A discontinuous SNAP-25 C-terminal coil supports exocytosis.
Membrane fusion requires the formation of four-helical bundles comprised of the SNARE proteins syntaxin, vesicle-associated membrane protein (VAMP), and the synaptosomal-associated protein of 25 kDa (SNAP-25). Botulinum neurotoxin E cleaves the C-terminal coil of SNAP-25, inhibiting exocytosis of norepinephrine from permeabilized PC12 cells. Addition of a 26-mer peptide comprising the C terminus of SNAP-25 that is cleaved by the toxin restores exocytosis, demonstrating that continuity of the SNAP-25 C-terminal helix is not critical for its function. By contrast, vesicle-associated membrane protein peptides could not rescue botulinum neurotoxin D-treated cells, suggesting that helix continuity is critical for VAMP function. Much higher concentrations of the SNAP-25 C-terminal peptide are required for rescuing exocytosis (K(assembly) = approximately 460 microm) than for binding to other SNAREs in vitro (Kd < 5 microm). Each residue of the peptide was mutated to alanine to assess its functional importance. Whereas most mutants rescue exocytosis with lower efficiency than the wild type peptide, D186A rescues with higher efficiency, and kinetic analysis suggests this is because of higher affinity for the cellular binding site. This is consistent with Asp-186 contributing to negative regulation of the fusion process.
Chen
Y A
YA
Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, California 94305-5345, USA.
Scales
S J
SJ
Jagath
J R
JR
Scheller
R H
RH
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D001905:Botulinum Toxins / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005089:Exocytosis; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000502:physiology*; D009638:Norepinephrine / Q000378:metabolism; D016716:PC12 Cells; D010455:Peptides / Q000737:chemistry; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D013696:Temperature; D013997:Time Factors; D033921:Vesicular Transport Proteins
D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010455:Peptides; D011994:Recombinant Proteins; D050600:SNARE Proteins; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins; D001224:Aspartic Acid; D001905:Botulinum Toxins; D000409:Alanine; C096326:botulinum toxin type E; D009638:Norepinephrine