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PMID: 11726512 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

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

The EMBO journal ·Vol. 20 ·No. 23 ·2001-12-03 ·Pages 6761-71

Rao SS, Stewart BA, Rivlin PK, Vilinsky I, Watson BO, Lang C, Boulianne G, Salpeter MM, Deitcher DL

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.

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,metabolism,physiology Microscopy, Electron Models, Molecular Molecular Sequence Data Mutagenesis, Site-Directed Mutation Nerve Tissue Proteins/genetics,metabolism Neuromuscular Junction/embryology,metabolism,ultrastructure Neurons/metabolism,physiology,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
Chemicals
Drosophila Proteins Membrane Proteins Nerve Tissue Proteins Neurotransmitter Agents SNARE Proteins Snap25 protein, Drosophila Synaptosomal-Associated Protein 25 Vesicular Transport Proteins Calcium
Authors & Affiliations
9 authors, click to expand affiliations / ORCID
Rao S S
Department of Neurobiology and Behavior, Cornell University, Ithaca, NY 14853, USA.
Stewart B A
Rivlin P K
Vilinsky I
Watson B O
Lang C
Boulianne G
Salpeter M M
Deitcher D L
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Article Info
Journal
The EMBO journal
Abbr.
EMBO J
ISSN
0261-4189
DOI
10.1093/emboj/20.23.6761
Published
2001-12-03
Pages
6761-71
Language
English
Region
England
NLM ID
8208664
PMCID
PMC125330
Subset
IM
Grants
NIGMS NIH HHS · T32 GM007469 · United States
NIGMS NIH HHS · 5T32GM07469 · United States
NINDS NIH HHS · NS09315 · United States
External Links
PubMed source DOI: 10.1093/emboj/20.23.6761 PMC Full Text View journal details
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