Ca2+–synaptotagmin directly regulates t-SNARE function during reconstituted membrane fusion

Bhalla, Akhil; Chicka, Michael C.; Tucker, Ward C.; Chapman, Edwin R.

doi:10.1038/nsmb1076

Cited by 174 publications

(244 citation statements)

References 60 publications

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“…There is ample evidence that synaptotagmin-1 binds to the SNARE complex or to individual SNAREs through interactions with one or both C2-domains (Bennett et al, 1992;Sollner et al, 1993;Chapman et al, 1995;Schiavo et al, 1997;Zhang et al, 2002;Rickman and Davletov, 2003;Shin et al, 2003;Bai et al, 2004;Bhalla et al, 2006;Pang et al, 2006;Lynch et al, 2007). It is possible that the SNAP-25 linker participates in synaptotagmin binding in situ.…”

Section: Discussionmentioning

confidence: 99%

“…Finally, the linker might affect stages of SNARE complex assembly, which could change both vesicle priming and fusion reactions , possibly by stabilizing an intermediate conformation of the SNARE complex (An and Almers, 2004). The picture emerging is that SNAREs, synaptotagmins, and lipids form an integrated fusion machine (Bhalla et al, 2006;Pang et al, 2006;Dai et al, 2007), whose calcium dependence is set by the properties of the entire assembly. The SNAP-25 linker must be seen as an integral part of this machine and it is tempting to speculate that the fusion of the Qb and Qc-SNARE motifs might have evolved as an adaptation toward calcium triggering of exocytosis.…”

Section: Discussionmentioning

confidence: 99%

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The SNAP-25 Linker as an Adaptation Toward Fast Exocytosis

Nagy

Milošević

Mohrmann

et al. 2008

MBoC

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The assembly of four soluble N-ethylmaleimide-sensitive factor attachment protein receptor domains into a complex is essential for membrane fusion. In most cases, the four SNARE-domains are encoded by separate membrane-targeted proteins. However, in the exocytotic pathway, two SNARE-domains are present in one protein, connected by a flexible linker. The significance of this arrangement is unknown. We characterized the role of the linker in SNAP-25, a neuronal SNARE, by using overexpression techniques in synaptosomal-associated protein of 25 kDa (SNAP-25) null mouse chromaffin cells and fast electrophysiological techniques. We confirm that the palmitoylated linker-cysteines are important for membrane association. A SNAP-25 mutant without cysteines supported exocytosis, but the fusion rate was slowed down and the fusion pore duration prolonged. Using chimeric proteins between SNAP-25 and its ubiquitous homologue SNAP-23, we show that the cysteine-containing part of the linkers is interchangeable. However, a stretch of 10 hydrophobic and charged amino acids in the C-terminal half of the SNAP-25 linker is required for fast exocytosis and in its absence the calcium dependence of exocytosis is shifted toward higher concentrations. The SNAP-25 linker therefore might have evolved as an adaptation toward calcium triggering and a high rate of execution of the fusion process, those features that distinguish exocytosis from other membrane fusion pathways. INTRODUCTIONThe soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are central to vesicular trafficking (Fasshauer, 2003;Jahn and Scheller, 2006;Rizo et al., 2006) and characterized by a stretch of 60 -70 amino acids, known as the SNARE motif (Terrian and White, 1997;Weimbs et al., 1997). The assembly of four SNARE domains into a coiled-coil bundle is a general mechanism in fusion of intracellular membrane compartments. The bundle is held together by layers of hydrophobic interaction, with the exception of the middle layer (layer "0"), which is formed by an arginine and three glutamines (Sutton et al., 1998). The SNARE domains can be subdivided into four homologous groups, named after their contribution to the zero layer: R, Qa, Qb, and Qc (Fasshauer et al., 1998b). SNARE assembly requires the participation of one domain from each group, resulting in a certain degree of specificity Scales et al., 2000a).In most cases, the four SNARE-domains are encoded by separate membrane-targeted proteins, but the SNAREs driving the fusion of vesicles with the plasma membrane (exocytosis) are special in that three proteins provide the four domains (Weimbs et al., 1998;Fukuda et al., 2000). One of the SNAREs in this pathway, exemplified by the best-known isoform synaptosomal-associated protein of 25 kDa (SNAP-25), seems to have been created by fusion of the Qb and Qc SNAREs. This arrangement necessitates a flexible linker, which runs back along the complex from the C-terminal end of the first (Qb) SNARE-domain and connects to the Nterminal end of the second SN...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

The SNAP-25 Linker as an Adaptation Toward Fast Exocytosis

Nagy

Milošević

Mohrmann

et al. 2008

MBoC

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“…Syt-1 mutations that eliminate Ca 2ϩ -dependent SNARE binding abrogate Ca 2ϩ -triggered dense-core vesicle exocytosis in PC12 cells , whereas mutations that enhance SNARE binding facilitate Ca 2ϩ -triggered synaptic vesicle exocytosis in neurons (Pang et al, 2006). The Ca 2ϩ regulation of liposome fusion mediated by Syt-1 requires Syt-1-SNARE interactions (Bhalla et al, 2006). These findings indicate an essential role for SNARE binding in determining Ca 2ϩ -dependent vesicle fusion probabilities.…”

Section: Introductionmentioning

confidence: 87%

Synaptotagmin-1 Utilizes Membrane Bending and SNARE Binding to Drive Fusion Pore Expansion

Lynch

Gerona

Kielar

et al. 2008

MBoC

118

147

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In regulated vesicle exocytosis, SNARE protein complexes drive membrane fusion to connect the vesicle lumen with the extracellular space. The triggering of fusion pore formation by Ca 2؉ is mediated by specific isoforms of synaptotagmin (Syt), which employ both SNARE complex and membrane binding. Ca 2؉ also promotes fusion pore expansion and Syts have been implicated in this process but the mechanisms involved are unclear. We determined the role of Ca 2؉ -dependent Syt-effector interactions in fusion pore expansion by expressing Syt-1 mutants selectively altered in Ca 2؉ -dependent SNARE binding or in Ca 2؉ -dependent membrane insertion in PC12 cells that lack vesicle Syts. The release of differentsized fluorescent peptide-EGFP vesicle cargo or the vesicle capture of different-sized external fluorescent probes was used to assess the extent of fusion pore dilation. We found that PC12 cells expressing partial loss-of-function Syt-1 mutants impaired in Ca 2؉ -dependent SNARE binding exhibited reduced fusion pore opening probabilities and reduced fusion pore expansion. Cells with gain-of-function Syt-1 mutants for Ca 2؉ -dependent membrane insertion exhibited normal fusion pore opening probabilities but the fusion pores dilated extensively. The results indicate that Syt-1 uses both Ca 2؉ -dependent membrane insertion and SNARE binding to drive fusion pore expansion. INTRODUCTIONNeurotransmitter and peptide hormone secretion is mediated by the Ca 2ϩ -dependent exocytosis of vesicles at the plasma membrane. Vesicle fusion proceeds through membrane intermediates of stalk formation, fusion pore opening, and fusion pore expansion (Lindau and Alvarez de Toledo, 2003). In classical modes of vesicle exocytosis, fusion pores expand to the point where the vesicle membrane flattens on the plasma membrane (full fusion), leading to complete luminal contents release (full release). However, vesicle exocytosis can utilize alternative modes in which the fusion pore either abruptly closes (kiss-and-run) or in which the fusion pore dilates but subsequently recloses (cavicapture; Henkel and Almers, 1996). These transient modes of vesicle exocytosis lead to the partial release of luminal contents depending on the size and diffusibility of the cargo (Alvarez de Toledo et al., 1993;Barg et al., 2002;Taraska et al., 2003). Ca 2ϩ levels regulate fusion pore expansion as well as fusion pore formation (Fernandez-Chacon and Alvarez de Toledo, 1995;Hartmann and Lindau, 1995;Wang et al., 2006), but the mechanisms underlying fusion pore expansion, which is the more energetically demanding step, are poorly understood (Cohen and Melikyan, 2004).A Ca 2ϩ -dependent fusion machinery mediates the triggered formation of fusion pores. Three SNARE proteins (VAMP-2 on the vesicle with SNAP25 and syntaxin-1 on the plasma membrane) form trans complexes that promote close membrane apposition and membrane fusion (Weber et al., 1998). Members of the synaptotagmin (Syt) protein family that localize to vesicles function as Ca 2ϩ sensors that couple Ca 2ϩ rises ...

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“…Regulation of SNARE-dependent membrane fusion by the SNARE-interacting protein Sec1/Munc18 (SM) (14,15) and the SNARE-binding proteins synaptotagmin and complexin (16)(17)(18)(19)(20)(21)(22)(23)(24)(25) has also been reconstituted. We have recently reported reconstituted membrane fusion that requires yeast vacuolar SNAREs, regulatory lipids, and the homotypic vacuole fusion and protein sorting (HOPS)/class C vacuole protein sorting (class C Vps) complex, a six-subunit effector and GEF for the yeast vacuolar Rab GTPase Ypt7p (26)(27)(28).…”

mentioning

confidence: 99%

Minimal membrane docking requirements revealed by reconstitution of Rab GTPase-dependent membrane fusion from purified components

Stroupe

Hickey

Mima

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

106

139

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Rab GTPases and their effectors mediate docking, the initial contact of intracellular membranes preceding bilayer fusion. However, it has been unclear whether Rab proteins and effectors are sufficient for intermembrane interactions. We have recently reported reconstituted membrane fusion that requires yeast vacuolar SNAREs, lipids, and the homotypic fusion and vacuole protein sorting (HOPS)/class C Vps complex, an effector and guanine nucleotide exchange factor for the yeast vacuolar Rab GTPase Ypt7p. We now report reconstitution of lysis-free membrane fusion that requires purified GTP-bound Ypt7p, HOPS complex, vacuolar SNAREs, ATP hydrolysis, and the SNARE disassembly catalysts Sec17p and Sec18p. We use this reconstituted system to show that SNAREs and Sec17p/Sec18p, and Ypt7p and the HOPS complex, are required for stable intermembrane interactions and that the three vacuolar Q-SNAREs are sufficient for these interactions.biochemical reconstitution ͉ Rab effector

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Ca2+–synaptotagmin directly regulates t-SNARE function during reconstituted membrane fusion

Cited by 174 publications

References 60 publications

The SNAP-25 Linker as an Adaptation Toward Fast Exocytosis

The SNAP-25 Linker as an Adaptation Toward Fast Exocytosis

Synaptotagmin-1 Utilizes Membrane Bending and SNARE Binding to Drive Fusion Pore Expansion

Minimal membrane docking requirements revealed by reconstitution of Rab GTPase-dependent membrane fusion from purified components

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