Immunoisolation of two synaptic vesicle pools from synaptosomes: a proteomics analysis.
The nerve terminal proteome governs neurotransmitter release as well as the structural and functional dynamics of the presynaptic compartment. In order to further define specific presynaptic subproteomes we used subcellular fractionation and a monoclonal antibody against the synaptic vesicle protein SV2 for immunoaffinity purification of two major synaptosome-derived synaptic vesicle-containing fractions: one sedimenting at lower and one sedimenting at higher sucrose density. The less dense fraction contains free synaptic vesicles, the denser fraction synaptic vesicles as well as components of the presynaptic membrane compartment. These immunoisolated fractions were analyzed using the cationic benzyldimethyl-n-hexadecylammonium chloride (BAC) polyacrylamide gel system in the first and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. Protein spots were subjected to analysis by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI TOF MS). We identified 72 proteins in the free vesicle fraction and 81 proteins in the plasma membrane-containing denser fraction. Synaptic vesicles contain a considerably larger number of protein constituents than previously anticipated. The plasma membrane-containing fraction contains synaptic vesicle proteins, components of the presynaptic fusion and retrieval machinery and numerous other proteins potentially involved in regulating the functional and structural dynamics of the nerve terminal.
Morciano
Marco
M
Neurochemistry, J.W. Goethe-University, Frankfurt am Main, Germany.
Burré
Jacqueline
J
Corvey
Carsten
C
Karas
Michael
M
Zimmermann
Herbert
H
Volknandt
Walter
W
D000818:Animals; D015153:Blotting, Western; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005233:Fatty Alcohols; D007120:Immunochemistry; D066298:In Vitro Techniques; D008854:Microscopy, Electron; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000276:immunology / Q000378:metabolism*; D040901:Proteomics; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D051381:Rats; D017208:Rats, Wistar; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013347:Subcellular Fractions / Q000276:immunology / Q000378:metabolism / Q000648:ultrastructure; D013572:Synaptic Vesicles / Q000276:immunology / Q000378:metabolism* / Q000648:ultrastructure; D013574:Synaptosomes / Q000276:immunology / Q000378:metabolism* / Q000648:ultrastructure
D005233:Fatty Alcohols; D009419:Nerve Tissue Proteins; D000644:Quaternary Ammonium Compounds; C012817:cetalkonium chloride