Ca2+-Dependent Synaptotagmin Binding to SNAP-25 Is Essential for Ca2+-Triggered Exocytosis

Zhang, Xiaodong; Kim-Miller, Mindy J.; Fukuda, Mitsunori; Kowalchyk, Judith A.; Martin, Thomas F.J.

doi:10.1016/s0896-6273(02)00671-2

Cited by 222 publications

(230 citation statements)

References 66 publications

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“…Third, the short linker between the two C2 domains would limit the orientations that the C2A domain can adopt, enhancing the rapid response to the calcium signal. In this orientation, C2A may respond to calcium entry either through binding electrostatically to the phospholipid membrane (Fernandez-Chacon et al, 2001) or, as has also been suggested, through the inducible, calcium-dependent, binding to SNAREs (Zhang et al, 2002;Bai et al, 2004). This inducible SYT/SNARE interaction exhibits properties very different from those characteristic of the constitutive association.…”

Section: Discussionmentioning

confidence: 89%

Conserved Prefusion Protein Assembly in Regulated Exocytosis

Rickman

Jiménez

Graham

et al. 2006

MBoC

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The regulated release of hormones and neurotransmitters is a fundamental process throughout the animal kingdom. The short time scale for the calcium triggering of vesicle fusion in regulated secretion suggests that the calcium sensor synaptotagmin and the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) membrane fusion machinery are well ordered before the calcium signal. To gain insight into the organization of the prefusion protein assembly in regulated exocytosis, we undertook a structural/functional study of the vesicular synaptotagmin1 and the plasma membrane SNARE proteins, which copurify from the brain in the absence of calcium. Based on an evolutionary analysis, mutagenesis screens, and a computational protein docking approach, we now provide the first testable description of the supramolecular prefusion assembly. Perturbing the determined synaptotagmin/SNARE-interacting interface in several models of regulated exocytosis altered the secretion of hormones and neurotransmitters. These mutations also disrupted the constitutive synaptotagmin/SNARE link in full agreement with our model. We conclude that the interaction of synaptotagmin with preassembled plasma membrane SNARE proteins, before the action of calcium, can provide a precisely organized "tethering" scaffold that underlies regulated secretion throughout evolution. INTRODUCTIONIn regulated secretion, fusion of docked secretory vesicles with the plasma membrane is triggered by the rapid elevation of the intracellular calcium concentration (Katz and Miledi, 1967). The membrane fusion itself is brought about by the action of the three soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, whereas the vesicular protein synaptotagmin (SYT) acts as the calcium sensor (Sollner et al., 1993;Sudhof and Scheller, 2001;Bonifacino and Glick, 2004). In neuroendocrine cells, the principal SNAREs are syntaxin1 and synaptosome-associated protein of 25 kDa (SNAP-25) on the plasma membrane (so-called target SNAREs or t-SNAREs), and vesicular synaptobrevin, also known as VAMP. Interaction between synaptobrevin and the two t-SNAREs leads to the formation of the ternary SNARE complex, a twisted parallel fourhelical bundle that probably drives membrane fusion (Sutton et al., 1998). With all the major players involved in vesicle fusion identified, it is becoming possible to tackle a central problem of this cellular process-how does calcium trigger vesicle fusion? Arguably, to understand the action of calcium it is essential to know 1) the extent of the assembly of the SNARE fusion proteins and 2) their organization in relation to the calcium sensor, SYT, before calcium-triggered events.It would be reasonable to assume that the SNAREs are at least partially preassembled, and the plasma membrane syntaxin and SNAP-25 are the first obvious candidates for being in such a ready state (An and Almers, 2004;Rickman et al., 2004b). Importantly, synaptobrevin binds syntaxin and SNAP-25 with high-affinity only when the two...

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Section: Discussionmentioning

confidence: 89%

Conserved Prefusion Protein Assembly in Regulated Exocytosis

Rickman

Jiménez

Graham

et al. 2006

MBoC

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“…refs. 40,42,54 ), yet there were contradictory data on these interactions and it was unclear how they might be coupled with Ca 2+ -phospholipid binding to synaptotagmin 1. The results described here now shed light on these questions, showing that synaptotagmin 1 forms an intimate quaternary complex with the SNAREs, Ca 2+ and phospholipids, and suggesting that membrane-anchoring of SNARE complexes increases the specificity of their interactions with synaptotagmin 1.…”

Section: Discussionmentioning

confidence: 99%

“…refs. [36][37][38][39][40]. Moreover, synaptotagmin 1 and the SNAREs are sticky proteins that have been reported to bind to many different targets (e.g.…”

Section: Introductionmentioning

confidence: 99%

A Quaternary SNARE–Synaptotagmin–Ca2+–Phospholipid Complex in Neurotransmitter Release

Han

Shen

Araç

et al. 2007

Journal of Molecular Biology

116

203

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SummaryThe function of synaptotagmin as a Ca 2+ sensor in neurotransmitter release involves Ca 2+ -dependent phospholipid binding to its two C 2 domains, but this activity alone does not explain why Ca 2+ binding to the C 2 B domain is more critical for release than Ca 2+ binding to the C 2 A domain. Synaptotagmin also binds to SNARE complexes, which are central components of the membrane fusion machinery, and displaces complexins from the SNAREs. However, it is unclear how phospholipid binding to synaptotagmin is coupled to SNARE binding and complexin displacement. Using supported lipid bilayers deposited within microfluidic channels, we now show that Ca 2+ induces simultaneous binding of synaptotagmin to phospholipid membranes and SNARE complexes, resulting in an intimate quaternary complex that we name SSCAP complex. Mutagenesis experiments show that Ca 2+ binding to the C 2 B domain is critical for SSCAP complex formation and displacement of complexin, providing a clear rationale for the preponderant role of the C 2 B domain in release. This and other correlations between the effects of mutations on SSCAP complex formation and their functional effects in vivo suggest a key role for this complex in release. We propose a model whereby the highly positive electrostatic potential at the tip of the SSCAP complex helps to induce membrane fusion during release.

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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%

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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Ca2+-Dependent Synaptotagmin Binding to SNAP-25 Is Essential for Ca2+-Triggered Exocytosis

Cited by 222 publications

References 66 publications

Conserved Prefusion Protein Assembly in Regulated Exocytosis

Conserved Prefusion Protein Assembly in Regulated Exocytosis

A Quaternary SNARE–Synaptotagmin–Ca2+–Phospholipid Complex in Neurotransmitter Release

The SNAP-25 Linker as an Adaptation Toward Fast Exocytosis

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