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

Loranger, Stephanie S.; Linder, Maurine E.

doi:10.1074/jbc.m202125200

Cited by 41 publications

(37 citation statements)

References 36 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).…”

Section: Discussionsupporting

confidence: 86%

“…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.…”

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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: Discussionsupporting

confidence: 86%

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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“…3c). We further determined that SNAP-25 can traffic to the plasma membrane in CHO cells (data not shown), consistent with previous observation that SNAP-25 trafficking is independent of syntaxin (26). In INS-1 cells, co-expression of SNAP-25 with t1-t3 mutants resulted in comparable surface fractions as those from cells without SNAP-25 co-transfection (Fig.…”

Section: Snap-25 Interacts With Exposed Snare Motif Of Stx1a and Transupporting

confidence: 91%

Domain Requirement for the Membrane Trafficking and Targeting of Syntaxin 1A

Yang

Xiao

et al. 2006

Journal of Biological Chemistry

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Syntaxin plays a key role in intracellular membrane fusion in eukaryotic cells. The function of syntaxin relies on its proper trafficking to and targeting at the target membrane. The mechanisms underlying the trafficking and targeting of syntaxin to its physiological sites remain poorly understood. Here we have analyzed the trafficking of syntaxin 1A in INS-1 and CHO cells. We have identified the transmembrane domain together with several flanking positivecharged amino acids as the minimal domain required for the membrane delivery. Interestingly, we found that SNARE motif-exposed syntaxin 1A mutants were retained in endoplasmic reticulum (ER) and failed to transport to the cell surface in the absence of SNAP-25, suggesting that the exposure of the SNARE motif causes ER retention and complexation with SNAP-25 helps the ER escape. Finally, our data propose two key roles for the H abc domain: to protect nonspecific interaction by masking the SNARE motif and to participate in the clustering of syntaxin 1A to the fusion sites in the plasma membrane. SNARE4 (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins play a central role in the process of intracellular membrane fusion in eukaryotic cells (for recent reviews, see Refs. 1-3). Intracellular SNAREs can be divided into two categories: the v-SNAREs located on carrier vesicles and the t-SNAREs present on target compartments (4, 5). The interaction between SNAREs present on two opposing membranes is generally believed to provide the driving force to initiate membrane fusion. Syntaxin 1A (Stx1A) is a t-SNARE predominantly located at the plasma membrane and has been shown to be essential for vesicle fusion (6). The essential requirement of Stx1A for exocytosis has been demonstrated in that deletion of its homologues leads to complete loss of synaptic transmission in Drosophila (7) and complete paralysis in Caenorhabditis elegans (8).To function appropriately, the SNAREs themselves must be correctly sorted and delivered to destination compartments. The intracellular trafficking of t-SNARE syntaxin is particularly intriguing as syntaxin belongs to a family of proteins that are "tail-anchored," also called Type IV membrane proteins (9). Such proteins have an N-terminal cytoplasmic domain that is membrane-bound by virtue of a single C-terminal hydrophobic domain. These proteins lack an N-terminal signal sequence, and their membrane-interacting region is so close to the C terminus that it emerges from the ribosome only upon termination of translation. The trafficking and targeting mechanism of these tail-anchored proteins have largely remained obscure.The trafficking of syntaxin may require the interaction with "accessory" proteins. For instance, it has been demonstrated that neural specific Stx1A sorting from the Golgi complex and delivery to the plasma membrane required Munc18-1 (10). However, this hypothesis has been challenged by another research demonstrating that syntaxin transportation to the membrane is independent of unc18 protein in C. e...

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“…The GluR1-GFP, GluR2-GFP, GluR3-GFP, GluR4 -GFP constructs (Shi et al, 2001) were kindly provided by Dr. Robert Malinow (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), synaptophysin-GFP (Scalettar et al, 2002) was generously provided by Dr. Bethe Scalettar (Department of Physics, Lewis and Clark College, Portland, OR), and the SNAP-25-GFP plasmid (Loranger and Linder, 2002) was a generous gift from Dr. Maurine Linder (Washington University School of Medicine, St. Louis, MO). The plasmid DNA was amplified in DH5␣ Escherichia coli, selected with the appropriate antibiotics, and subsequently purified using the Qiagen QIAprep Spin Miniprep Kit (catalog # 27106).…”

Section: Methodsmentioning

confidence: 99%

Reciprocal Regulation of Presynaptic and Postsynaptic Proteins in Bipolar Spiral Ganglion Neurons by Neurotrophins

Flores‐Otero¹,

Xue²,

Davis³

2007

J. Neurosci.

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A unifying principle of sensory system organization is feature extraction by modality-specific neuronal maps in which arrays of neurons show systematically varied response properties and receptive fields. Only beginning to be understood, however, are the mechanisms by which these graded systems are established. In the peripheral auditory system, we have shown previously that the intrinsic firing features of spiral ganglion neurons are influenced by brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3). We now show that is but a part of a coordinated package of neurotrophin actions that also includes effects on presynaptic and postsynaptic proteins, thus encompassing the input, transmission, and output functions of the spiral ganglion neurons. Using immunocytochemical methods, we determined that proteins targeted to opposite ends of the neuron were organized and regulated in a reciprocal manner. AMPA receptor subunits GluR2 and GluR3 were enriched in base neurons compared with their apex counterparts. This distribution pattern was enhanced by exposure to BDNF but reduced by NT-3. SNAP-25 and synaptophysin were distributed and regulated in the mirror image: enriched in the apex, enhanced by NT-3 and reduced by BDNF. Moreover, we used a novel coculture to identify potential endogenous sources of neurotrophins by showing that sensory receptors from different cochlear regions were capable of altering presynaptic and postsynaptic protein levels in these neurons. From these studies, we suggest that BDNF and NT-3, which are systematically distributed in complementary gradients, are responsible for orchestrating a comprehensive set of electrophysiological specializations along the frequency contour of the cochlea.

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

Cited by 41 publications

References 36 publications

The SNAP-25 Linker as an Adaptation Toward Fast Exocytosis

The SNAP-25 Linker as an Adaptation Toward Fast Exocytosis

Domain Requirement for the Membrane Trafficking and Targeting of Syntaxin 1A

Reciprocal Regulation of Presynaptic and Postsynaptic Proteins in Bipolar Spiral Ganglion Neurons by Neurotrophins

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