Synaptic vesicle membrane proteins interact to form a multimeric complex.
Potential interactions between membrane components of rat brain synaptic vesicles were analyzed by detergent solubilization followed by size fractionation or immunoprecipitation. The behavior of six synaptic vesicle membrane proteins as well as a plasma membrane protein was monitored by Western blotting. Solubilization of synaptic vesicle membranes in CHAPS resulted in the recovery of a large protein complex that included SV2, p65, p38, vesicle-associated membrane protein, and the vacuolar proton pump. Solubilization in octylglucoside resulted in the preservation of interactions between SV2, p38, and rab3A, while solubilization of synaptic vesicles with Triton X-100 resulted in two predominant interactions, one involving p65 and SV2, and the other involving p38 and vesicle-associated membrane protein. The multicomponent complex preserved with CHAPS solubilization was partially reconstituted following octylglucoside solubilization and subsequent dialysis against CHAPS. Reduction of the CHAPS concentration by gel filtration chromatography resulted in increased recovery of the multicomponent complex. Examination of the large complex isolated from CHAPS-solubilized vesicles by negative stain EM revealed structures with multiple globular domains, some of which were specifically labeled with gold-conjugated antibodies directed against p65 and SV2. The protein interactions defined in this report are likely to underlie aspects of neurotransmitter secretion, membrane traffic, and the spatial organization of vesicles within the nerve terminal.
Bennett
M K
MK
Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University Medical Center, California 94305.
Calakos
N
N
Kreiner
T
T
Scheller
R H
RH
D000818:Animals; D002499:Centrifugation, Density Gradient; D002793:Cholic Acids; D002850:Chromatography, Gel; D003902:Detergents; D005960:Glucosides; D008297:Male; D008565:Membrane Proteins / Q000737:chemistry*; D008854:Microscopy, Electron; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D017830:Octoxynol; D011092:Polyethylene Glycols; D011233:Precipitin Tests; D050683:R-SNARE Proteins; D051381:Rats; D011919:Rats, Inbred Strains; D012995:Solubility; D013572:Synaptic Vesicles / Q000737:chemistry* / Q000648:ultrastructure
D002793:Cholic Acids; D003902:Detergents; D005960:Glucosides; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050683:R-SNARE Proteins; C018619:octyl-beta-D-glucoside; D011092:Polyethylene Glycols; D017830:Octoxynol; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate