In vivo analysis of conserved C. elegans tomosyn domains.
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.
Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois, United States of America.
Anna O
AO
Burdina
Susan M
SM
Klosterman
Ludmila
L
Shtessel
Shawn
S
Ahmed
Janet E
JE
Richmond
D000818:Animals; D030801:Animals, Genetically Modified; D017173:Caenorhabditis elegans / Q000235:genetics / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry* / Q000378:metabolism*; D017124:Conserved Sequence; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D050600:SNARE Proteins / Q000378:metabolism; D013569:Synapses / Q000378:metabolism
D029742:Caenorhabditis elegans Proteins; D050505:Mutant Proteins; D050600:SNARE Proteins; C513301:tom-1 protein, C elegans