Multiple palmitoylation of synaptotagmin and the t‐SNARE SNAP‐25

Veit, Michael; Söllner, Thomas H.; Rothman, James E.

doi:10.1016/0014-5793(96)00362-6

Cited by 235 publications

(222 citation statements)

References 27 publications

(10 reference statements)

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“…We analyzed the role of the palmitoylated cysteines in a null background, by expression in Snap-25 knockout chromaffin cells. Our data confirm the function of the cysteines in targeting SNAP-25 to the plasma membrane (Hess et al, 1992;Veit et al, 1996;Lane and Liu, 1997;Gonzalo et al, 1999;Vogel and Roche, 1999;Gonelle-Gispert et al, 2000;Koticha et al, 2002;Loranger and Linder, 2002;Kammer et al, 2003), in which it acts in exocytosis by binding to the Q-SNARE partner syntaxin-1. Removing single cysteines reduced the amount of SNAP-25 on the plasma membrane to Ͻ50%, but it did not impair secretion, as previously shown in insulin-secreting cells (Gonelle-Gispert et al, 2000).…”

Section: Discussionsupporting

confidence: 86%

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: Discussionsupporting

confidence: 86%

The SNAP-25 Linker as an Adaptation Toward Fast Exocytosis

Nagy

Milošević

Mohrmann

et al. 2008

MBoC

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“…Synaptobrevin and syntaxin both contain a single transmembrane domain at the C terminus (1,2). SNAP-25 does not contain a transmembrane domain but carries palmitoyl side chains attached to cysteine residues in the middle of the sequence (3,4). Homologues of these proteins have been identified in many eukaryotic cells including yeast, suggesting that fusion of trafficking vesicles with their respective target membranes is mediated by a conserved mechanism (2,(5)(6)(7)(8).…”

mentioning

confidence: 99%

Structural Changes Are Associated with Soluble N-Ethylmaleimide-sensitive Fusion Protein Attachment Protein Receptor Complex Formation

Fasshauer

Otto

Eliason

et al. 1997

Journal of Biological Chemistry

342

372

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SNAP-25, syntaxin, and synaptobrevin play a key role in the regulated exocytosis of synaptic vesicles, but their mechanism of action is not understood. In vitro, the proteins spontaneously assemble into a ternary complex that can be dissociated by the ATPase N-ethylmaleimide-sensitive fusion protein and the cofactors ␣-, ␤-, and ␥-SNAP. Since the structural changes associated with these reactions probably form the basis of membrane fusion, we have embarked on biophysical studies aimed at elucidating such changes in vitro using recombinant proteins. All proteins were purified in a monomeric form. Syntaxin showed significant ␣-helicity, whereas SNAP-25 and synaptobrevin exhibited characteristics of largely unstructured proteins. Formation of the ternary complex induced dramatic increases in ␣-helicity and in thermal stability. This suggests that structure is induced in SNAP-25 and synaptobrevin upon complex formation. In addition, the stoichiometry changed from 2:1 in the syntaxin-SNAP-25 complex to 1:1:1 in the ternary complex. We propose that the transition from largely unstructured monomers to a tightly packed, energetically favored ternary complex connecting two membranes is a key step in overcoming energy barriers for membrane fusion.Neurons release their neurotransmitters by the Ca 2ϩ -dependent exocytosis of synaptic vesicles. In recent years, several membrane proteins have been identified which are required for exocytotic membrane fusion. These proteins include the synaptic vesicle protein synaptobrevin (also referred to as VAMP) 1 and the synaptic membrane proteins syntaxin and SNAP-25, collectively referred to as SNAREs. Synaptobrevin and syntaxin both contain a single transmembrane domain at the C terminus (1, 2). SNAP-25 does not contain a transmembrane domain but carries palmitoyl side chains attached to cysteine residues in the middle of the sequence (3, 4). Homologues of these proteins have been identified in many eukaryotic cells including yeast, suggesting that fusion of trafficking vesicles with their respective target membranes is mediated by a conserved mechanism (2,(5)(6)(7)(8).While the evidence linking synaptobrevin, SNAP-25, and syntaxin to exocytosis is compelling, their precise role is unknown. In detergent extracts of brain membranes, the three proteins form a tight complex (9, 10). A ternary complex with properties similar to the native complex can be formed using recombinant proteins lacking their transmembrane anchors (11). Both native and recombinant complexes can be disassembled by the concerted action of the ATPase NSF and the protein ␣-SNAP (9, 10, 12, 13). The latter two proteins are soluble, abundant, and highly conserved through evolution. They are essential for the fusion of trafficking vesicles with their target membranes (9, 10).The assembly and disassembly of the SNARE proteins has not yet been integrated into a coherent picture of exocytosis. However, any interference with these reactions seriously inhibits membrane fusion (6 -8). To further understand these essential reac...

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“…SNAP-25 is an abundant highly conserved membrane-associated protein (Oyler et al, 1989;Risinger et al, 1993). It is post-translationally modified by palmitoyl side chains that mediate membrane binding (Hess et al, 1992;Veit et al, 1996).Syntaxin is a similarly abundant small integral membrane protein with a C-terminal transmembrane domain (Bennett et al, 1992).In detergent extracts of brain membranes, synaptobrevin, SNAP-25, and syntaxin are associated in a stable 7S complex (Söllner et al, 1993a,b). This synaptic SNAP receptor complex (SNARE) binds ␣-SNAP and NSF to form a 20S particle.…”

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confidence: 99%

Inhibition of Transmitter Release Correlates with the Proteolytic Activity of Tetanus Toxin and Botulinus Toxin A in Individual Cultured Synapses ofHirudo medicinalis

et al. 1997

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We have studied the effects of tetanus toxin and botulinus toxin A on neurotransmitter release in the Retzius3 P-cell synapse of the leech and exploited the unique properties of this system, which allow for combined physiological and biochemical analyses in single-cell pairs. The sequences of Hirudo medicinalis synaptobrevin and synaptosomal-associated protein of 25 kDa , deduced by cDNA cloning, are 61 and 55% identical, respectively, to their corresponding mammalian homologs. Whereas Hirudo synaptobrevin is proteolyzed by tetanus toxin, its SNAP-25 isoform is resistant to botulinus toxin A cleavage because of amino acid substitutions within and around the putative cleavage site. In close correlation, microinjection of tetanus toxin into the presynaptic neuron produced a block of transmitter release, whereas botulinus toxin A had no effect on synaptic transmission. Subsequent immunoblotting of single-cell pairs demonstrated directly that the tetanus toxinmediated block of exocytosis is accompanied by cleavage of synaptobrevin in the injected neuron, resulting in the generation of a detectable C-terminal cleavage product. Immunoblotting also confirmed the resistance of SNAP-25 to botulinus toxin A cleavage in vivo. Using recombinant proteins, we show that the N-terminal fragment of synaptobrevin released by tetanus toxin, but not its C-terminal membrane-anchored cleavage product, participates with syntaxin and SNAP-25 in synaptic SNAP receptor (SNARE) ternary complex formation in Hirudo. Our data demonstrate a direct correlation between the inhibition of transmitter release and the ability of the neurotoxin to proteolyze its target protein and support the view that SNARE ternary complex formation is an important step leading to synaptic vesicle exocytosis. Key words: transmitter release; synaptobrevin; VAMP; SNAP-25; SNARE; tetanus toxin; botulinus toxin A; single-cell immunoblotting; Hirudo medicinalisCalcium-regulated exocytosis of synaptic vesicles is the primary means of communication between neurons. A complex of conserved cytosolic proteins known as NSF (N-ethylmaleimidesensitive fusion protein) and SNAP proteins (soluble NSF attachment proteins) is required for various intracellular fusion events in eukaryotic cells (Ferro-Novick and Jahn, 1994;Südhof, 1995;Calakos and Scheller, 1996;Rothman and Wieland, 1996). The search for neuronal membrane receptors (SNAREs, SNAP receptors) for these soluble "fusion" factors led to the identification of the vesicle protein synaptobrevin (VAMP) and the plasma membrane proteins, syntaxin and SNAP-25 (synaptosomal-associated protein of 25 kDa) (Söllner et al., 1993a).Synaptobrevins are class II integral membrane proteins that expose most of their sequence at the cytoplasmic face of secretory vesicles (Südhof, 1995). SNAP-25 is an abundant highly conserved membrane-associated protein (Oyler et al., 1989;Risinger et al., 1993). It is post-translationally modified by palmitoyl side chains that mediate membrane binding (Hess et al., 1992;Veit et al., 1996).Syntaxin is a simi...

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Multiple palmitoylation of synaptotagmin and the t‐SNARE SNAP‐25

Cited by 235 publications

References 27 publications

The SNAP-25 Linker as an Adaptation Toward Fast Exocytosis

The SNAP-25 Linker as an Adaptation Toward Fast Exocytosis

Structural Changes Are Associated with Soluble N-Ethylmaleimide-sensitive Fusion Protein Attachment Protein Receptor Complex Formation

Inhibition of Transmitter Release Correlates with the Proteolytic Activity of Tetanus Toxin and Botulinus Toxin A in Individual Cultured Synapses ofHirudo medicinalis

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