Analysis of the mutant Drosophila N-ethylmaleimide sensitive fusion-1 protein in comatose reveals molecular correlates of the behavioural paralysis

NEM-sensitive fusion protein (NSF) is an ATPase required for many intracell ular membrane trafficking steps. Recent studies have suggested that NSF alt ers the conformation of the SNAP receptors (SNAREs) to permit their interac tion, or to uncouple them after they interact. Most organisms have a single NSF gene product but Drosophila express two highly related isoforms, dNSF- 1 and dNSF-2. dNSF-1 is encoded by the gene comatose (comt), first identifi ed as the locus of a temperature-sensitive paralytic mutation. Here we show that dNSF-1 is most abundant in the nervous system and can be detected in larval and adult CNS. Subcellular fractionation revealed that dNSF-1 was en riched in a Vesicle fraction along with the synaptic vesicle protein synapt otagmin. comt flies maintained at the non-permissive temperature rapidly ac cumulate sodium dodecyl sulfate (SDS)-resistant SNARE complexes at the rest rictive temperature, with concomitant translocation of dNSF-1 from cytosol and membrane fractions into a Triton X-100 insoluble fraction. The long rec overy of comt flies after heat shock induced paralysis correlated with the irreversibility of this translocation. interestingly, while dNSF-1 also tra nslocates in comt(TP7) larvae, there is no associated neurophysiological ph enotype at the neuromuscular junction (nmj) or accumulation of SDS-resistan t complexes in the CNS. Together, these results suggest that dNSF-1 is requ ired for adult neuronal function, but that in the larval nmj function may b e maintained by other isoforms.