A multigene family encoding R-SNAREs in the ciliate Paramecium tetraurelia

SNARE proteins (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) mediate membrane interactions and are conventionally divided into Q-SNAREs and R-SNAREs according to the possession of a glutamine or arginine residue at the core of their SNARE domain. Here, we describe a set of R-SNAREs from the ciliate Paramecium tetraurelia consisting of seven families encoded by 12 genes that are expressed simultaneously. The complexity of the endomembrane system in Paramecium can explain this high number of genes. All P. tetraurelia synaptobrevins (PtSybs) possess a SNARE domain and show homology to the Longin family of R-SNAREs such as Ykt6, Sec22 and tetanus toxin-insensitive VAMP (TI-VAMP). We localized four exemplary PtSyb subfamilies with GFP constructs and antibodies on the light and electron microscopic level. PtSyb1-1, PtSyb1-2 and PtSyb3-1 were found in the endoplasmic reticulum, whereas PtSyb2 is localized exclusively in the contractile vacuole complex. PtSyb6 was found cytosolic but also resides in regularly arranged structures at the cell cortex (parasomal sacs), the cytoproct and oral apparatus, probably representing endocytotic compartments. With gene silencing, we showed that the R-SNARE of the contractile vacuole complex, PtSyb2, functions to maintain structural integrity as well as functionality of the osmoregulatory system but also affects cell division.

Molecular identification of 26 syntaxin genes and their assignment to the different trafficking pathways in Paramecium

SNARE proteins have been classified as vesicular (v)- and target (t)-SNAREs and play a central role in the various membrane interactions in eukaryotic cells. Based on the Paramecium genome project, we have identified a multigene family of at least 26 members encoding the t-SNARE syntaxin (PtSyx) that can be grouped into 15 subfamilies. Paramecium syntaxins match the classical build-up of syntaxins, being 'tail-anchored' membrane proteins with an N-terminal cytoplasmic domain and a membrane-bound single C-terminal hydrophobic domain. The membrane anchor is preceded by a conserved SNARE domain of approximately 60 amino acids that is supposed to participate in SNARE complex assembly. In a phylogenetic analysis, most of the Paramecium syntaxin genes were found to cluster in groups together with those from other organisms in a pathway-specific manner, allowing an assignment to different compartments in a homology-dependent way. However, some of them seem to have no counterparts in metazoans. In another approach, we fused one representative member of each of the syntaxin isoforms to green fluorescent protein and assessed the in vivo localization, which was further supported by immunolocalization of some syntaxins. This allowed us to assign syntaxins to all important trafficking pathways in Paramecium.