ata comprehensive and quantitative molecular descriptionof synaptic vesicles (SVs) as model trafficking organelles.SVs are concentrated in the presynaptic nerve terminals ofevery neuron. They store neurotransmitters and undergoCa2+-dependent exocytosis upon the arrival of an actionpotential. After exocytosis, SVs are retrieved by clathrin-dependent endocytosis and are locally recycled to regen-erate exocytosis-competent vesicles. Although the inter-mediate steps in the recycling pathway are still debated,it is clear that nerve terminals contain endosomes andthat the SV cycle may involve endosomal intermediates,although not necessarily during each recycling event(Su¨dhof, 2004).SVs can be purified to apparent homogeneity in largequantities, making them amenable to biochemical studies(Nagy et al., 1976; Huttner et al., 1983). Indeed, the proteincomposition of SVs is, at present, better understood thanthat of any other trafficking organelle, and several proteinsfirst identified in SVs turned out to be founding membersof conserved protein families involved in all traffickingsteps (Jahn and Su¨dhof, 1994). In addition, SVs containthe machinery required for the uptake and storage of neu-rotransmitters including vesicular transporters, ion chan-nels, and the vacuolar H+ATPase that fuels the trans-porters (Ahnert-Hilger et al., 2003). Recently, proteomeanalyses of enriched fractions of both SVs and brain-derived clathrin-coated vesicles have been performed, re-sulting in the identification of many of the known vesicleproteins (Morciano et al., 2005; Blondeau et al., 2004;see also Discussion).In contrast to our advanced knowledge about individualSV proteins, we lack even elementary quantitative in-formation on the composition of the whole vesicle. Witha diameter of approximately 40 nm, SVs are small organ-elles, making