These authors contributed equally to this work 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 membranebound single C-terminal hydrophobic domain. The membrane anchor is preceded by a conserved SNARE domain of $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.

No abstract

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 facilitated by the availability of specific inhibitors, e.g., by Clostridium botulinum neurotoxins (BoNTs), that specifically cleave certain neuronal SNAREs (46). BoNTs are zinc-dependent proteases which, by cleaving SNARE proteins, inhibit neurotransmitter release. The structural basis for the specificity of SNAP-25 cleavage by BoNT/A and BoNT/E has been solved, and the interacting amino acid...

No abstract

3,3'-Dihydroxyisorenieratene (DHIR) is a structurally unusual carotenoid exhibiting bifunctional antioxidant properties. It is synthesized by Brevibacterium linens, used in dairy industry for the production of red smear cheeses. The compound protects cellular structures against photo-oxidative damage and inhibits the UV-dependent formation of thymidine dimers. Here we show that DHIR prevents a UV-induced intracellular release of zinc ions from proteins in human dermal fibroblasts. The effect is correlated with a decreased formation of intracellular reactive oxygen species. In contrast, zinc release from cellular proteins induced by hyperthermia is not affected by pretreatment of cells with the antioxidant DHIR. It is suggested that the intracellular zinc release upon UV irradiation is due to oxidative modifications of the zinc ligands in proteins (e.g. cysteine) and that protection by DHIR is due to intracellular scavenging of reactive oxygen species generated in photo-oxidation.