SNAP-25 is a component of the membrane fusion apparatus required for neurotransmitter release. SNAP-25 has been proposed to act as a plasma membrane receptor in vesicle docking and fusion and to facilitate axonal growth during neural development. The conservation of SNAP-25 during evolution prompted us to evaluate the molecular and biological functions of SNAP-25 in C. elegans. We cloned a homolog of SNAP-25 by PCR with the degenerate primers used by C. Risinger and D. Larhammer to clone a Drosophila SNAP-25 homolog. Comparison of the amino acid sequence showed that C. elegans SNAP-25 is about 57% identical to the fly homolog and 52% identical to the human homolog. It also has limited similarity to the yeast Sec9 protein (36% identity in a stretch of 50 amino acids). We physically mapped SNAP-25 to LG V near
ric-4 by YAC grid filter hybridization. The resistance of
ric-4 mutants to aldicarb, a cholines-terase inhibitor, suggested that
ric-4 may encode SNAP-25. DNA transformation rescue experiments and DNA sequence analysis of
ric-4 mutations confirmed our surmise. The available alleles of
ric-4 do not completely eliminate gene function, since
ric-4/Df trans heterozygotes are inviable and die as uncoordinated L1s, suggesting that the null phenotype of SNAP-25 is lethality rather than simply resistance to aldicarb. The lethality of
ric-4/Df heterozygotes is rescued by a cosmid containing the SNAP-25 gene. Immunohistochemistry using polyclonal antibodies raised against C. elegans SNAP-25 showed that it is expressed in synaptic-rich regions including the nerve ring and the dorsal and ventral cords, but not secretory cells. Consistent with SNAP-25 being a plasma membrane receptor is our observation that
unc-104 mutants that prevent the transport of synaptic vesicles exhibit wild-type staining with anti-SNAP-25 antibodies. This wild-type pattern is in contrast to the pattern of
unc-104 mutants stained with antibodies to the synaptic vesicle protein synaptotagmin, which exhibit restricted staining to cell bodies rather than processes. Analysis of the partial loss of function
ric-4 mutants has thus far failed to reveal any defects in axonal outgrowth, but null mutants must be isolated and examined before such a conclusion is warranted. Efforts to identify and characterize null mutations will also be presented.