Two distinct effects on neurotransmission in a temperature-sensitive SNAP-25 mutant

Vesicle fusion in eukaryotic cells is mediated by SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). In neurons, the t-SNARE SNAP-25 is essential for synaptic vesicle fusion but its exact role in this process is unknown. We have isolated a SNAP-25 temperature-sensitive paralytic mutant in Drosophila, SNAP-25(ts). The mutation causes a Gly50 to Glu change in SNAP-25's first amphipathic helix. A similar mutation in the yeast homologue SEC9 also results in temperature sensitivity, implying a conserved role for this domain in secretion. In vitro-generated 70 kDa SNARE complexes containing SNAP-25's are thermally stable but the mutant SNARE multimers (of similar to 120 kDa) rapidly dissociate at 37 degreesC. The SNAP-25(ts) mutant has two effects on neurotransmitter release depending upon temperature. At 22 degreesC, evoked release of neurotransmitter in SNAP-25(ts) larvae is greatly increased, and at 37 degreesC, the release of neurotransmitter is reduced as compared with controls. Our data suggest that at 22 degreesC the mutation causes the SNARE complex to be more fusion competent but, at 37 degreesC the same mutation leads to SNARE multimer instability and fusion incompetence.