Botulinum neurotoxin E-insensitive mutants of SNAP-25 fail to bind VAMP but support exocytosis.
Neurotransmitter release from synaptic vesicles is mediated by complex machinery, which includes the v- and t-SNAP receptors (SNAREs), vesicle-associated membrane protein (VAMP), synaptotagmin, syntaxin, and synaptosome-associated protein of 25 kDa (SNAP-25). They are essential for neurotransmitter exocytosis because they are the proteolytic substrates of the clostridial neurotoxins tetanus neurotoxin and botulinum neurotoxins (BoNTs), which cause tetanus and botulism, respectively. Specifically, SNAP-25 is cleaved by both BoNT/A and E at separate sites within the COOH-terminus. We now demonstrate, using toxin-insensitive mutants of SNAP-25, that these two toxins differ in their specificity for the cleavage site. Following modification within the COOH-terminus, the mutants completely resistant to BoNT/E do not bind VAMP but were still able to form a sodium dodecyl sulfate-resistant complex with VAMP and syntaxin. Furthermore, these mutants retain function in vivo, conferring BoNT/E-resistant exocytosis to transfected PC12 cells. These data provide information on structural requirements within the C-terminal domain of SNAP-25 for its function in exocytosis and raise doubts about the significance of in vitro binary interactions for the in vivo functions of synaptic protein complexes.
Washbourne
P
P
Centro CNR Biomembrane and Department of Biomedical Sciences, University of Padova, Italy.
Bortoletto
N
N
Graham
M E
ME
Wilson
M C
MC
Burgoyne
R D
RD
Montecucco
C
C
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D001905:Botulinum Toxins / Q000494:pharmacology*; D019556:COS Cells; D002135:Calcium-Binding Proteins; D002522:Chlorocebus aethiops; D004351:Drug Resistance; D005089:Exocytosis / Q000187:drug effects*; D013006:Growth Hormone / Q000378:metabolism; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D016716:PC12 Cells; D011485:Protein Binding; D050683:R-SNARE Proteins; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013572:Synaptic Vesicles / Q000187:drug effects / Q000378:metabolism; D050825:Synaptosomal-Associated Protein 25; D050857:Synaptotagmins
D002135:Calcium-Binding Proteins; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; D050857:Synaptotagmins; D012967:Sodium Dodecyl Sulfate; D013006:Growth Hormone; D001905:Botulinum Toxins; C096326:botulinum toxin type E