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(ts) are thermally stable but the mutant SNARE multimers (of approximately 120 kDa) rapidly dissociate at 37 degrees C. The SNAP-25(ts) mutant has two effects on neurotransmitter release depending upon temperature. At 22 degrees C, evoked release of neurotransmitter in SNAP-25(ts) larvae is greatly increased, and at 37 degrees C, the release of neurotransmitter is reduced as compared with controls. Our data suggest that at 22 degrees C the mutation causes the SNARE complex to be more fusion competent but, at 37 degrees C the same mutation leads to SNARE multimer instability and fusion incompetence.
Rao
S S
SS
Department of Neurobiology and Behavior, Cornell University, Ithaca, NY 14853, USA.
Stewart
B A
BA
Rivlin
P K
PK
Vilinsky
I
I
Watson
B O
BO
Lang
C
C
Boulianne
G
G
Salpeter
M M
MM
Deitcher
D L
DL
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000494:pharmacology; D003433:Crosses, Genetic; D004305:Dose-Response Relationship, Drug; D004330:Drosophila; D029721:Drosophila Proteins; D004594:Electrophysiology; D005808:Genes, Recessive; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D008854:Microscopy, Electron; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism*; D009469:Neuromuscular Junction / Q000196:embryology / Q000378:metabolism / Q000648:ultrastructure; D009474:Neurons / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D018377:Neurotransmitter Agents / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050600:SNARE Proteins; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D050825:Synaptosomal-Associated Protein 25; D013696:Temperature; D013997:Time Factors; D014170:Transformation, Genetic; D033921:Vesicular Transport Proteins
D029721:Drosophila Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D018377:Neurotransmitter Agents; D050600:SNARE Proteins; C494207:Snap25 protein, Drosophila; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins; D002118:Calcium