Tomosyn Binds t-SNARE Proteins via a VAMP-like Coiled Coil

nature of the interaction was not determined, tomosyn Tomosyn Binds t-SNARE Proteins was found to interact with the coiled coil domain of via a VAMP-like Coiled Coil syntaxin-1a. Furthermore, it was also suggested that tomosyn would be later replaced by VAMP to form the Exocytosis requires the fusion of vesicular and plasma SNARE complex required for membrane fusion, since the characterization of the SNARE proteins present in membranes and results in the release of vesicle contents into the extracellular space. This membrane fusion event different high molecular complexes showed that the presence of tomosyn and VAMP was mutually exclusive is mediated in large part by specific interactions of proteins on the vesicle membrane (v-SNAREs) with proteins (Fujita et al., 1998). In this way, tomosyn may play a key role in regulating the generation of fusion-competent on the target membrane (t-SNAREs) (Rothman, 1994; Bajjalieh and Scheller, 1995; Sudhof, 1995). In neuroSNARE complexes. We have started, among several approaches, to transmitter release, for example, the vesicle protein VAMP (also called synaptobrevin) forms a tight SDSsearch for proteins that interact with SNARE proteins from mast cells using a yeast two-hybrid system in order resistant protein complex with two target membrane proteins: syntaxin and SNAP-25. This trimeric protein to identify and map regulatory components of the exocytic process in these cells. Interestingly, using SNAPcomplex has been proposed to constitute part of the minimal machinery required for membrane fusion (Jahn 23 (the counterpart for SNAP-25 in mast cells) as a bait, we found 3 different interacting clones among 3 3 10 and Hanson, 1998). SNARE complexes assemble into a four-helix bundle clones screened which encoded the carboxyl terminus of tomosyn. Similarly, 6 different clones encoding the formed by the 70 membrane-proximal residues of SNARE proteins. The minimal core consists of two helical docarboxyl terminus of tomosyn were recovered among 3 3 10 clones screened using syntaxin-4 as a bait. mains from SNAP-25 and one from syntaxin arranged in parallel with one helical domain from VAMP (FassThese results indicate that tomosyn, like VAMP, has the capacity to bind not only syntaxin-1a but also synhauer et al., 1998). Importantly, the generation of the SNARE corecomplexes ishighly regulated. Despite their taxin-4 and SNAP-23. The results also show that tomosyn is expressed in mast cells, specifically the murine ability to form SDS-resistant complexes, most VAMP, syntaxin, and SNAP-25 proteins are not bound to each mast cell line MC-9, and suggest that, as in neurons and PC12 cells, tomosyn plays a role in the regulation other under steady state conditions. Instead, it appears that the availability of syntaxin and VAMP proteins to of mast cell exocytosis. Altogether, the 9 cDNA clones encoded peptides conform SNARE complexes is limited by their interactions with other proteins, including Munc18 (also called rSec1) taining thecarboxy-terminal 82–142 amino acid residues of tomosyn. This defines the carboxyl terminus as the and synaptophysin, respectively. Munc18 in vitro competitively inhibits the binding of SNAP-25 or VAMP to domain of tomosyn that interacts with the coiled coil domains of syntaxin and SNAP-23. Analysis of this carsyntaxin-1a (Pevsner et al., 1994); yet, how these complexes are rearranged during the priming and triggering boxy-terminal interacting domain of tomosyn with the COILS program (21 amino acid window size) predicted of exocytosis remains to be determined. Recently, a new syntaxin-1–binding protein, tomosyn, a coiled coil centered on arginine 1188 with a probability of 0.9. In a blast search of the nonredundant database, was shown to have the ability to displace Munc18 from syntaxin-1 and subsequently to form a new complex two VAMP homologs are the fourth and fifth highest scores behind tomosyn itself and previously discussed with syntaxin, SNAP-25 and synaptotagmin (a syntaxinbinding protein) (Fujita et al., 1998). Although the exact tumor suppressor–related genes. In addition, the VAMP1