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 intracellular membrane trafficking steps. Recent studies have suggested that NSF alters the conformation of the SNAP receptors (SNAREs) to permit their interaction, 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 identified 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 enriched in a vesicle fraction along with the synaptic vesicle protein synaptotagmin. comt flies maintained at the non-permissive temperature rapidly accumulate sodium dodecyl sulfate (SDS)-resistant SNARE complexes at the restrictive temperature, with concomitant translocation of dNSF-1 from cytosol and membrane fractions into a Triton X-100 insoluble fraction. The long recovery of comt flies after heat shock induced paralysis correlated with the irreversibility of this translocation. Interestingly, while dNSF-1 also translocates in comt(TP7) larvae, there is no associated neurophysiological phenotype at the neuromuscular junction (nmj) or accumulation of SDS-resistant complexes in the CNS. Together, these results suggest that dNSF-1 is required for adult neuronal function, but that in the larval nmj function may be maintained by other isoforms.
Mohtashami
M
M
Programme in Cell Biology, Developmental Biology, Hospital for Sick Children, Toronto, Ontario, Canada.
Stewart
B A
BA
Boulianne
G L
GL
Trimble
W S
WS
D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000032:analysis / Q000235:genetics*; D004330:Drosophila; D004594:Electrophysiology; D019706:Excitatory Postsynaptic Potentials / Q000187:drug effects; D007150:Immunohistochemistry; D007814:Larva; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009154:Mutation / Q000235:genetics; D052066:N-Ethylmaleimide-Sensitive Proteins; D009469:Neuromuscular Junction / Q000187:drug effects; D010243:Paralysis / Q000235:genetics*; D050600:SNARE Proteins; D013347:Subcellular Fractions / Q000737:chemistry / Q000378:metabolism; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D008565:Membrane Proteins; D050600:SNARE Proteins; D033921:Vesicular Transport Proteins; C498732:comt protein, Drosophila; D052066:N-Ethylmaleimide-Sensitive Proteins