Neurotoxins as Tools in Dissecting the Exocytic Machinery - Fusion of Biological Membranes and Related Problems - page 56

SNAREs either by in vitro translation systems or by expression in E. coli
was used to explore their mutual interactions, their stability and their
assembly properties. Over-expressed SNAREs were used initially to verify
the exact cleavage site in the target protein in isolated situation (Binz et al.,
1994; Blasi et al., 1993a; Schiavo et al., 1993).
In vitro mixing of all three over-expressed SNAREs allowed formation
of a stable SDS-resistant complex. Application of the clostridial toxins to
the already assembled trimeric complex did not result in its disassembly.
These results suggested that also in vivo, clostridial toxins will preferably
attack the uncomplexed proteins (or at least not in their trimeric stable
complex state). VAMP was also protected from proteolysis by TeNT under
conditions that stabilize VAMP in the 7S protein complex (consists of
SNAREs protein and synaptotagmin) (Sollner et al., 1993b) or in the
complex that also contains the NSF and SNAP proteins (also known as
the 20s complex) (Hayashi et al., 1994; Pellegrini et al., 1994; Pellegrini
et al., 1995).
An in vitro simulation assay to reveal the effects of these neurotoxins
on the SNAREs' assembly was performed. The results indicated that pro-
teolysis of the proteins by different clostridial neurotoxins has inhibitory
effects on the formation of SNARE complexes, but these effects depended
on the type of toxin used. For example, the cleaved SNAP-25 by BoNT/A
and BoNT/E shows reduced binding to VAMP and not to syntaxin
(Chapman et al., 1994; Hayashi et al., 1995) but VAMP is able to participate
in the 20s complex (Hayashi et al., 1994; Otto et al., 1995). Such simulations
were also performed for the assembly of dimeric compositions (Hayashi et
al., 1994).
Recombinant v- and t-SNARE proteins were reconstituted into lipid
bilayer vesicles. This combination induced the assembly of SNARE com-
plexes and ultimately led to lipid mixing between separate liposomes. In
these studies, the introduction of clostridial toxin abolished the formation
of the essential SNARE complex, ensuring the role of the formation of a
stable SNARE complex for fusion, even in such in vitro, artificial conditions
(Weber et al., 1998).
Most structural studies took advantage of the available expressing
systems and the potential of following structural changes by combining
SNARE molecules in a test tube. Proteins were expressed with or without
a tag (i.e., hexa-histidine or GST-fusion proteins). The expressed proteins
were purified by an affinity matrix and used for in vitro binding assays.
Based on the knowledge that each of the clostridial toxins alter (to a lesser
extend) the stability of the SNARE complex, a set of experiments using
recombinant truncated SNAREs were performed. For example, the use of