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

EMBO J. 2001 Dec 3;20(23):6761-71. doi: 10.1093/emboj/20.23.6761.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Blotting, Western
  • Calcium / pharmacology
  • Crosses, Genetic
  • Dose-Response Relationship, Drug
  • Drosophila
  • Drosophila Proteins
  • Electrophysiology
  • Genes, Recessive
  • Immunohistochemistry
  • Membrane Proteins / genetics*
  • Membrane Proteins / metabolism*
  • Membrane Proteins / physiology
  • Microscopy, Electron
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Mutation*
  • Nerve Tissue Proteins / genetics*
  • Nerve Tissue Proteins / metabolism*
  • Neuromuscular Junction / embryology
  • Neuromuscular Junction / metabolism
  • Neuromuscular Junction / ultrastructure
  • Neurons / metabolism
  • Neurons / physiology*
  • Neurons / ultrastructure
  • Neurotransmitter Agents / metabolism
  • Plasmids / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • SNARE Proteins
  • Sequence Analysis, DNA
  • Sequence Homology, Amino Acid
  • Synaptosomal-Associated Protein 25
  • Temperature
  • Time Factors
  • Transformation, Genetic
  • Vesicular Transport Proteins*

Substances

  • Drosophila Proteins
  • Membrane Proteins
  • Nerve Tissue Proteins
  • Neurotransmitter Agents
  • SNARE Proteins
  • Snap25 protein, Drosophila
  • Synaptosomal-Associated Protein 25
  • Vesicular Transport Proteins
  • Calcium