Molecular identification of a SNAP-25-like SNARE protein in Paramecium.

Using database searches of the completed Paramecium tetraurelia macronuclear genome with the metazoan SNAP-25 homologues, we identified a single 21-kDa Qb/c-SNARE in this ciliated protozoan, named P. tetraurelia SNAP (PtSNAP), containing the characteristic dual heptad repeat SNARE motifs of SNAP-25. The presence of only a single Qb/c class SNARE in P. tetraurelia is surprising in view of the multiple genome duplications and the high number of SNAREs found in other classes of this organism. As inferred from the subcellular localization of a green fluorescent protein (GFP) fusion construct, the protein is localized on a variety of intracellular membranes, and there is a large soluble pool of PtSNAP. Similarly, the PtSNAP that is detected with a specific antibody in fixed cells is associated with a number of intracellular membrane structures, including food vacuoles, the contractile vacuole system, and the sites of constitutive endo- and exocytosis. Surprisingly, using gene silencing, we could not assign a role to PtSNAP in the stimulated exocytosis of dense core vesicles (trichocysts), but we found an increased number of food vacuoles in PtSNAP-silenced cells. In conclusion, we identify PtSNAP as a Paramecium homologue of metazoan SNAP-25 that shows several divergent features, like resistance to cleavage by botulinum neurotoxins. Membrane trafficking in eukaryotic cells involves budding of vesicles from a donor compartment and transport to and fusion with the acceptor compartment. The soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are of central importance in the mediation of membrane fusions (32). The crystal structure of the synaptic SNARE complex has been resolved (70). The ternary synaptic SNARE complex consists of the SNARE motifs of synaptobrevin-2 (VAMP2) and syntaxin-1A and the two SNARE motifs from the synaptosome-associated protein of 25 kDa (SNAP-25). Structures of different SNARE complexes revealed a highly conserved four-helix structure, with the difference that the positions of the two SNARE motifs from SNAP-25 can be contributed by two different SNARE proteins (7). The highly conserved pattern of SNARE pairing has led to the so-called 3Q-plus-1R rule (21). According to this rule, fusogenic SNARE complexes always contain three SNARE motifs containing a glutamine residue in the center of the SNARE motif (Q-SNARE) and one SNARE displaying an arginine at the same position (R-SNARE). Furthermore, Qa-, Qb-, Qc-, and R-SNAREs can be recognized by specific sequence features (40). Identification of the SNARE components of the synaptic SNARE complex and functional analysis have been greatly