Different Effects on Fast Exocytosis Induced by Synaptotagmin 1 and 2 Isoforms and Abundance But Not by Phosphorylation

Nagy, Gábor; Kim, Jun Hee; Pang, Zhiping P.; Matti, Ulf; Rettig, Jens; Südhof, Thomas C.; Sørensen, Jakob B.

doi:10.1523/jneurosci.2589-05.2006

Cited by 111 publications

(107 citation statements)

References 73 publications

(102 reference statements)

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“…A previous study demonstrated that Syt T112A does not affect vesicle release in chromaffin cells (21). Our results underscore that, despite many similarities, key differences exist between the release machineries of chromaffin cells and synapses (see also refs.…”

Section: Discussionsupporting

confidence: 77%

“…The Syt1 homologs Syt7 and Syt12 contain similar phosphorylation sites in the linker between TM and C2A, and their phosphorylation potentiates Ca 2+ -induced vesicle fusion (19,20). Unexpectedly, a nonphosphorylatable Syt1 variant had no effect on vesicle fusion in mouse embryonic chromaffin cells (21). However, we previously observed only limited contribution of PKC in mouse embryonic chromaffin cells compared with adult bovine chromaffin cells and neurons (8,22), suggesting that the role of PKC may differ between model systems.…”

mentioning

confidence: 79%

“…28 and 29). Although PMA potentiates release from embryonic chromaffin cells (21,30), potentiation persists after inhibition of PKC (22), deletion of Munc18-1 (30), or rescue of Syt1-KO with Syt T112A (21). In addition, the relative contribution of PKC to DAG-induced potentiation differs among synapses, as PKC activation is strictly required in hippocampal (6,8), but not in Calyceal synapses (7,11).…”

Section: Discussionmentioning

confidence: 99%

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Phosphorylation of synaptotagmin-1 controls a post-priming step in PKC-dependent presynaptic plasticity

Jong

Meijer

Saarloos

et al. 2016

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

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Presynaptic activation of the diacylglycerol (DAG)/protein kinase C (PKC) pathway is a central event in short-term synaptic plasticity. Two substrates, Munc13-1 and Munc18-1, are essential for DAGinduced potentiation of vesicle priming, but the role of most presynaptic PKC substrates is not understood. Here, we show that a mutation in synaptotagmin-1 (Syt1 T112A ), which prevents its PKCdependent phosphorylation, abolishes DAG-induced potentiation of synaptic transmission in hippocampal neurons. This mutant also reduces potentiation of spontaneous release, but only if alternative Ca 2+ sensors, Doc2A/B proteins, are absent. However, unlike mutations in Munc13-1 or Munc18-1 that prevent DAG-induced potentiation, the synaptotagmin-1 mutation does not affect pairedpulse facilitation. Furthermore, experiments to probe vesicle priming (recovery after train stimulation and dual application of hypertonic solutions) also reveal no abnormalities. Expression of synaptotagmin-2, which lacks a seven amino acid sequence that contains the phosphorylation site in synaptotagmin-1, or a synaptotagmin-1 variant with these seven residues removed (Syt1 Δ109-116 ), supports normal DAG-induced potentiation. These data suggest that this seven residue sequence in synaptotagmin-1 situated in the linker between the transmembrane and C2A domains is inhibitory in the unphosphorylated state and becomes permissive of potentiation upon phosphorylation. We conclude that synaptotagmin-1 phosphorylation is an essential step in PKC-dependent potentiation of synaptic transmission, acting downstream of the two other essential DAG/PKC substrates, Munc13-1 and Munc18-1.P resynaptic strength changes rapidly during repetitive stimulation [short-term plasticity (STP)] and activation of intracellular signal transduction pathways (1, 2). The diacylglycerol (DAG)/protein kinase C (PKC) cascade is one of the most potent pathways at the presynaptic terminal. Its activation leads to 50-100% potentiation of spontaneous and action potential (AP)-evoked release (3-5), and is critical for multiple forms of presynaptic plasticity (6-8). DAG directly activates the vesicle priming factor Munc13-1 (9, 10) and indirectly activates downstream effectors via PKC. Activation of both Munc13-1 and PKC is essential for this pathway to operate (Fig. 1A) (8, 11). We previously identified Munc18-1 as an essential PKC substrate, because a nonphosphorylatable Munc18-1 mutant completely inhibits PKC-dependent STP (8, 12). Importantly, a phosphomimetic mutation of Munc18-1 cannot fully bypass the requirement for PKC activation, indicating that other PKC substrates must contribute to this form of plasticity (8). These substrates have not been identified to date.Among other presynaptic PKC substrates is synaptotagmin-1 (Syt1, ref. 13). Syt1 is the vesicular Ca 2+ sensor that mediates fast AP-evoked release in the hippocampus (14) and drives a large fraction of spontaneous release (15). In the latter case, Syt1 competes with alternative sensors, in particular Doc2s, for SNARE binding ...

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Section: Discussionsupporting

confidence: 77%

mentioning

confidence: 79%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Phosphorylation of synaptotagmin-1 controls a post-priming step in PKC-dependent presynaptic plasticity

Jong

Meijer

Saarloos

et al. 2016

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

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“…Indeed, it is striking that the only molecular manipulations that are known-or are likely based on published literature-to change the intracellular calcium-dependence of exocytosis triggering are mutations in synaptotagmins and SNAP-25 (Sorensen et al, 2003a;Wang et al, 2003;Chieregatti et al, 2004;Rhee et al, 2005;Wang et al, 2005;Nagy et al, 2006;Pang et al, 2006;Sorensen et al, 2006). In contrast, manipulations of proteins involved in exocytosis more often lead to changes in pool sizes and/or recruitment in the absence of a change in triggering: examples include tomosyn (Yizhar et al, 2004), Munc13 (Ashery et al, 2000), Munc18 (Voets et al, 2001;Gulyas-Kovacs et al, 2007), CAPS1 (Speidel et al, 2005), SV2 (Xu and Bajjalieh, 2001), Snapin (Tian et al, 2005), synaptobrevin (Borisovska et al, 2005), and ␣-SNAP/NSF .…”

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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“…Of the synaptotagmin family, synaptotagmin-2 exhibits the highest degree of homology with synaptotagmin-1. Indeed, synaptotagmin-2 can rescue synaptotagmin-1 deficiency, and its expression overlaps partially but not completely with synaptotagmin-1 (Geppert et al, 1994;Nagy et al, 2006).…”

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confidence: 99%