[
International Worm Meeting,
2005]
The neuronal SNARE protein syntaxin (UNC-64) contains an autoinhibitory domain (the Habc domain) that binds syntaxins SNARE domain and prevents SNARE complex formation (closed syntaxin). A mutant syntaxin in which the autoinhibitory domain does not bind the SNARE domain (open syntaxin) can rescue the L1 lethal phenotype of null syntaxin mutants. However, we found that a mutant syntaxin with the autoinhibitory domain deleted (naked syntaxin) cannot rescue the syntaxin null. Naked syntaxin might fail to rescue because it does not support release, or because it supports constitutive release at lethal levels. To determine the effect on release of naked syntaxin in neurons that lack wild-type syntaxin, we rescued the L1 lethal phenotype of the syntaxin null by expressing syntaxin in a limited number of neurons. In neurons that lacked wild-type syntaxin, the presence of naked syntaxin had no effect on morphology or on synaptic vesicle localization and distribution. Constitutive release is likely to result in a reduction in synaptic vesicles. Thus, these data suggest that naked syntaxin is unable to support exocytosis. However, in the wild-type background, naked syntaxin increased Aldicarb sensitivity, showing that it can promote release in combination with full-length syntaxin. These data suggest that the Habc domain may promote release. To test the effect of the Habc domain on release, we used two mutant syntaxins: one that contained only the Habc domain (free Habc), and one that contained the Habc domain tethered to syntaxins trans-membrane domain by a bacterial alpha-helix (tethered Habc). Free Habc had no effect under any condition tested. However, tethered Habc could rescue the syntaxin null when co-expressed with naked syntaxin, but not when expressed alone. To assess synaptic function in rescued animals, we measured miniature post-synaptic potentials (minis) at the neuromuscular junction. We found that rescued animals have high levels of neurotransmitter release. Together, these results demonstrate that interactions between the Habc domain and the SNARE domain have an essential function in promoting syntaxin-mediated vesicle release. Further, these interactions can function in trans, but depend on membrane localization. We are currently testing whether the Habc domain functions during syntaxin transport and localization or at the synapse itself.