Modification of a Hydrophobic Layer by a Point Mutation in Syntaxin 1A Regulates the Rate of Synaptic Vesicle Fusion

Lagow, Robert D.; Bao, Hong; Cohen, Evan N.; Daniels, Richard W.; Zuzek, Aleksej; Williams, Wade H.; Macleod, Gregory T.; Sutton, R. Bryan; Zhang, Bing

doi:10.1371/journal.pbio.0050072

Cited by 46 publications

(61 citation statements)

References 64 publications

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“…In the original paper (Littleton et al, 1998), the syx 3-69 T254I mutation was shown to block SV exocytosis because of a failure to form 7S SNARE complexes. In stark contrast, the new syx study (Lagow et al, 2007) utterly contradicts the earlier report, showing that the mutant T-I substitution confers a dominant positive effect on Syntaxin-1A, promoting 7S-SNARE-complex formation and increasing SV fusion. This is not at all compatible with an additive defect with rbo ts in blocking SV exocytosis.…”

Section: Discussioncontrasting

confidence: 94%

“…Moreover, rbo ts ;syx 3-69 double mutants displayed a severe synergistic defect in FM endocytosis at permissive temperature. These new results, together with those of Lagow et al (Lagow et al, 2007) lead us to conclude that there is a requirement for RBO in endocytosis, which becomes more demanding in rbo ts ;syx 3-69 double mutants owing to an increased need to recycle SVs to keep up with increased fusion rate.…”

Section: Discussionmentioning

confidence: 56%

“…Doublehomozygous mutants paralyze at a lower temperature than either single mutant, suggesting that RBO and Syntaxin-1A act synergistically in SV exocytosis. However, a recently published analysis of syx revealed that this allele is in fact a dominant positive mutation causing elevated Syntaxin-1A function (Lagow et al, 2007). This revelation forced us to question our hypothesis.…”

Section: Introductionmentioning

confidence: 91%

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Rolling blackout is required for bulk endocytosis in non-neuronal cells and neuronal synapses

Vijayakrishnan

Woodruff

Broadie

2009

Journal of Cell Science

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Rolling blackout (RBO) is a Drosophila EFR3 integral membrane lipase. A conditional temperature-sensitive (TS) mutant (rbots) displays paralysis within minutes following a temperature shift from 25°C to 37°C, an impairment previously attributed solely to blocked synaptic-vesicle exocytosis. However, we found that rbots displays a strong synergistic interaction with the Syntaxin-1A TS allele syx3-69, recently shown to be a dominant positive mutant that increases Syntaxin-1A function. At neuromuscular synapses, rbots showed a strong defect in styryl-FM-dye (FM) endocytosis, and rbots;syx3-69 double mutants displayed a synergistic, more severe, endocytosis impairment. Similarly, central rbots synapses in primary brain culture showed severely defective FM endocytosis. Non-neuronal nephrocyte Garland cells showed the same endocytosis defect in tracer-uptake assays. Ultrastructurally, rbots displayed a specific defect in tracer uptake into endosomes in both neuronal and non-neuronal cells. At the rbots synapse, there was a total blockade of endosome formation via activity-dependent bulk endocytosis. Clathrin-mediated endocytosis was not affected; indeed, there was a significant increase in direct vesicle formation. Together, these results demonstrate that RBO is required for constitutive and/or bulk endocytosis and/or macropinocytosis in both neuronal and non-neuronal cells, and that, at the synapse, this mechanism is responsive to the rate of Syntaxin-1A-dependent exocytosis.

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Section: Discussioncontrasting

confidence: 94%

Section: Discussionmentioning

confidence: 56%

Section: Introductionmentioning

confidence: 91%

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Rolling blackout is required for bulk endocytosis in non-neuronal cells and neuronal synapses

Vijayakrishnan

Woodruff

Broadie

2009

Journal of Cell Science

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“…A structure-function study of a SNARE complex with a single site mutation near the C-terminal end of the syntaxin SNARE motif is also consistent with a lower stability and folding rate of the C-terminal compared to the N-terminal end of SNARE complex formation (35). The layer ϩ7 T254I mutation enhances constitutive and evoked fusion and also changes the structure to one that is more similar to SNARE complexes associated with constitutive fusion.…”

Section: Helix I As a Nucleation Site For Trans-snare Complex Formation?mentioning

confidence: 57%

Dynamic structure of lipid-bound synaptobrevin suggests a nucleation-propagation mechanism for trans-SNARE complex formation

Ellena

Liang

Wiktor

et al. 2009

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

107

130

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The synaptic vesicle protein synaptobrevin engages with syntaxin and SNAP-25 to form the SNARE complex, which drives membrane fusion in neuronal exocytosis. In the SNARE complex, the SNARE motif of synaptobrevin forms a 55-residue helix, but it has been assumed to be mostly unstructured in its prefusion form. NMR data for full-length synaptobrevin in dodecylphosphocholine micelles reveals two transient helical segments flanked by natively disordered regions and a third more stable helix. Transient helix I comprises the most Nterminal part of the SNARE motif, transient helix II extends the SNARE motif into the juxtamembrane region, and the more stable helix III is the transmembrane domain. These helices may have important consequences for SNARE complex folding and fusion: helix I likely forms a nucleation site, the C-terminal disordered SNARE motif may act as a folding arrest signal, and helix II likely couples SNARE complex folding and fusion.structure ͉ dynamics ͉ membrane fusion ͉ SNARE proteins ͉ NMR S ynaptic release of neurotransmitter requires the docking and fusion of synaptic vesicles at the presynaptic membrane. The fusion reaction is thought to be mediated by the formation of the SNARE complex from its components syntaxin-1a and SNAP-25 in the target membrane and synaptobrevin-2 (Syb) in the vesicle membrane (1). These three proteins together form a complex that consists of a thermally very stable coiled-coil four-helix bundle, as revealed by the crystal structure of the soluble SNARE core complex (2). SNAP-25 contributes two helices to this complex and syntaxin and synaptobrevin each contribute one. More recently, the structure of the postfusion cis-SNARE complex with its C-terminal transmembrane (TM) domain extensions was solved at 3.4 Å resolution (3). Most interestingly, the helices continue through the juxtamembrane linker region and into the membrane, suggesting that force could be transmitted through this region into the membrane leading to membrane bending and eventually membrane fusion. This interesting finding immediately raises the question: how exactly is the assembly and folding of the four-helix bundle coupled into the membrane and how does the energy derived from this reaction ultimately fuse two different membranes into one? A common notion is that an initial trans-SNARE complex forms by pairing vesicle and target membrane SNAREs from their Nterminal ends and progressively folds in a zipper-like fashion toward the C-terminal TM domains. This reaction is thought to pull the two membranes into closer contact until, at some stage they merge into a single membrane. However, structural data on a trans-SNARE complex do not yet exist. It is also not yet known whether zipperfolding of trans-SNARE complexes progresses smoothly and continuously into the membrane or whether this reaction is discontinuous and segmented in some fashion.To understand how the SNARE complex is formed and to find possible reaction intermediates, several structural studies of SNARE proteins in isolation or in binary comp...

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“…The ϩ7 layer of the SNARE complex has been proposed to be one of the key differences between SNARE complexes that fuse constitutively and those that undergo triggered fusion (24). For Drosophila SNAREs, the T254I mutation enhances both constitutive and evoked neurotransmitter release, suggesting that the tighter packing of isoleucine promotes SNARE formation and fusion, whereas the presence of threonine prevents good packing (Fig.…”

Section: Resultsmentioning

confidence: 99%

The Structure of the Yeast Plasma Membrane SNARE Complex Reveals Destabilizing Water-filled Cavities

Strop

Kaiser

Vrljic

et al. 2008

Journal of Biological Chemistry

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SNARE proteins form a complex that leads to membrane fusion between vesicles, organelles, and plasma membrane in all eukaryotic cells. We report the 1.7 Å resolution structure of the SNARE complex that mediates exocytosis at the plasma membrane in the yeast Saccharomyces cerevisiae. Similar to its neuronal and endosomal homologues, the S. cerevisiae SNARE complex forms a parallel four-helix bundle in the center of which is an ionic layer. The S. cerevisiae SNARE complex exhibits increased helix bending near the ionic layer, contains waterfilled cavities in the complex core, and exhibits reduced thermal stability relative to mammalian SNARE complexes. Mutagenesis experiments suggest that the water-filled cavities contribute to the lower stability of the S. cerevisiae complex.Membrane fusion is a fundamental and highly regulated process that is required for the transport of proteins, lipids, and metabolites in all eukaryotes. Highly conserved SNARE 2 (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins play a key role in the fusion of a transport vesicle with its target membrane (1, 2). Specific sets of SNAREs located on vesicle and target membranes form a complex that draws together SNARE transmembrane domains, leading to juxtaposition and fusion of the two lipid membranes. SNARE-mediated fusion processes are either constitutive or triggered, and they require additional SNARE-interacting factors, such as Munc13, Sec1/Munc 18-like proteins, synaptotagmins, and complexins (3, 4).Neuronal SNAREs play a key role in the fusion of synaptic vesicles with the plasma membrane, a process that is critical for neurotransmission (5). Synaptic vesicles dock at the plasma membrane and upon cell depolarization and Ca 2ϩ entry fuse with the plasma membrane, thus releasing neurotransmitters into the synaptic cleft. The neuronal SNARE complex is composed of synaptobrevin 2, which is primarily localized to synaptic vesicles, and syntaxin 1A and SNAP-25, which are primarily associated with the plasma membrane.The family of yeast SNAREs mediates constitutive fusion between transport vesicles and intracellular organelles or the plasma membrane (6, 7). Yeast has been a valuable system for studying membrane fusion and vesicular transport because of the ease of genetic and biochemical manipulations. Yeast is also one of the first organisms that evolved to utilize intracellular membrane fusion and therefore provides a valuable snapshot of the fusion machinery in lower eukaryotes. The Saccharomyces cerevisiae SNARE proteins involved in exocytosis at the plasma membrane (the synaptobrevin homologue Snc1p, the syntaxin homologue Sso1p, and the SNAP-25 homologue Sec9p) show assembly properties similar to their neuronal and endosomal homologues. Relative to the neuronal SNARE complex the S. cerevisiae SNARE complex exhibits decreased thermal stability, and prior to SNARE complex formation, Sso1p interacts with Sec9p with 1:1 stoichiometry (as opposed to a mixture of 1:1 and 1:2 for the neuronal SNAREs) (8, 9).Several...

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Modification of a Hydrophobic Layer by a Point Mutation in Syntaxin 1A Regulates the Rate of Synaptic Vesicle Fusion

Cited by 46 publications

References 64 publications

Rolling blackout is required for bulk endocytosis in non-neuronal cells and neuronal synapses

Rolling blackout is required for bulk endocytosis in non-neuronal cells and neuronal synapses

Dynamic structure of lipid-bound synaptobrevin suggests a nucleation-propagation mechanism for trans-SNARE complex formation

The Structure of the Yeast Plasma Membrane SNARE Complex Reveals Destabilizing Water-filled Cavities

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