SNAP-25 Is Targeted to the Plasma Membrane through a Novel Membrane-binding Domain

Gonzalo, Susana; Greentree, Wendy K.; Linder, Maurine E.

doi:10.1074/jbc.274.30.21313

Cited by 109 publications

(123 citation statements)

References 35 publications

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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).…”

supporting

confidence: 73%

“…This stretch begins immediately C-terminal of the minimal domain required for palmitoylation of SNAP-25 in vivo (Gonzalo et al, 1999) and did not affect expression level or membrane targeting. It consists of hydrophobic and charged amino acids, whereas the stretch in SNAP-23 is hydrophilic, but uncharged.…”

Section: Discussionmentioning

confidence: 99%

“…The only function so far ascribed to the linker domain is the membrane-targeting of SNAP-25 and SNAP-23 through the palmitoylation of four to five cysteine residues in the N-terminal end of the linker (Gonzalo et al, 1999;Loranger and Linder, 2002). However, other members of the family (SNAP-29, SNAP-46, Sec9, and SPO20) lack linker cysteines.…”

Section: Introductionmentioning

confidence: 99%

“…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 SNARE-domain (Qc). This antiparallel linker is a special feature of exocytotic SNARE complexes in eukaryotic organisms from yeast to human.The only function so far ascribed to the linker domain is the membrane-targeting of SNAP-25 and SNAP-23 through the palmitoylation of four to five cysteine residues in the N-terminal end of the linker (Gonzalo et al, 1999;Loranger and Linder, 2002). However, other members of the family (SNAP-29, SNAP-46, Sec9, and SPO20) lack linker cysteines.…”

mentioning

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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supporting

confidence: 73%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

mentioning

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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“…Moreover, palmitoylation and membrane association of the newly synthesized protein are sensitive to Brefeldin A, suggesting that these events are coupled and depend on an intact secretory pathway (15). Palmitoylation-defective mutants of SNAP-25 and SNAP-23 are found predominately in the cytoplasm of transfected cells (11,16,17). However, palmitoylation-defective forms of SNAP-25 can be targeted to membranes when syntaxin 1A is present (18, 19).…”

mentioning

confidence: 99%

SNAP-25 Traffics to the Plasma Membrane by a Syntaxin-independent Mechanism

Loranger¹,

Linder

2002

Journal of Biological Chemistry

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Membrane trafficking is a fundamental cellular process that must operate with high fidelity to maintain organelle identity, cell function, and viability. Integral membrane proteins referred to as SNAREs 1 (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are important elements in this process, participating in the docking and fusion of vesicles with target membranes (1). The hallmark of SNARE proteins is the presence of one or more ␣-helices in the cytoplasmic domain, which have a propensity to form coiled-coils (reviewed in Refs. 2 and 3). Pairing between cognate SNAREs on opposing membranes results in the formation of a parallel four-helix bundle that brings the membranes together to allow fusion (4, 5). At the neuronal synapse, the SNARE complex is formed between SNAP-25 and syntaxin, localized on the presynaptic membrane, and synaptobrevin/VAMP, localized on the vesicle membrane (1). The central role that SNAP-25 plays in this complex is evident from the impairment of neuronal secretion by botulinum neurotoxin A/E, a toxin that proteolytically inactivates SNAP-25 (6). Moreover, loss of SNAP-25 expression in mice results in embryonic lethality late in gestation with evoked neurotransmitter release absent at the neuromuscular junction and central synapses (7).Each membrane compartment appears to be associated with a unique set of SNAREs, thereby contributing to the specificity of membrane fusion. The mechanisms by which SNAREs are targeted to their resident membranes are only beginning to be understood. Most syntaxin and synaptobrevin/VAMP family members are classified as tail-anchored proteins based on the presence of a transmembrane segment at the C terminus. Studies of synaptobrevin and other vesicle-associated SNAREs (vSNAREs) demonstrate that these proteins are initially targeted to the endoplasmic reticulum and then sorted to their ultimate destinations (8, 9).The SNAP-25 family of SNAREs, SNAP-25a and b, SNAP-23, and SNAP-29, is structurally distinct from the tail-anchored SNAREs (3) and thus uses different mechanisms for membrane localization. The tail-anchored SNARES have a single cytoplasmic coiled-coil domain or SNARE motif, whereas SNAP-25 family members have two SNARE motifs that are connected by an interhelical domain (5). SNAP-25 and SNAP-23 are palmitoylated at a cluster of cysteine residues in the interhelical domain (10 -12). Many soluble proteins rely on fatty acylation for membrane association (13,14), and there is evidence to support a similar functional role for SNAP-25 palmitoylation. Our study of the native protein in PC12 cells revealed that palmitoylation of newly synthesized SNAP-25 coincides temporally with its membrane association (15). Moreover, palmitoylation and membrane association of the newly synthesized protein are sensitive to Brefeldin A, suggesting that these events are coupled and depend on an intact secretory pathway (15). Palmitoylation-defective mutants of SNAP-25 and SNAP-23 are found predominately in the cytoplasm of transfected cells (11,16,1...

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Glucose‐stimulated signaling pathways in biphasic insulin secretion

Straub

Sharp

2002

Diabetes Metabolism Res

364

366

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Glucose-stimulated biphasic insulin secretion involves at least two signaling pathways, the KATP channel-dependent and KATP channel-independent pathways, respectively. In the former, enhanced glucose metabolism increases the cellular adenosine triphosphate/adenosine diphosphate (ATP/ADP) ratio, closes KATP channels and depolarizes the cell. Activation of voltage-dependent Ca(2+) channels increases Ca(2+) entry and [Ca(2+)]i and stimulates insulin release. The KATP channel-independent pathways augment the response to increased [Ca(2+)]i by mechanisms that are currently unknown. However, they affect different pools of insulin-containing granules in a highly coordinated manner. The beta-cell granule pools can be minimally described as reserve, morphologically docked, readily and immediately releasable. Activation of the KATP channel-dependent pathway results in exocytosis of an immediately releasable pool that is responsible for the first phase of glucose-stimulated insulin release. Following glucose metabolism, the rate-limiting step for the first phase lies in the rate of signal transduction between sensing the rise in [Ca(2+)]i and exocytosis of the immediately releasable granules. The immediately releasable pool of granules can be enlarged by previous exposure to glucose (by time-dependent potentiation, TDP), and by second messengers such as cyclic adenosine monophosphate (cyclic AMP) and diacylglycerol (DAG). The second phase of glucose-stimulated insulin secretion is due mainly to the KATP channel-independent pathways acting in synergy with the KATP channel-dependent pathway. The rate-limiting step here is the conversion of readily releasable granules to the state of immediate releasability, following which, in an activated cell they will undergo exocytosis. In the rat and human beta-cell the KATP channel-independent pathways induce a time-dependent increase in the rate of this step that results in the typical rising second-phase response. In the mouse beta-cell the rate appears not to be changed much by glucose. Potential intermediates involved in controlling the rate-limiting step include increases in cytosolic long-chain acyl-CoA levels, adenosine triphosphate (ATP) and guanosine triphosphate (GTP), DAG binding proteins, including some isoforms of protein kinase (PKC), and protein acyl transferases. Agonists that can change the rate-limiting steps for both phases of insulin release include those like glucagon-like peptide 1 (GLP-1) that raise cyclic AMP levels and those like acetylcholine that act via DAG.

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SNAP-25 Is Targeted to the Plasma Membrane through a Novel Membrane-binding Domain

Cited by 109 publications

References 35 publications

The SNAP-25 Linker as an Adaptation Toward Fast Exocytosis

The SNAP-25 Linker as an Adaptation Toward Fast Exocytosis

SNAP-25 Traffics to the Plasma Membrane by a Syntaxin-independent Mechanism

Glucose‐stimulated signaling pathways in biphasic insulin secretion

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