An α-Helical Minimal Binding Domain within the H3 Domain of Syntaxin Is Required for SNAP-25 Binding

Zhong, Pingyu; Chen, Yu A.; Tam, Deborah D.; Chung, David; Scheller, Richard H.; Miljanich, George P.

doi:10.1021/bi9625408

Cited by 40 publications

(32 citation statements)

References 21 publications

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“…Data were measured both at pH 8.0 and 5.5, to reflect the purification and crystallization conditions, respectively; pH 5.5 was as close to the crystallization pH as was obtainable without causing the protein to precipitate. Consistent with previous studies (25,26), the CD spectra indicate that the H3 polypeptide is mostly helical. The average T m for the wild type H3 oligomer is 47°C at pH 8.0 and 58°C at pH 5.5 (Fig.…”

Section: H3 Forms Oligomers In Solution-supporting

confidence: 91%

Self-association of the H3 Region of Syntaxin 1A

Misura¹,

Scheller²,

Weis³

2001

Journal of Biological Chemistry

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Intracellular membrane fusion requires SNARE proteins found on the vesicle and target membranes. SNAREs associate by formation of a parallel four-helix bundle, and it has been suggested that formation of this complex promotes membrane fusion. The membrane proximal region of the cytoplasmic domain of the SNARE syntaxin 1A, designated H3, contributes one of the four helices to the SNARE complex. In the crystal structure of syntaxin 1A H3, four molecules associate as a homotetramer composed of two pairs of parallel helices that are anti-parallel to each other. The H3 oligomer observed in the crystals is also found in solution, as assessed by gel filtration and chemical cross-linking studies. The crystal structure reveals that the highly conserved Phe-216 packs against conserved Gln-226 residues present on the anti-parallel pair of helices. Modeling indicates that Phe-216 prevents parallel tetramer formation. Mutation of Phe-216 to Leu appears to allow formation of parallel tetramers, whereas mutation to Ala destabilizes the protein. These results indicate that Phe-216 has a role in preventing formation of stable parallel helical bundles, thus favoring the interaction of the H3 region of syntaxin 1a with other proteins involved in membrane fusion.Eukaryotic cells transport cargo between different intracellular compartments, and release selective cargo into the extracellular space. This task requires that transport vesicles bud from the membranes of organelles and fuse specifically with target membranes. An extensively studied example of this process is synaptic vesicle exocytosis, in which neurotransmitter-filled vesicles fuse with the plasma membrane of a presynaptic neuron to release neurotransmitter into the synaptic cleft (1). The process of directed docking and fusion of vesicles is mediated by several proteins; these include the soluble NSF 1 attachment proteins (SNAPs), the SNAP receptor proteins (SNAREs) NSF, Sec1 proteins, and the Rab small GTPases. In neurons, the SNARE proteins involved in this process are VAMP2, syntaxin 1a, and SNAP-25.The SNARE proteins are believed to be directly involved in the intracellular membrane fusion event. SNAREs are associated with vesicle (v-SNARE) and target (t-SNARE) membranes and can interact with each other by forming stable four-helix bundles (SNARE complexes). The synaptic vesicle v-SNARE VAMP2 contains a small cytoplasmic domain followed by a C-terminal transmembrane anchor, and the cytoplasmic domain is largely unstructured in isolation (2, 3). The t-SNARE SNAP-25 is attached to the plasma membrane via palmitoylation of four cysteine residues and displays a significant amount of disorder in isolation in solution (2, 4). However, both VAMP2 and SNAP-25 adopt helical conformations when part of the SNARE complex. The t-SNARE syntaxin 1a consists of an N-terminal domain termed Habc, a region of ϳ9 kDa termed H3, and a C-terminal transmembrane anchor. The Habc domain is a three-helix bundle (5, 6), and the H3 region forms an amphipathic helix that associates with either ...

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Section: H3 Forms Oligomers In Solution-supporting

confidence: 91%

Self-association of the H3 Region of Syntaxin 1A

Misura¹,

Scheller²,

Weis³

2001

Journal of Biological Chemistry

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“…Injections of TAX74 also reversibly inhibited transmitter release (Table 1). A decreased potency of TAX74 probably reflects additional sequence in TAX86 that help stabilize H3 domain structure (28). In contrast to the above, microinjecting buffer alone (not shown) or the truncated H3 domain TAX50 to a concentration at which TAX86 produced maximal effects (Fig.…”

Section: Molecular Characterization Of Squid Syntaxinmentioning

confidence: 83%

Disruption of syntaxin-mediated protein interactions blocks neurotransmitter secretion

O’Connor

Heuss

Bello

et al. 1997

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

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The membrane protein syntaxin participates in several protein-protein interactions that have been implicated in neurotransmitter release. To probe the physiological importance of these interactions, we microinjected into the squid giant presynaptic terminal botulinum toxin C1, which cleaves syntaxin, and the H3 domain of syntaxin, which mediates binding to other proteins. Both reagents inhibited synaptic transmission yet did not affect the number or distribution of synaptic vesicles at the presynaptic active zone. Recombinant H3 domain inhibited the interactions between syntaxin and SNAP-25 that underlie the formation of stable SNARE complexes in vitro. These data support the notion that syntaxin-mediated SNARE complexes are necessary for docked synaptic vesicles to fuse.Synaptic transmission is based on the rapid exocytosis of neurotransmitters from synaptic vesicles (1). Exocytosis occurs at active zones, where vesicles are docked for Ca 2ϩ -triggered fusion with the presynaptic plasma membrane (2). The exocytotic event is coupled to an endocytotic cascade that recycles fused synaptic vesicles for additional rounds of regulated fusion (3). Several proteins of the synaptic vesicle, plasma membrane, and presynaptic cytosol have been implicated in neurotransmitter release (4, 5). Syntaxin-1A, a neuronal homologue of a family of proteins involved in membrane fusion (6-8), plays a key role in exocytosis. First, this plasma membrane protein is a substrate of botulinum C1 (Bot-C1), a toxin that inhibits transmitter release by the proteolytic activity of its disulfide-linked light chain (9). Second, Drosophila lacking syntaxin suffer embryonic lethality and a loss of synaptic transmission (10, 11).In vitro, syntaxin 1A participates in a number of interactions that are believed to be essential for neurotransmitter release. Syntaxin-binding partners include Ca 2ϩ channels, complexin, and synaptotagmin, a putative Ca 2ϩ receptor for transmitter release, as well as mammalian homologues of the Unc13 and secϪ1 protein families (4,5,12). Syntaxin also binds SNAP-25 (synaptosomal associated protein of 25 kDa), a peripheral plasma membrane protein, and synaptobrevin, a major integral membrane protein of synaptic vesicles (13,14). Together these proteins form a stable ternary complex of 7S (15) that resists dissociation by SDS (14). This 7S complex has been proposed to mediate the docking and fusion of synaptic vesicles at the presynaptic plasma membrane, because it binds the fusion catalyzing proteins SNAP (soluble NSF attachment protein) and NSF (for N-ethylmaleimide-sensitive fusion protein) (15, 16). The resulting higher-order SNARE (SNAP receptor) complex of 20S dissociates upon hydrolysis of ATP by NSF, and this dissociation is thought to promote membrane fusion (15-17).To investigate how syntaxin's interactions with presynaptic proteins contribute to the trafficking events that underlie neurotransmitter release, we have perturbed syntaxin function in vivo by microinjecting Bot-C1 and recombinant fragments of synt...

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“…The soluble coiled coil domain of syntaxin 1a (the H3 domain) is known to directly interact with SNAP-25, forming a binary complex (21,28). It was shown previously that addition of a syntaxin 1a H3 peptide construct inhibits fusion of norepinephrine-containing granules with the plasma membrane of PC12 cells (28), presumably by competing with the endogenous protein.…”

Section: Resultsmentioning

confidence: 99%

Syntaxin 3 is required for cAMP-induced acid secretion: streptolysin O-permeabilized gastric gland model

Ammar

Zhou

Forte

et al. 2002

American Journal of Physiology-Gastrointestinal and Liver Physi

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-ATPase translocation from cytoplasmic tubulovesicles to apical plasma membrane in parietal cells, resulting in HCl secretion. We studied the mechanisms involved in tubulovesicle translocation with a permeabilized gland system. Streptolysin O (SLO)-treated glands were permeabilized such that exogenous fluorescently labeled actin incorporated into cytoskeleton in a pattern mimicking endogenous F-actin. As shown by accumulation of the weak base aminopyrine (AP), SLO-permeabilized glands are stimulated to secrete acid by addition of cAMP and ATP and inhibited by proton pump inhibitors. Direct visualization with the fluorescent pH probe Lysosensor showed acid accumulation in glandular lumen and parietal cell canaliculi. ME-3407, an antiulcer drug with inhibitory action implicated to involve ezrin, inhibited AP uptake in and effectively released ezrin from intact and SLOpermeabilized glands. In contrast, wortmannin, an effective secretion inhibitor in intact glands, had minimal effects on ezrin or AP accumulation in SLO-permeabilized glands. The finding that SNARE protein syntaxin 3 is associated with H ϩ ,K ϩ -ATPase-containing tubulovesicles suggested that it is involved in membrane fusion. Addition of recombinant syntaxin 3, but not syntaxin 5 or heat-denatured syntaxin 3, dose-dependently inhibited acid secretion. Our studies are consistent with a membrane recycling hypothesis that activation of protein kinase cascades leads to SNARE-mediated fusion of H ϩ ,K ϩ -ATPase-containing tubulovesicles to apical plasma membrane. SNARE hypothesis; membrane fusion; ezrin; wortmannin; hydrogen,potassium-adenosinetriphosphatase THE DEVELOPMENT OF PERMEABILIZED cell preparations has provided a valuable experimental tool linking in vitro studies that use subcellular fractions with direct assays of cellular function. In parietal cell physiology, a number of important functions have been elucidated through the study of gastric glands permeabilized with detergents (reviewed in Ref. 13). One of the major drawbacks with many detergent-like molecules (digitonin or saponin) was that the treated glands were unable to transform from the resting to the stimulated state, although preparations stimulated before treatment could be maintained to secrete acid (10, 15). It was then discovered that pore-forming bacterial toxins permeabilize gastric glands while allowing the transition of resting to a stimulated state (22). These toxins have been valuable in generating permeable cell systems that facilitate better understanding of the parietal cell activation process (19,21,26). Depending on the size of the plasma membrane pore, one can introduce a variety of molecules into the cell to facilitate or interfere with normal functional activity. In previous work, we found ␣-toxin to be a superior permeabilization agent over digitonin and other pore-forming bacterial toxins. We determined (19) that stimulation was dependent on addition of cAMP, that addition of ATP led to synthesis of cAMP and therefore could trigger stimulation, and that addition o...

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An α-Helical Minimal Binding Domain within the H3 Domain of Syntaxin Is Required for SNAP-25 Binding

Cited by 40 publications

References 21 publications

Self-association of the H3 Region of Syntaxin 1A

Self-association of the H3 Region of Syntaxin 1A

Disruption of syntaxin-mediated protein interactions blocks neurotransmitter secretion

Syntaxin 3 is required for cAMP-induced acid secretion: streptolysin O-permeabilized gastric gland model

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