Now that whole genome sequences are available for many eukaryotic organisms from yeast to man, we can form broad hypotheses on the basis of the relative expansion of protein families. To investigate the molecular mechanisms responsible for the organization of membrane compartments, we identified members of the SNARE, coat complex, Rab and Sec1 protein families in four eukaryotic genomes. Of these families only the Rab family expanded from the unicellular yeast to the multicellular fly and worm. All families were expanded in humans, where we find 35 SNAREs, 60 Rabs and 53 coat complex subunits. In addition, we were able to resolve the SNARE class of proteins into four distinct subfamilies.

Soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) proteins of the vesicle-associated membrane protein (VAMP) and syntaxin families play a central role in vesicular trafficking through the formation of complexes between proteins present on vesicle and target membranes. Formation of these complexes is proposed to mediate aspects of the specificity of vesicle trafficking and to promote fusion of the lipid bilayers. In order to further understand the molecular mechanisms that organize membrane compartments, we have characterized seven new mammalian proteins of the VAMP and syntaxin families. The proteins are broadly expressed; however, syntaxin 13 is enriched in brain and VAMP 8 in kidney. The seven novel SNAREs localize in distinct patterns overlapping with Golgi, endosomal, or lysosomal markers. Our studies support the hypothesis that evolutionary radiation of these two gene families gave rise to sets of proteins whose differential expression and combinatorial associations define and organize the membrane compartments of cells.The distribution and restriction of molecules to membrane compartments is an essential process of eukaryotic cells. Distinct organelles of the secretory pathway are synthesized and maintained by budding of transport vesicles from a donor compartment followed by fusion of these vesicles with an acceptor membrane (1). The molecular mechanisms responsible for vesicle biogenesis, protein sorting, and membrane fusion are not yet fully understood. While yeast genetics, in vitro biochemistry, and studies of synaptic vesicles have identified many of the components essential for these processes (2-4), the full repertoire of important proteins and their mechanisms of action are yet to be determined. One particularly interesting issue is how a vesicle loaded with specific cargo recognizes the appropriate target. It is becoming clear that several independent mechanisms contribute to the specificity of vesicle trafficking, and it is the sum of these multiple layers of specificity that results in a process with high fidelity (5).A vesicle-target membrane recognition event mediated by interaction of integral membrane proteins of the vesicle (vSNAREs) 1 and target (t-SNAREs) membranes represents one layer of targeting specificity, acting at the final step of membrane fusion (6, 7). This process has been extensively studied in the mammalian presynaptic nerve terminal, where formation of a heterotrimeric complex between the v-SNARE, VAMPs 1 or 2, and the t-SNAREs syntaxin 1 and SNAP-25 is thought to serve as a membrane recognition mechanism and may drive fusion of the lipid bilayers (8, 9). These proteins have subsequently been found to be prototypic members of gene families that span species as well as membrane compartments (6). For example, syntaxin homologs in yeast have been localized to the Golgi (Sed5p) (10), endosomes (Pep12p) (11), lysosomes (Vam3p) (12), and the plasma membrane (Sso1p and Sso2p) (13). In particular, the SNAREs present on yeast vacuoles have been extensivel...

The specific transfer of vesicles between organelles is critical in generating and maintaining the organization of membrane compartments within cells. Syntaxin 6 is a recently discovered member of the syntaxin family, whose constituents are required components of several vesicle trafficking pathways. To better understand the function of syntaxin 6, we generated a panel of monoclonal antibodies that specifically recognize different epitopes of the protein. Immunoelectron microscopy shows syntaxin 6 primarily on the trans-Golgi network (TGN), where is partially colocalizes with the TGN adapter protein AP-1 on clathrin-coated membranes. Additional label is present on small vesicles in the vicinity of endosome-like structures. Immunoprecipitation of syntaxin 6 revealed that it is present in a complex or complexes with alpha-soluble NSF attachment protein, vesicle-associated membrane protein 2, or cellubrevin and a mammalian homologue of VPS45, which is a member of the sec1 family implicated in Golgi to prevacuolar compartment trafficking in yeast. We show that mammalian VPS45 is found in multiple tissues, is partially membrane associated, and is enriched in the Golgi region. Converging lines of evidence suggest that syntaxin 6 mediates a TGN trafficking event, perhaps targeting to endosomes in mammalian cells.

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...