SNAREpin Assembly by Munc18-1 Requires Previous Vesicle Docking by Synaptotagmin 1

159

The synaptic vesicle protein synaptotagmin-1 (SYT) is required to couple calcium influx to the membrane fusion machinery. However, the structural mechanism underlying this process is unclear.Here we report an unexpected circular arrangement (ring) of SYT's cytosolic domain (C2AB) formed on lipid monolayers in the absence of free calcium ions as revealed by electron microscopy. Rings vary in diameter from 18-43 nm, corresponding to 11-26 molecules of SYT. Continuous stacking of the SYT rings occasionally converts both lipid monolayers and bilayers into protein-coated tubes. Helical reconstruction of the SYT tubes shows that one of the C2 domains (most likely C2B, based on its biochemical properties) interacts with the membrane and is involved in ring formation, and the other C2 domain points radially outward. SYT rings are disrupted rapidly by physiological concentrations of free calcium but not by magnesium. Assuming that calcium-free SYT rings are physiologically relevant, these results suggest a simple and novel mechanism by which SYT regulates neurotransmitter release: The ring acts as a spacer to prevent the completion of the soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) complex assembly, thereby clamping fusion in the absence of calcium. When the ring disassembles in the presence of calcium, fusion proceeds unimpeded. S ynaptotagmin-1 (SYT) is the calcium (Ca 2+ ) sensor that triggers synchronous release of neurotransmitters for synaptic transmission (1-4). It is a transmembrane protein, localized to the synaptic vesicles (1, 5), with tandem cytosolic C2 domains (C2A and C2B) that bind phospholipids in both a Ca 2+ -independent and a Ca 2+ -dependent manner (1, 6). The membrane-distal C2B domain interacts with acidic lipids such as phosphatidylserine (PS) and phosphatidylinositol 4,5-bisphosphate (PIP 2 ) to mediate efficient docking of the synaptic vesicles (7-10) before the influx of Ca 2+ ions. Recent studies (7,8,11,12) have located this calciumindependent membrane-binding site to a polybasic patch on the C2B domain (site I in Fig. 1A). The binding of Ca 2+ to the calcium-coordination pocket of the C2B domain (site II in Fig. 1A) triggers the insertion of flanking portions of site II into the plasma membrane, and this Ca 2+ -triggered membrane insertion is absolutely required for neurotransmitter release (13-16).The C2B domain also mediates the Ca 2+ -independent binding of SYT to neuronal soluble N-ethylmaleimide-sensitive factor activating protein receptor on the plasma membrane (t-SNARE) [syntaxin/ synaptosomal-associated protein 25 (SNAP25)], most likely via its interaction with SNAP25 (17-21). This interaction is believed to position SYT on the prefusion SNARE complexes to trigger rapid exocytosis in response to Ca 2+ (20,22). How the insertion of site II into the membrane bilayer is coupled to the completion of the SNARE assembly to release neurotransmitter is still unclear, although some key points have been established. In the prefusion state, the SNARE complexes...

Section: Discussionmentioning

confidence: 55%

mentioning

confidence: 99%

Calcium sensitive ring-like oligomers formed by synaptotagmin

Bello¹,

Auclair²,

Wang³

et al. 2014

159

“…These results suggest that the Munc18-2 R65Q mutant stabilizes the assembly of SNARE complexes at an intermediate state before lipid mixing and interferes with complete zippering of the SNARE complex. Therefore, the Munc18-2 R65Q mutant seems to dissociate two distinct functions attributed to SM proteins: the stimulatory effect on membrane fusion observed in ensemble lipid mixing assays (43,53,54) and the inhibitory function recently described in single molecule experiments (55). Order-of-addition experiments show that Munc18-2 accelerates the formation cdV8-resistant SNARE complexes, therefore supporting a role of Munc18-2 in proof-reading trans-SNARE complexes (52,(55)(56)(57).…”

Section: Discussionmentioning

confidence: 93%

SM protein Munc18-2 facilitates transition of Syntaxin 11-mediated lipid mixing to complete fusion for T-lymphocyte cytotoxicity

Spessott

Sanmillan

McCormick

et al. 2017

The atypical lipid-anchored Syntaxin 11 (STX11) and its binding partner, the Sec/Munc (SM) protein Munc18-2, facilitate cytolytic granule release by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. Patients carrying mutations in these genes develop familial hemophagocytic lymphohistiocytosis, a primary immunodeficiency characterized by impaired lytic granule exocytosis. However, whether a SNARE such as STX11, which lacks a transmembrane domain, can support membrane fusion in vivo is uncertain, as is the precise role of Munc18-2 during lytic granule exocytosis. Here, using a reconstituted "flipped" cell-cell fusion assay, we show that lipidanchored STX11 and its cognate SNARE proteins mainly support exchange of lipids but not cytoplasmic content between cells, resembling hemifusion. Strikingly, complete fusion is stimulated by addition of wild-type Munc18-2 to the assay, but not of Munc18-2 mutants with abnormal STX11 binding. Our data reveal that Munc18-2 is not just a chaperone of STX11 but also directly contributes to complete membrane merging by promoting SNARE complex assembly. These results further support the concept that SM proteins in general are part of the core fusion machinery. This fusion mechanism likely contributes to other cell-type-specific exocytic processes such as platelet secretion.SNARE | Syntaxin 11 | Munc18-2 | CTL | SM protein

“…This function is coupled to membrane fusion through the neuronal soluble N-ethylmaleimide sensitive factor adaptor protein receptor (SNARE) proteins (6,7), which bring the synaptic vesicle and plasma membranes together by forming SNARE complexes (2,3). Synaptotagmin-1 function also depends on a tight interplay with complexins (8)(9)(10) and other key proteins of the release machinery (11,12). The synaptotagmin-1 C 2 domains bind three or two Ca 2+ ions through loops at the top of β-sandwich structures (13)(14)(15) (Fig.…”

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confidence: 99%

Prevalent mechanism of membrane bridging by synaptotagmin-1

Seven¹,

Brewer²,

Shi

et al. 2013

Synaptotagmin-1 functions as a Ca 2+ sensor in neurotransmitter release through its two C 2 domains (the C 2 A and C 2 B domain). The ability of synaptotagmin-1 to bridge two membranes is likely crucial for its function, enabling cooperation with the soluble Nethylmaleimide sensitive factor adaptor protein receptors (SNAREs) in membrane fusion, but two bridging mechanisms have been proposed. A highly soluble synaptotagmin-1 fragment containing both domains (C 2 AB) was shown to bind simultaneously to two membranes via the Ca 2+ -binding loops at the top of both domains and basic residues at the bottom of the C 2 B domain (direct bridging mechanism). In contrast, a longer fragment including a linker sequence (lnC 2 AB) was found to aggregate in solution and was proposed to bridge membranes through trans interactions between lnC 2 AB oligomers bound to each membrane via the Ca 2+ -binding loops, with no contact of the bottom of the C 2 B domain with the membranes. We now show that lnC 2 AB containing impurities indeed aggregates in solution, but properly purified lnC 2 AB is highly soluble. Moreover, cryo-EM images reveal that a majority of lnC 2 AB molecules bridge membranes directly. Fluorescence spectroscopy indicates that the bottom of the C 2 B domain contacts the membrane in a sizeable population of molecules of both membranebound C 2 AB and membrane-bound lnC 2 AB. NMR data on nanodiscs show that a fraction of C 2 AB molecules bind to membranes with antiparallel orientations of the C 2 domains. Together with previous studies, these results show that direct bridging constitutes the prevalent mechanism of membrane bridging by both C 2 AB and lnC 2 AB, suggesting that this mechanism underlies the function of synaptotagmin-1 in neurotransmitter release.membrane bending | Ca2+ sensing | exocytosis | synaptic transmission N eurotransmitter release is a key event in interneuronal communication that is acutely triggered by Ca 2+ influx into a presynaptic terminal (1). The synaptic vesicle protein synaptotagmin-1 acts as a Ca 2+ sensor in fast release through the two C 2 domains that form most of its cytoplasmic region (the C 2 A and C 2 B domains) (2-5). This function is coupled to membrane fusion through the neuronal soluble N-ethylmaleimide sensitive factor adaptor protein receptor (SNARE) proteins (6, 7), which bring the synaptic vesicle and plasma membranes together by forming SNARE complexes (2, 3). Synaptotagmin-1 function also depends on a tight interplay with complexins (8-10) and other key proteins of the release machinery (11, 12). The synaptotagmin-1 C 2 domains bind three or two Ca 2+ ions through loops at the top of β-sandwich structures (13-15) (Fig. 1A). These top loops also mediate Ca 2+ -dependent phospholipid binding (15-17), which is crucial for synaptotagmin-1 function (5, 18). Moreover, the synaptotagmin-1 cytoplasmic region clusters chromaffin granules and liposomes (19), and a fragment containing its two C 2 domains was shown by cryo-EM to bind simultaneously to two membranes, bringing them i...