Amisyn, a Novel Syntaxin-binding Protein That May Regulate SNARE Complex Assembly

Abstract

The regulation of SNARE complex assembly likely plays an important role in governing the specificity as well as the timing of membrane fusion. Here we identify a novel brain-enriched protein, amisyn, with a tomosynand VAMP-like coiled-coil-forming domain that binds specifically to syntaxin 1a and syntaxin 4 both in vitro and in vivo, as assessed by co-immunoprecipitation from rat brain. Amisyn is mostly cytosolic, but a fraction cosediments with membranes. The amisyn coil domain can form SNARE complexes of greater thermostability than can VAMP2 with syntaxin 1a and SNAP-25 in vitro, but it lacks a transmembrane anchor and so cannot act as a v-SNARE in this complex. The amisyn coil domain prevents the SNAP-25 C-terminally mediated rescue of botulinum neurotoxin E inhibition of norepinephrine exocytosis in permeabilized PC12 cells to a greater extent than it prevents the regular exocytosis of these vesicles. We propose that amisyn forms nonfusogenic complexes with syntaxin 1a and SNAP-25, holding them in a conformation ready for VAMP2 to replace it to mediate the membrane fusion event, thereby contributing to the regulation of SNARE complex formation.The exocytosis of synaptic and dense core vesicles with the plasma membrane in neurons and neuroendocrine cells requires proteins of the SNARE 1 families. The vesicle-associated membrane protein, VAMP2, a v-or R-SNARE, forms a specific SNARE complex with the target membrane-associated t-or Q-SNAREs syntaxin 1 and SNAP-25, whose parallel orientation brings the two membranes in close enough proximity to fuse (1, 2). Structurally the SNARE complex is a four-helix bundle comprised of one coiled-coil-forming domain from each of syntaxin and VAMP and two from 4). The center of the bundle is made up of 15 hydrophobic layers from the "a" and "d" positions of the heptad repeats of these coiled-coilforming domains, whereas the central "ionic" layer is highly conserved and polar in nature, containing a glutamine residue in the three t-SNAREs and an arginine in the v-SNARE (3), hence the classification of v-and t-SNAREs as R-and QSNAREs, respectively (5). The parallel orientation and high stability of the SNARE bundle led to the proposal that its formation drives the mixing of the membrane bilayers by zippering up through the transmembrane domains of syntaxin and VAMP (1, 2), and there is some evidence to support this (6 -9). After the fusion event, the SNAREs are in a cis-complex in the same membrane and must be dissociated by the action of the ATPase NSF and ␣-SNAP, so that SNAREs can be regenerated for the next round of fusion (10 -13).In addition to mediating the fusion event, the SNARE proteins have also been implicated in the specificity of membrane fusion, the SNARE hypothesis stating that a particular v-SNARE on a transport vesicle should only form a specific complex with its cognate t-SNARE on the correct target membrane, thereby ensuring that the vesicles only fuse with the right compartment (14). Although this hypothesis was cast into doubt by the finding that soluble ...

Tomosyn Interacts with the t-SNAREs Syntaxin4 and SNAP23 and Plays a Role in Insulin-stimulated GLUT4 Translocation

“…This complex regulates a variety of exocytic transport events including the translocation of GLUT4 to the plasma membrane of adipocytes and the translocation of water channels to the cell surface of kidney cells (23,(37)(38)(39). Tomosyn binds to Syntaxin1A via a helical domain that is homologous to the VAMP-2 SNARE motif, and a recently described molecule, Amisyn, contains a Tomosyn-like SNARE motif and has further been shown to interact with both Syntaxin1A and Syntaxin4 from rat brain (18,20,40). Therefore, we set out to investigate whether Tomosyn binds to Syntaxin4 and whether it plays a similar role in destabilizing the Syntaxin4/Munc18c complex to that observed in neurons.…”

Position effect leading to haploinsufficiency in a mosaic ring chromosome 14 in a boy with autism

“…10,16 AMISYN is a brain-enriched syntaxin-binding protein (STXBP6) interacting with t-SNAREs syntaxin-1 and SNAP-25 through its C-terminal VAMP-like coiled-coil domain. 8 As a consequence, AMISYN and v-SNARE VAMP-2 are competitive in their binding to the t-SNARE complex syntaxin-1/SNAP-25, 8 which plays an important role in vesicle fusion. However, since the unique N-terminus of AMISYN lacks the hydrophobic stretch that may serve as a transmembrane anchor to the vesicle, 8 AMISYN functions as an inhibitory SNARE (i-SNARE).…”

“…8 As a consequence, AMISYN and v-SNARE VAMP-2 are competitive in their binding to the t-SNARE complex syntaxin-1/SNAP-25, 8 which plays an important role in vesicle fusion. However, since the unique N-terminus of AMISYN lacks the hydrophobic stretch that may serve as a transmembrane anchor to the vesicle, 8 AMISYN functions as an inhibitory SNARE (i-SNARE). 17,18 Therefore, AMISYN may be involved in fine-tuning vesicle fusion and secretion of vesicle content upon receipt of the appropriate stimulus, 8,19 making it a functional candidate for autism.…”

“…However, since the unique N-terminus of AMISYN lacks the hydrophobic stretch that may serve as a transmembrane anchor to the vesicle, 8 AMISYN functions as an inhibitory SNARE (i-SNARE). 17,18 Therefore, AMISYN may be involved in fine-tuning vesicle fusion and secretion of vesicle content upon receipt of the appropriate stimulus, 8,19 making it a functional candidate for autism. However, in a cohort of 227 persons with autism, we were unable to detect mutations in the amisyn gene.…”

“…8,9 Given the evidence that genes implicated in synaptic processes, such as synaptogenesis and regulated secretion in neurons, may be involved in autism; 6,10 -14 we investigated the expression of amisyn in the patient. The patient was heterozygous for SNP rs1052484, in the 3 0 UTR (Figure 1d).…”