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

Exocytosis requires the fusion of vesicular and plasma membranes and results in the release of vesicle contents into the extracellular space. This membrane fusion event is mediated in large part by specific interactions of proteins on the vesicle membrane (v-SNAREs) with proteins on the target membrane (t-SNAREs) (6xRothman, J.E. Nature. 1994; 372: 55–63CrossRef | PubMed | Scopus (1801)See all References, 1xBajjalieh, S.M. and Scheller, R.H. J. Biol. Chem. 1995; 270: 1971–1974CrossRef | PubMed | Scopus (165)See all References, 7xSudhof, T.C. Nature. 1995; 375: 645–653CrossRef | PubMedSee all References). In neurotransmitter release, for example, the vesicle protein VAMP (also called synaptobrevin) forms a tight SDS-resistant protein complex with two target membrane proteins: syntaxin and SNAP-25. This trimeric protein complex has been proposed to constitute part of the minimal machinery required for membrane fusion (Jahn and Hanson 1998xJahn, R. and Hanson, P.I. Nature. 1998; 393: 14–15CrossRef | PubMed | Scopus (71)See all ReferencesJahn and Hanson 1998).SNARE complexes assemble into a four-helix bundle formed by the 70 membrane-proximal residues of SNARE proteins. The minimal core consists of two helical domains from SNAP-25 and one from syntaxin arranged in parallel with one helical domain from VAMP (Fasshauer et al. 1998xFasshauer, D., Eliason, W.K., Brunger, A.T., and Jahn, R. Biochemistry. 1998; 37: 10354–10362CrossRef | PubMed | Scopus (175)See all ReferencesFasshauer et al. 1998). Importantly, the generation of the SNARE core complexes is highly regulated. Despite their ability to form SDS-resistant complexes, most VAMP, syntaxin, and SNAP-25 proteins are not bound to each other under steady state conditions. Instead, it appears that the availability of syntaxin and VAMP proteins to form SNARE complexes is limited by their interactions with other proteins, including Munc18 (also called rSec1) and synaptophysin, respectively. Munc18 in vitro competitively inhibits the binding of SNAP-25 or VAMP to syntaxin-1a (Pevsner et al. 1994xPevsner, J., Hsu, S.C., Braun, J.E., Calakos, N., Ting, A.E., Bennett, M.K., and Scheller, R.H. Neuron. 1994; 13: 353–361Abstract | Full Text PDF | PubMed | Scopus (473)See all ReferencesPevsner et al. 1994); yet, how these complexes are rearranged during the priming and triggering of exocytosis remains to be determined.Recently, a new syntaxin-1–binding protein, tomosyn, was shown to have the ability to displace Munc18 from syntaxin-1 and subsequently to form a new complex with syntaxin, SNAP-25 and synaptotagmin (a syntaxin-binding protein) (Fujita et al. 1998xFujita, Y., Shirataki, H., Sakisaka, T., Asakura, T., Ohya, T., Kotani, H., Yokoyama, S., Nishioka, H., Matsuura, Y., Mizoguchi, A. et al. Neuron. 1998; 20: 905–915Abstract | Full Text | Full Text PDF | PubMed | Scopus (188)See all ReferencesFujita et al. 1998). Although the exact nature of the interaction was not determined, tomosyn was found to interact with the coiled coil domain of syntaxin-1a. Furthermore, it was also suggested that tomosyn would be later replaced by VAMP to form the SNARE complex required for membrane fusion, since the characterization of the SNARE proteins present in different high molecular complexes showed that the presence of tomosyn and VAMP was mutually exclusive (Fujita et al. 1998xFujita, Y., Shirataki, H., Sakisaka, T., Asakura, T., Ohya, T., Kotani, H., Yokoyama, S., Nishioka, H., Matsuura, Y., Mizoguchi, A. et al. Neuron. 1998; 20: 905–915Abstract | Full Text | Full Text PDF | PubMed | Scopus (188)See all ReferencesFujita et al. 1998). In this way, tomosyn may play a key role in regulating the generation of fusion-competent SNARE complexes.We have started, among several approaches, to search for proteins that interact with SNARE proteins from mast cells using a yeast two-hybrid system in order to identify and map regulatory components of the exocytic process in these cells. Interestingly, using SNAP-23 (the counterpart for SNAP-25 in mast cells) as a bait, we found 3 different interacting clones among 3 × 106 clones screened which encoded the carboxyl terminus of tomosyn. Similarly, 6 different clones encoding the carboxyl terminus of tomosyn were recovered among 3 × 107 clones screened using syntaxin-4 as a bait. These results indicate that tomosyn, like VAMP, has the capacity to bind not only syntaxin-1a but also syntaxin-4 and SNAP-23. The results also show that tomosyn is expressed in mast cells, specifically the murine mast cell line MC-9, and suggest that, as in neurons and PC12 cells, tomosyn plays a role in the regulation of mast cell exocytosis.Altogether, the 9 cDNA clones encoded peptides containing the carboxy-terminal 82–142 amino acid residues of tomosyn. This defines the carboxyl terminus as the domain of tomosyn that interacts with the coiled coil domains of syntaxin and SNAP-23. Analysis of this carboxy-terminal interacting domain of tomosyn with the COILS program (21 amino acid window size) predicted a coiled coil centered on arginine 1188 with a probability of 0.9. In a blast search of the nonredundant database, two VAMP homologs are the fourth and fifth highest scores behind tomosyn itself and previously discussed tumor suppressor–related genes. In addition, the VAMP1 homology is 10 and the VAMP4 homology is 8 standard deviations above the mean randomized homology found between the sequences (Figure 1Figure 1). Comparison to several other known coiled coil sequences results in insignificant scores ranging from 0–4 standard deviations above the mean randomized homology. Interestingly, sensitive profile searches based on the coiled coil domains of the SNARE proteins have shown that all SNARE coiled coils are distantly related (Weimbs et al. 1998xWeimbs, T., Mostov, K., and Hui Low, S. Trends Cell Biol. 1998; 8: 260–262Abstract | Full Text | Full Text PDF | PubMed | Scopus (100)See all ReferencesWeimbs et al. 1998). Moreover, alignment of the coiled coil domains of the SNARE superfamily reveal the conservation of an arginine residue in the d position of a central heptad repeat in all VAMP-like SNAREs. A glutamine residue replaces the arginine in all other SNARE coiled coils (Weimbs et al. 1998xWeimbs, T., Mostov, K., and Hui Low, S. Trends Cell Biol. 1998; 8: 260–262Abstract | Full Text | Full Text PDF | PubMed | Scopus (100)See all ReferencesWeimbs et al. 1998). The tomosyn predicted coiled coil includes the conserved arginine at position d in the central heptad repeat of the α helix (Figure 1Figure 1). This finding thus argues that tomosyn, like VAMP proteins, is capable of forming trimeric complexes with t-SNAREs and is consistent with the proposed replacement of tomosyn by VAMP during the exocytic process. We propose that tomosyn is the first of a class of membrane trafficking regulators that utilizes a SNARE coil domain to substitute for one of the components of the four-helix bundle that makes up the core complex. Further characterization of the tomosyn molecule should shed light on the regulation of dissociation and association of SNARE complexes during the exocytic membrane fusion events.Figure 1Schematic Representation of Tomosyn and Sequence Alignment of Its Carboxy-Terminal Domain Residues with the Coiled Coil Sequences of Representative VAMP Family ProteinsThe amino acid sequence of the 82 residues from the smallest interacting domain of murine tomosyn was identical to the sequence from rat tomosyn (black box). Numbers indicating the position of tomosyn residues correspond to the rat sequence (ra Tomosyn, U92072). GenBank accession numbers for the other sequences are as follows: mu VAMP1, U61751; mu VAMP4, AF061516; and sc Snc1, U12980 (mu, murine; sc, S. cerevisiae). Black boxes indicate identical residues present in all sequences. White boxes indicate positions that show 75% match (3 out of 4) to similar amino acid residues. The black triangle marks the arginine (R) residue conserved in all VAMP family members. The a and d positions in the heptad repeats are marked by black dots below the alignment.View Large Image | View Hi-Res Image | Download PowerPoint Slide