The neuronal SNARE complex
Synaptic vesicle exocytosis: SNARE-complex functions
Yale University Medical School, Head of lab: Prof. R. Jahn
To investigate the dynamics of synaptic vesicle exocytosis, a cell-free membrane-fusion assay based on fluorescence dequenching or fluorescence resonance energy transfer (FRET) had to be developed.I recombinantly produced the proteins required for this assa: the SNARE (SNAP receptor) proteins synaptobrevin, SNAP-25 (synaptosome-associated protein of 25 kDa) and syntaxin as well as the chaperone NSF (NEM-sensitive fusion protein) and its co-chaperones alpha-SNAP and gamma-SNAP (soluble NSF-attachment proteins). The proteins were purified and reconstituted into proteoliposomes. In parallel, I tested the assembly and disassembly of the SNARE fusion complex (Otto et al. (1995)). Despite some hints that at a low-level slow SNARE-controlled membrane fusion occured, I did not succeed in convincingly establishing such an assay at that time. Therefore, the assembly and disassembly characteristics of the SNARE fusion complex had to be characterized more thoroughly.
In this context, I was able to show that a high amount of the ternary fusion complex does form spontaneously, when synaptosomes are solubilized by Triton X-100. These complexes are normally disassembled by NSF in the presence of ATP. Dr. Phyllis Hanson and I demonstrated that NSF induces this disassembly by acting on syntaxin, changing its conformation, which releases synaptobrevin and SNAP-25 from the complex (Hanson et al. (1995)). Finally, I reported that highly stable ternary SNARE fusion complexes consisting of synaptobrevin and the plasma membrane proteins exist also on synaptic vesicles. There, they can be likewise disassembled by NSF and ATP (Otto et al. (1997)). This study provided the first evidence that the SNARE complex exists in two configurations: trans-SNARE complexes between vesicle and plasma membrane, which support membrane fusion, and cis-SNARE complexes on either plasma membrane or vesicle membrane, which cannot initiate membrane fusion. Thus, NSF regenerates monomeric SNARE proteins from fusion-incompetent cis-SNARE complexes after membrane fusion, but is not involved in inducing membrane fusion, as had been indicated earlier.
For recent developments in the field of synaptic vesicle exocytosis you are kindly referred to the web sites of Reinhard Jahn's research group at the Max-Planck-Institute for Biophysical Chemistry in Göttingen.
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Henning Otto / updated: 12.04.2011
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