Synaptobrevin binding to synaptophysin: a potential mechanism for controlling the exocytotic fusion machine.
The synaptic vesicle protein synaptobrevin (VAMP) has recently been implicated as one of the key proteins involved in exocytotic membrane fusion. It interacts with the synaptic membrane proteins syntaxin I and synaptosome-associated protein (SNAP)-25 to form a complex which precedes exocytosis [Söllner et al. (1993b) Cell, 75, 409-418]. Here we demonstrate that the majority of synaptobrevin is bound to the vesicle protein synaptophysin in detergent extracts. No syntaxin I was found in this complex when synaptophysin-specific antibodies were used for immunoprecipitation. Conversely, no synaptophysin was associated with the synaptobrevin-syntaxin I complex when syntaxin-specific antibodies were used for immunoprecipitation. Thus, the synaptobrevin pool bound to synaptophysin is not available for binding to syntaxin I and SNAP-25, and vice versa. Synaptobrevin-synaptophysin binding was also demonstrated by chemical cross-linking in isolated nerve terminals. Furthermore, recombinant synaptobrevin II efficiently bound synaptophysin and its isoform synaptoporin, but not the more distantly related synaptic vesicle protein p29. Recombinant synaptobrevin I bound with similar efficiency, whereas the non-neuronal isoform cellubrevin displayed a lower affinity towards synaptophysin. Treatment with high NaCl concentrations resulted in a dissociation of the synaptobrevin-synaptophysin complex. In addition, the interaction of synaptobrevin with synaptophysin was irreversibly abolished by low amounts of SDS, while the interaction with syntaxin I was enhanced. We conclude that synaptophysin selectively interacts with synaptobrevin in a complex which excludes the t-SNAP receptors syntaxin I and SNAP-25, suggesting a role for synaptophysin in the control of exocytosis.
Edelmann
L
L
Howard Hughes Medical Institute, Yale University School of Medicine, New Haven CT 06510.
Hanson
P I
PI
Chapman
E R
ER
Jahn
R
R
D000595:Amino Acid Sequence; D000818:Animals; D002459:Cell Fusion; D003432:Cross-Linking Reagents; D005089:Exocytosis; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D050683:R-SNARE Proteins; D051381:Rats; D016708:Synaptophysin / Q000302:isolation & purification / Q000378:metabolism*
D003432:Cross-Linking Reagents; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050683:R-SNARE Proteins; D016708:Synaptophysin