A New Syntaxin Family Member Implicated in Targeting of Intracellular Transport Vesicles

Abstract

Despite the central role vesicular trafficking occupies in protein targeting, the molecular coding of the trafficking signals and the mechanism of vesicle docking and fusion are just beginning to be understood. We report here the cloning and initial characterization of a new member of the syntaxin family of vesicular transport receptors. Syntaxin 6 is a 255-amino acid protein with two domains predicted to form coiled-coils, as well as a carboxyl-terminal membrane anchor. Syntaxin 6 is broadly expressed and localizes in the region of the Golgi apparatus. In vitro binding studies established that syntaxin 6 binds to ␣-soluble NSF attachment protein (␣-SNAP). The sequence homology, topology, localization, and ␣-SNAP binding suggest that syntaxin 6 is involved in intracellular vesicle trafficking.Eukaryotic cells contain highly specialized organelles that are defined by their specific protein complements. The mechanism by which cells are able to route proteins along particular pathways to these various organelles has been the focus of intense investigation. While it was theorized since the 1970's that transport vesicles mediated this process (1), it was not until recently that a convergence of genetic, biochemical, and cell biological approaches permitted significant insights into the molecular mechanisms underlying this process (2, 3). The intersection of these approaches took place at the mammalian presynaptic nerve terminal, where a biochemical model for synaptic vesicle docking and fusion has been proposed (4). This model has been put forward as a paradigm for all intracellular vesicle trafficking (5).Current models suggest that synaptic vesicle proteins (vSNAREs) 1 interact specifically with plasma membrane-localized molecules (t-SNAREs), and that these complexes act as a scaffold for the assembly of the fusion apparatus (6, 7). At the synapse a promenade of protein-protein interactions mediate the docking, priming, and fusion of synaptic vesicles (8). The first step in defining this mechanism was the identification of the proteins involved. Syntaxin 1a and SNAP-25 are the tSNAREs present on the plasma membrane, while VAMP or synaptobrevin and synaptotagmin are the v-SNAREs on synaptic vesicles (4). A complex of these four membrane-bound proteins can be isolated in vivo and reconstituted in vitro (7,9,10). Concurrent with the dissociation of synaptotagmin, this complex binds the cytosolic protein ␣-SNAP, which was originally isolated through its requirement in a reconstituted Golgi transport vesicle fusion assay (11). The model predicts that the ATPase NSF then binds ␣-SNAP and, through the hydrolysis of ATP, primes the complex for the eventual calcium influx leading to vesicle fusion. Thus, it is the interaction of the t-SNAREs with their cognate v-SNAREs, which contributes to the specificity, and it is the binding of ␣-SNAP to the members of this complex that is essential to the transition from a docking state toward a fusion competent one.The fundamental question that we begin to address in this study is: ca...

OSM1/SYP61: A Syntaxin Protein in Arabidopsis Controls Abscisic Acid–Mediated and Non-Abscisic Acid–Mediated Responses to Abiotic Stress

“…those of other gene products in the database revealed that OSM1/SYP61 shares greatest sequence similarities with t-SNARE-type syntaxins from mammals ( Figure 12B) (Jahn and Südhof, 1999;Sanderfoot et al, 2000). The OSM1/SYP61 sequence also predicts structural characteristics similar to those of the syntaxin family of vesicle transport proteins (Bock et al, 1996;Sanderfoot et al, 2000). It has a predicted transmembrane domain (outside to inside helix, from amino acids 225 to 243; 85% probability) at the C-terminal end.…”

Three-dimensional structure of the amino-terminal domain of syntaxin 6, a SNAP-25 C homolog

“…Syntaxin 6 contains an N-terminal domain followed by a SNARE motif and a single C-terminal membrane anchor. This primary structure organization, as well as significant homology to several syntaxin family members in the SNARE motif, led to the classification of this protein as a syntaxin family member (9). Moreover, syntaxin 6 coprecipitates with vps45, a sec1-like protein (8).…”

“…Syntaxin 6 also shares significant sequence homology with the C-terminal SNARE region of SNAP-25 (9). A recent genomescale comparison of SNARE-motif sequences, coupled with the constraint that the four SNARE families be roughly equal in size, placed the syntaxin 6 SNARE motif in the S25C family rather than the syntaxin family (5).…”

Syntaxin 12, a Member of the Syntaxin Family Localized to the Endosome

“…Its localization to the plasma membrane and its interaction with the synaptic vesicle v-SNARE synaptobrevin point to its function as a t-SNARE. Subsequently, a family of syntaxin-related molecules that shares 23-84% amino acid identity has been identified (6,9). These syntaxins are more ubiquitous in their expressions in various tissues, which is indicative of their possible functions in other vesicular transport steps in the cell.…”

“…These syntaxins also display a variety of cellular localizations within the secretory pathway. Whereas syntaxins 2, 3, and 4 are apparently cell surface proteins (6, 10 -11), syntaxin 5 and syntaxin 6 are localized to the Golgi region (6,9).…”