In vivo analysis of conserved C. elegans tomosyn domains

• Published in: PloS one 2011-10, Vol.6 (10), p.e26185-e26185
• Main Authors: Burdina, Anna O, Klosterman, Susan M, Shtessel, Ludmila, Ahmed, Shawn, Richmond, Janet E
• Format: Article
• Language: English
• Subjects: Analysis; Animals; Animals, Genetically Modified; Assembly; Binding; Biology; Caenorhabditis elegans - genetics; Caenorhabditis elegans - metabolism; Caenorhabditis elegans Proteins - chemistry; Caenorhabditis elegans Proteins - metabolism; Conserved Sequence; Macromolecules; Mutant Proteins - chemistry; Mutant Proteins - metabolism; Mutants; Mutation - genetics; Nematodes; Neurons; Neurosecretion; Neurotransmitter release; Protein Structure, Tertiary; Protein Transport; Proteins; Recovery of function; Regulation; Rodents; SNAP receptors; SNAP-25 protein; SNARE Proteins - metabolism; Synapses; Synapses - metabolism; Synaptic transmission; Synaptobrevin; Syntaxin; Tomosyn; Trends
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0026185

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.