TheCaenorhabditis elegans unc-64Locus Encodes a Syntaxin That Interacts Genetically with Synaptobrevin

Abstract

We describe the molecular cloning and characterization of the unc-64 locus of Caenorhabditis elegans. unc-64 expresses three transcripts, each encoding a molecule with 63-64% identity to human syntaxin 1A, a membrane-anchored protein involved in synaptic vesicle fusion. Interestingly, the alternative forms of syntaxin differ only in their Cterminal hydrophobic membrane anchors. The forms are differentially expressed in neuronal and secretory tissues; genetic evidence suggests that these forms are not functionally equivalent. A complete loss-of-function mutation in unc-64 results in a worm that completes embryogenesis, but arrests development shortly thereafter as a paralyzed L1 larva, presumably as a consequence of neuronal dysfunction. The severity of the neuronal phenotypes of C. elegans syntaxin mutants appears comparable to those of Drosophila syntaxin mutants. However, nematode syntaxin appears not to be required for embryonic development, for secretion of cuticle from the hypodermis, or for the function of muscle, in contrast to Drosophila syntaxin, which appears to be required in all cells. Less severe viable unc-64 mutants exhibit a variety of behavioral defects and show strong resistance to the acetylcholinesterase inhibitor aldicarb. Extracellular physiological recordings from pharyngeal muscle of hypomorphic mutants show alterations in the kinetics of transmitter release. The lesions in the hypomorphic alleles map to the hydrophobic face of the H3 coiled-coil domain of syntaxin, a domain that in vitro mediates physical interactions with similar coiled-coil domains in SNAP-25 and synaptobrevin. Furthermore, the unc-64 syntaxin mutants exhibit allele-specific genetic interactions with mutants carrying lesions in the coiled-coil domain of synaptobrevin, providing in vivo evidence for the significance of these domains in regulating synaptic vesicle fusion. INTRODUCTIONCommunication between neurons at synaptic contacts occurs through the regulated release of chemical neurotransmitters. This release process is mediated by fusion of transmitter-filled vesicles with the plasma membrane. Through both biochemical and genetic studies, several dozen proteins have been implicated in this fusion process in neurons Sollner and Rothman, 1994;Sudhof, 1995). A central player is the integral membrane protein syntaxin (Bennett et al., 1992). In neurons, syntaxin is found in abundance on the plasma membrane and also in low levels in synaptic vesicles (Bennett et al., 1992;Galli et al., 1995;Walch-Solimena et al., 1995). A vast array of experiments all indicate that syntaxin plays a critical role in regulating neurotransmitter release in neurons. For example, serotype C neurotoxin from Clostridium botulinum, which is a potent inhibitor of synaptic transmission, is a metalloprotease with high specificity for cleaving syntaxin (Blasi et al., 1993;Schiavo et al., 1995) Moreover, physiological recordings from Drosophila syntaxin mutants reveal the absence of both induced and spontaneous fusion events, * Corresponding author.© ...

Rabphilin Potentiates SolubleN-Ethylmaleimide Sensitive Factor Attachment Protein Receptor Function Independently of rab3

“…However, the null phenotype has not been strictly defined. snb-1(md247), snb-1( js17), snb-1( js44), unc-64( js21) unc-64(e246), ric-4( js20), and ric-4(md1088) are hypomorphic alleles based on molecular, genetic, and immunohistochemical criteria Saifee et al, 1998) (Y. Lee, J. Rand, M. L. Nonet, B. J. Meyer, and J. Lee, unpublished observations). Double and triple mutants were constructed using standard methods.…”

“…Electrophysiolog y. Electropharyngeograms (EPGs) were performed as described by Saifee et al (1998). Bath solution consisted of Dent's saline with 2-5 mM serotonin to stimulate pumping.…”

“…The rab-3 phenotype is also characterized by aberrant synaptic physiology that can be assessed using the EPG, a simple extracellular recording technique developed by Raizen and Avery (1994) and Avery et al (1995). Mutations in several genes implicated in synaptic transmission significantly alter the EPG trace (Iwasaki et al, 1997;Nonet et al, 1997Nonet et al, , 1998Saifee et al, 1998). In contrast to rab-3, which exhibits EPG defects , the EPGs of most rbf-1 worms were not distinguishable from those of wild-type animals (Fig.…”

“…We paid particular attention to mutations in genes with homologs that had been implicated in docking and fusion of synaptic vesicles, including the membrane-associated SNARE proteins (Sollner et al, 1993). snb-1, ric-4, and unc-64 encode synaptobrevin/VAMP, synaptosomal-associated protein of 25 kDa (SNAP-25), and syntaxin, respectively Saifee et al, 1998) (Lee, Rand, Nonet, Meyer, and Lee, unpublished observations). We built and characterized double mutant combinations with various C. elegans SNARE homologs, as well as rab-3 and its putative regulators ae x -3 and Rim.…”

In VivoAnalysis of Membrane Fusion

“…The early steps of hemifusion require less energy than the later steps of fusion pore formation and expansion (Chernomordik and Kozlov, 2005;Cohen and Melikyan, 2004;Kuzmin et al, 2001). When the SNARE transmembrane is replaced by an artificial lipid anchor or when it is truncated, fusion is markedly reduced and in many cases no longer proceeds (Giraudo et al, 2005;Grote et al, 2000;McNew et al, 2000b;Nonet et al, 1998;Saifee et al, 1998;Xu et al, 2005). However, these perturbations do lead to a state in which lipids can exchange -a hallmark of hemifusion (Chernomordik and Kozlov, 2005;Giraudo et al, 2005;Grote et al, 2000;Xu et al, 2005).…”