The SNARE protein SNAP-25 is linked to fast calcium triggering of exocytosis (original) (raw)
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Structural determinants for SNARE-mediated neurosecretion
2010
14 synaptobrevin2) and the synaptic plasma membrane proteins syntaxin1A and SNAP-25 [44-47]. A molecular model of SNARE-mediated vesicle exocytosis has emerged within the last 30 years [2, 48] (Figure 4). This model of regulated exocytosis consists of a series of transition steps that are controlled by additional late regulatory proteins including synaptotagmin, complexin, tomosyn, Munc-13, syntaphilin and snapin [49-55]. The process begins when syntaxin1A and SNAP-25, which are organized in clusters at the plasma membrane [56, 57], assemble together to form a binary complex called acceptor complex [58-60]. The acceptor complex provides a binding interface for the vesicular SNARE VAMP2, thus forming a ternary complex. Figure 4. Model for SNARE-mediated neuronal exocytosis. The neuronal t-SNAREs SNAP-25 and syntaxin1A (labelled in green and red, respectively), assemble together to form the acceptor complex followed by binding of the v-SNARE partner VAMP2 (in blue). The three SNARE proteins form the trans-SNARE complex that brings opposing membranes into close proximity awaiting a Ca 2+ signal. Additional proteins such as synaptotagmin (purple) and complexin (pink) bind to this trans-SNARE complex with possibly distinct outcomes. Ca 2+ entry triggers membrane fusion, followed by the generation of cis-SNARE complexes, which are disassembled by NSF and α-SNAP upon ATP-hydrolysis This ternary complex proceeds from a loose state (in which only the Nterminal part is assembled) as studied in vivo [61-63] and in vitro [64] to a tight Ca + Calcium influx Membrane fusion Synaptobrevin/Vamp
Journal of Cell Biology, 1998
Cortical vesicles (CV) possess components critical to the mechanism of exocytosis. The homotypic fusion of CV centrifuged or settled into contact has a sigmoidal Ca 2 ϩ activity curve comparable to exocytosis (CV-PM fusion). Here we show that Sr 2 ϩ and Ba 2 ϩ also trigger CV-CV fusion, and agents affecting different steps of exocytotic fusion block Ca 2 ϩ , Sr 2 ϩ , and Ba 2 ϩtriggered CV-CV fusion. The maximal number of active fusion complexes per vesicle, Max , was quantified by NEM inhibition of fusion, showing that CV-CV fusion satisfies many criteria of a mathematical analysis developed for exocytosis. Both Max and the Ca 2 ϩ sensitivity of fusion complex activation were comparable to that determined for CV-PM fusion. Using Ca 2 ϩinduced SNARE complex disruption, we have analyzed the relationship between membrane fusion (CV-CV and CV-PM) and the SNARE complex. Fusion and complex disruption have different sensitivities to Ca 2 ϩ , Sr 2 ϩ , and Ba 2 ϩ , the complex remains Ca 2 ϩ -sensitive on fusion-incompetent CV, and disruption does not correlate with the quantified activation of fusion complexes. Under conditions which disrupt the SNARE complex, CV on the PM remain docked and fusion competent, and isolated CV still dock and fuse, but with a markedly reduced Ca 2 ϩ sensitivity. Thus, in this system, neither the formation, presence, nor disruption of the SNARE complex is essential to the Ca 2 ϩ -triggered fusion of exocytotic membranes. Therefore the SNARE complex alone cannot be the universal minimal fusion machine for intracellular fusion. We suggest that this complex modulates the Ca 2 ϩ sensitivity of fusion.
The primed SNARE–complexin–synaptotagmin complex for neuronal exocytosis
Nature, 2017
Synaptotagmin, complexin and neuronal SNARE proteins mediate evoked synchronous neurotransmitter release, but the molecular mechanisms mediating the cooperation between these molecules remain unclear. Here, we determined crystal structures of the primed pre-fusion SNARE-complexin-synaptotagmin-1 complex. These structures reveal an unexpected tripartite interface between synaptotagmin-1 and both the SNARE complex and complexin. Simultaneously, a second synaptotagmin-1 molecule interacted with the other side of the SNARE complex via the previously identified primary interface. Mutations that disrupt either interface in solution also severely impaired evoked synchronous release in neurons, suggesting that both interfaces are essential for the primed pre-fusion state. Ca 2+ binding to the synaptotagmin-1 molecules unlocks the complex, allows full zippering of the SNARE complex, and triggers membrane fusion. The tripartite SNARE-complexin-synaptotagmin-1 complex at a synaptic vesicle docking site has to be unlocked for triggered fusion to commence, explaining the cooperation between complexin and synaptotagmin-1 in synchronizing evoked release on the sub-millisecond timescale. During synaptic transmission, Ca 2+ influx into a presynaptic terminal triggers fusion of neurotransmitter-filled synaptic vesicles with the presynaptic plasma membrane 1,2. The SNARE (for Soluble N-ethylmaleimide sensitive factor Attachment protein REceptor) proteins synaptobrevin-2/VAMP2 on the synaptic vesicle and syntaxin-1A and SNAP-25 on the plasma membrane initiate vesicle fusion by forming a trans-SNARE complex before Ca 2+ triggering 3,4. In addition to SNAREs, synaptotagmin-1 (Syt1) is vital for Ca 2+triggered synaptic vesicle fusion 5,6. Syt1 contains a single transmembrane-spanning domain Reprints and permissions information is available at www.nature.com/reprints.
A novel site of action for alpha-SNAP in the SNARE conformational cycle controlling membrane fusion
Molecular biology of the cell, 2008
Regulated exocytosis in neurons and neuroendocrine cells requires the formation of a stable soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex consisting of synaptobrevin-2/vesicle-associated membrane protein 2, synaptosome-associated protein of 25 kDa (SNAP-25), and syntaxin 1. This complex is subsequently disassembled by the concerted action of alpha-SNAP and the ATPases associated with different cellular activities-ATPase N-ethylmaleimide-sensitive factor (NSF). We report that NSF inhibition causes accumulation of alpha-SNAP in clusters on plasma membranes. Clustering is mediated by the binding of alpha-SNAP to uncomplexed syntaxin, because cleavage of syntaxin with botulinum neurotoxin C1 or competition by using antibodies against syntaxin SNARE motif abolishes clustering. Binding of alpha-SNAP potently inhibits Ca(2+)-dependent exocytosis of secretory granules and SNARE-mediated liposome fusion. Membrane clustering and inhibition of both exocy...
v-SNAREs control exocytosis of vesicles from priming to fusion
The EMBO Journal, 2005
SNARE proteins (soluble NSF-attachment protein receptors) are thought to be central components of the exocytotic mechanism in neurosecretory cells, but their precise function remained unclear. Here, we show that each of the vesicle-associated SNARE proteins (v-SNARE) of a chromaffin granule, synaptobrevin II or cellubrevin, is sufficient to support Ca 2 þ -dependent exocytosis and to establish a pool of primed, readily releasable vesicles. In the absence of both proteins, secretion is abolished, without affecting biogenesis or docking of granules indicating that v-SNAREs are absolutely required for granule exocytosis. We find that synaptobrevin II and cellubrevin differentially control the pool of readily releasable vesicles and show that the v-SNARE's amino terminus regulates the vesicle's primed state. We demonstrate that dynamics of fusion pore dilation are regulated by v-SNAREs, indicating their action throughout exocytosis from priming to fusion of vesicles.
SNARE Conformational Changes that Prepare Vesicles for Exocytosis
Cell Metabolism, 2010
When cells release hormones and neurotransmitters through exocytosis, cytosolic Ca 2+ triggers the fusion of secretory vesicles with the plasma membrane. It is well known that this fusion requires assembly of a SNARE protein complex. However, the timing of SNARE assembly relative to vesicle fusion-essential for understanding exocytosis-has not been demonstrated. To investigate this timing, we constructed a probe that detects the assembly of two plasma membrane SNAREs, SNAP25 and syntaxin-1A, through fluorescence resonance energy transfer (FRET). With two-photon imaging, we simultaneously measured FRET signals and insulin exocytosis in b cells from the pancreatic islet of Langerhans. In some regions of the cell, we found that the SNARE complex was preassembled, which enabled rapid exocytosis. In other regions, SNARE assembly followed Ca 2+ influx, and exocytosis was slower. Thus, SNARE proteins exist in multiple stable preparatory configurations, from which Ca 2+ may trigger exocytosis through distinct mechanisms and with distinct kinetics.
Molecular biology of the cell, 2007
Synaptotagmins contain tandem C2 domains and function as Ca(2+) sensors for vesicle exocytosis but the mechanism for coupling Ca(2+) rises to membrane fusion remains undefined. Synaptotagmins bind SNAREs, essential components of the membrane fusion machinery, but the role of these interactions in Ca(2+)-triggered vesicle exocytosis has not been directly assessed. We identified sites on synaptotagmin-1 that mediate Ca(2+)-dependent SNAP25 binding by zero-length cross-linking. Mutation of these sites in C2A and C2B eliminated Ca(2+)-dependent synaptotagmin-1 binding to SNAREs without affecting Ca(2+)-dependent membrane binding. The mutants failed to confer Ca(2+) regulation on SNARE-dependent liposome fusion and failed to restore Ca(2+)-triggered vesicle exocytosis in synaptotagmin-deficient PC12 cells. The results provide direct evidence that Ca(2+)-dependent SNARE binding by synaptotagmin is essential for Ca(2+)-triggered vesicle exocytosis and that Ca(2+)-dependent membrane binding...