The multisubunit structure of synaptophysin. Relationship between disulfide bonding and homo-oligomerization.
Synaptophysin, a major membrane protein of synaptic vesicles, contains four transmembrane regions and two intravesicular loops. Synaptophysin monomers associate into homopolymers that have the potential to form channels in the synaptic vesicle membrane. Here we show that in native synaptophysin, homopolymers are linked by noncovalent forces. The molecule contains unstable intramolecular disulfide bonds that undergo disulfide exchange during solubilization, thereby covalently cross-linking neighboring synaptophysin molecules. The locations of the intramolecular disulfide bonds in synaptophysin were determined, revealing that each of the two intravesicular loops of synaptophysin is circularized by a single disulfide bond. Cross-linking of synaptophysin by disulfide bonds can be triggered in synaptic vesicles and in intact cells by a cycle of reduction and oxidation, suggesting that native synaptophysin is a homomultimer in situ. In addition, chemical cross-linking of native synaptophysin demonstrates that a low molecular weight protein is specifically associated with synaptophysin complexes and is lost upon reduction of the intramolecular disulfide bonds. These data suggest that native synaptophysin forms a noncovalent homomultimeric complex whose structure and interaction with other proteins are dependent on the integrity of its intramolecular disulfide bonds and phospholipid environment.
Johnston
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Howard Hughes Medical Institute, University of Texas Southwestern Medical School, Dallas 75235-9050.
Südhof
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TC
D000310:Adrenal Gland Neoplasms; D000818:Animals; D000906:Antibodies; D002460:Cell Line; D003432:Cross-Linking Reagents / Q000378:metabolism; D004120:Dimethyl Suberimidate / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000378:metabolism*; D008961:Models, Structural; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D010673:Pheochromocytoma; D013572:Synaptic Vesicles / Q000378:metabolism*; D016708:Synaptophysin
D000906:Antibodies; D003432:Cross-Linking Reagents; D004220:Disulfides; D046911:Macromolecular Substances; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D016708:Synaptophysin; D004120:Dimethyl Suberimidate