Abstract
Neurosecretion is critically dependent on the assembly of a macromolecular complex between the SNARE proteins syntaxin, SNAP-25 and synaptobrevin. Evidence indicates that the binding of tomosyn to syntaxin and SNAP-25 interferes with this assembly, thereby negatively regulating both synaptic transmission and peptide release. Tomosyn has two conserved domains: an N-terminal encompassing multiple WD40 repeats predicted to form two β-propeller structures and a C-terminal SNARE-binding motif. To assess the function of each domain, we performed an in vivo analysis of the N- and C- terminal domains of C. elegans tomosyn (TOM-1) in a tom-1 mutant background. We verified that both truncated TOM-1 constructs were transcribed at levels comparable to rescuing full-length TOM-1, were of the predicted size, and localized to synapses. Unlike full-length TOM-1, expression of the N- or C-terminal domains alone was unable to restore inhibitory control of synaptic transmission in tom-1 mutants. Similarly, co-expression of both domains failed to restore TOM-1 function. In addition, neither the N- nor C-terminal domain inhibited release when expressed in a wild-type background. Based on these results, we conclude that the ability of tomosyn to regulate neurotransmitter release in vivo depends on the physical integrity of the protein, indicating that both N- and C-terminal domains are necessary but not sufficient for effective inhibition of release in vivo.
Highlights
Synaptic vesicles undergo a priming process in which they become competent to fuse in response to a calcium signal [1]
Our results indicate that the integrity of C. elegans TOM-1 is critical for its inhibitory function, as neither TOM-1A SNARE nor DSNARE were able to restore TOM-1 function, when expressed separately or together in tom-1 mutants
Biochemical evidence strongly implicates the tomosyn SNARE domain in the regulation of SNARE complex formation [6,12,16], the inhibitory capacity of this domain depends on the experimental context [6,17,18]
Summary
Synaptic vesicles undergo a priming process in which they become competent to fuse in response to a calcium signal [1]. Several SNARE-interacting proteins have been shown to regulate priming, including the syntaxin-binding partner tomosyn, which acts as a negative regulator [5] This conclusion is based on the inhibitory effects of tomosyn over-expression on release in several cell types [5,6,7,8] and on enhanced synaptic transmission in both C. elegans and mouse mutants [9,10,11,12]. The tomosyn SNARE domain can substitute for synaptobrevin, forming a 4-alpha helical bundle with syntaxin and SNAP-25, which closely resembles the crystal structure of the fusogenic SNARE complex [5,6,16] Based on these observations, inhibition by tomosyn is thought to involve assembly of nonfusogenic tomosyn SNARE complexes at the expense of fusogenic
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