Eukaryotic membrane trafficking is a conserved process under tight temporal and spatial regulation in which the fusion of membranes is driven by the formation of the ternary SNARE complex. Syntaxin 1a, a core component of the exocytic SNARE complex in neurons and neuroendocrine cells, is regulated directly by munc18-1, its cognate Sec1p/munc18 (SM) protein. SM proteins show remarkable structural conservation throughout evolution, indicating a common binding mechanism and function. However, SM proteins possess disparate binding mechanisms and regulatory effects with munc18-1, the major brain isoform, classed as atypical in both its binding specificity and its mode. We now show that munc18-1 interacts with syntaxin 1a through two mechanistically distinct modes of binding, both in vitro and in living cells, in contrast to current models. Furthermore, these functionally divergent interactions occur at distinct cellular locations. These findings provide a molecular explanation for the multiple, spatially distinct roles of munc18-1.In neuronal and neuroendocrine cells, exocytosis is mediated by the plasma membrane proteins (t-SNAREs) 2 syntaxin and SNAP-25 (synaptosome-associated protein 25 kDa) and the vesicular protein synaptobrevin (v-SNARE) (1, 2). The cytoplasmic regions of these three proteins interact to form a trimeric, four-helical complex, the generation of which drives fusion of the two opposing bilayers (3). This process is regulated by a conserved set of accessory proteins that operate throughout the trafficking pathway. The Sec1p/munc18 (SM) protein family represents one such set of modulators with SM protein mutations characterized by a severe disruption of general secretion or neurotransmitter release (4 -7). The mammalian SM protein munc18-1 was originally isolated as a syntaxin 1-binding protein that binds to the monomeric form of syntaxin 1, rendering the t-SNARE unable to form the SDS-resistant ternary SNARE complex (8, 9). Syntaxin 1 can act as a molecular switch, adopting two structurally distinct forms (10). In the open form, the SNARE helix does not interact with the N-terminal three-helical regulatory domain (termed Habc) and has been shown to not interact with munc18-1 (10). In contrast, the closed form of syntaxin 1, in which the N-terminal Habc domain interacts with the SNARE helix, exhibits a high affinity for munc18-1. Association of syntaxin 1 with its SNARE partners to form the ternary SNARE complex, which prevents syntaxin from adopting the closed conformation, has also been shown to preclude munc18-1 binding (11). However, a recent finding by Zilly et al. (12) using lysed cellular membrane sheets provided evidence that munc18-1 may interact with syntaxin 1 when in the binary SNARE complex (a heterodimer of syntaxin and SNAP-25).The interaction of munc18-1 with its cognate syntaxin is in sharp contrast to the specificity of its yeast homologue Sec1p, which binds its cognate syntaxin, Sso1p, in the ternary SNARE complex and not in the monomeric state (13). This binding specificity has, ...

Release of neurotransmitter occurs when synaptic vesicles fuse with the plasma membrane. This neuronal exocytosis is triggered by calcium and requires three SNARE (soluble-N-ethylmaleimide-sensitive factor attachment protein receptors) proteins: synaptobrevin (also known as VAMP) on the synaptic vesicle, and syntaxin and SNAP-25 on the plasma membrane. Neuronal SNARE proteins form a parallel four-helix bundle that is thought to drive the fusion of opposing membranes. As formation of this SNARE complex in solution does not require calcium, it is not clear what function calcium has in triggering SNARE-mediated membrane fusion. We now demonstrate that whereas syntaxin and SNAP-25 in target membranes are freely available for SNARE complex formation, availability of synaptobrevin on synaptic vesicles is very limited. Calcium at micromolar concentrations triggers SNARE complex formation and fusion between synaptic vesicles and reconstituted target membranes. Although calcium does promote interaction of SNARE proteins between opposing membranes, it does not act by releasing synaptobrevin from synaptic vesicle restriction. Rather, our data suggest a mechanism in which calcium-triggered membrane apposition enables syntaxin and SNAP-25 to engage synaptobrevin, leading to membrane fusion.

NMDA receptors (NMDARs) mediate ischemic brain damage, for which interactions between the C termini of NR2 subunits and PDZ domain proteins within the NMDAR signaling complex (NSC) are emerging therapeutic targets. However, expression of NMDARs in a non-neuronal context, lacking many NSC components, can still induce cell death. Moreover, it is unclear whether targeting the NSC will impair NMDAR-dependent prosurvival and plasticity signaling. We show that the NMDAR can promote death signaling independently of the NR2 PDZ ligand, when expressed in non-neuronal cells lacking PSD-95 and neuronal nitric oxide synthase (nNOS), key PDZ proteins that mediate neuronal NMDAR excitotoxicity. However, in a non-neuronal context, the NMDAR promotes cell death solely via c-Jun N-terminal protein kinase (JNK), whereas NMDAR-dependent cortical neuronal death is promoted by both JNK and p38. NMDARdependent pro-death signaling via p38 relies on neuronal context, although death signaling by JNK, triggered by mitochondrial reactive oxygen species production, does not. NMDAR-dependent p38 activation in neurons is triggered by submembranous Ca 2ϩ , and is disrupted by NOS inhibitors and also a peptide mimicking the NR2B PDZ ligand (TAT-NR2B9c). TAT-NR2B9c reduced excitotoxic neuronal death and p38-mediated ischemic damage, without impairing an NMDAR-dependent plasticity model or prosurvival signaling to CREB or Akt. TAT-NR2B9c did not inhibit JNK activation, and synergized with JNK inhibitors to ameliorate severe excitotoxic neuronal loss in vitro and ischemic cortical damage in vivo. Thus, NMDAR-activated signals comprise pro-death pathways with differing requirements for PDZ protein interactions. These signals are amenable to selective inhibition, while sparing synaptic plasticity and prosurvival signaling.

Synaptotagmins are membrane proteins that possess tandem C2 domains and play an important role in regulated membrane fusion in metazoan organisms. Here we show that both synaptotagmins I and II, the two major neuronal isoforms, can interact with the syntaxin/ synaptosomal-associated protein of 25 kDa (SNAP-25) dimer, the immediate precursor of the soluble NSF attachment protein receptor (SNARE) fusion complex. A stretch of basic amino acids highly conserved throughout the animal kingdom is responsible for this calciumindependent interaction. Inositol hexakisphosphate modulates synaptotagmin coupling to the syntaxin/ SNAP-25 dimer, which is mirrored by changes in chromaffin cell exocytosis. Our results shed new light on the functional importance of the conserved polybasic synaptotagmin motif, suggesting that synaptotagmin interacts with the t-SNARE dimer to up-regulate the probability of SNARE-mediated membrane fusion.Cell-cell communication relies on the regulated release of transmitter molecules from secretory vesicles. These vesicles fuse with the plasma membrane in a calcium-dependent manner to release the transmitter molecules (1, 2). Despite identification of the major players involved in intracellular membrane fusion (3, 4), the molecular steps leading to vesicle fusion are still not fully understood. Synaptotagmin I, a calciumphospholipid binding protein, is essential for synchronous synaptic vesicle exocytosis, whereas membrane fusion itself relies on the three SNARE 1 proteins: synaptobrevin, also known as vesicle-associated membrane protein or VAMP, on the vesicular membrane, and syntaxin and SNAP-25, on the target plasma membrane (5-8). The three SNARE proteins form a four-helical bundle that likely drives membrane fusion (9), with the syntaxin/SNAP-25 dimer (t-SNARE dimer) being an important intermediate in this process (10, 11).Current models of calcium-triggered exocytosis depict the calcium sensor, synaptotagmin, being physically linked to the SNARE fusion machinery in anticipation of the calcium entry (12-15). Indeed, many independent studies have demonstrated that synaptotagmin I, the major brain isoform, can interact specifically with the neuronal SNAREs in the absence of calcium, as evidenced by pull-downs and affinity chromatography approaches followed by Coomassie staining (16 -20). Using the brain-purified SNAREs, syntaxin1 and SNAP-25, we were recently able to show that synaptotagmin I binds specifically and with high affinity the t-SNARE dimer (but not the monomeric SNARE proteins) in a calcium-independent manner (18). Furthermore, we found that the majority of syntaxin and SNAP-25 in neuroendocrine cells likely exist as stable t-SNARE dimers on the plasma membrane (11), suggesting that interaction of vesicular synaptotagmin with this entity may take place during the SNARE-mediated fusion of the two membranes.Because it is well established that synaptotagmin I plays a critical role in SNARE-mediated membrane fusion (3, 6, 7), the molecular basis for the observed calcium-independent physic...