13 research outputs found
Correction: Modification of a Hydrophobic Layer by a Point Mutation in Syntaxin 1A Regulates the Rate of Synaptic Vesicle Fusion
Modification of a Hydrophobic Layer by a Point Mutation in Syntaxin 1A Regulates the Rate of Synaptic Vesicle Fusion
Both constitutive secretion and Ca(2+)-regulated exocytosis require the assembly of the soluble N-ethylmaleimideâsensitive factor attachment protein receptor (SNARE) complexes. At present, little is known about how the SNARE complexes mediating these two distinct pathways differ in structure. Using the Drosophila neuromuscular synapse as a model, we show that a mutation modifying a hydrophobic layer in syntaxin 1A regulates the rate of vesicle fusion. Syntaxin 1A molecules share a highly conserved threonine in the C-terminal +7 layer near the transmembrane domain. Mutation of this threonine to isoleucine results in a structural change that more closely resembles those found in syntaxins ascribed to the constitutive secretory pathway. Flies carrying the I254 mutant protein have increased levels of SNARE complexes and dramatically enhanced rate of both constitutive and evoked vesicle fusion. In contrast, overexpression of the T254 wild-type protein in neurons reduces vesicle fusion only in the I254 mutant background. These results are consistent with molecular dynamics simulations of the SNARE core complex, suggesting that T254 serves as an internal brake to dampen SNARE zippering and impede vesicle fusion, whereas I254 favors fusion by enhancing intermolecular interaction within the SNARE core complex
The Assembly of SDS-Resistant SNARE Complexes Is Increased in <i>syx<sup>3â69</sup></i> Mutant Flies at Permissive Temperatures
<div><p>(A and B) The amount of SDS-resistant 7S complex as well as the multimeric complex is significantly increased in homozygous <i>syx<sup>3â69</sup></i> mutant flies at 22 °C. A representative Western blot shows the syntaxin 1A monomer, the 7S SNARE complex, and the multimeric complex obtained from heads of the wild type (+/+) and the mutant (<i>syx</i> [A]). The relative level of total proteins loaded in the lanes is illustrated by the intensity of tubulin. Histograms of ratios of the 7S and multimeric complexes to monomer in wild-type and <i>syx<sup>3â69</sup></i> mutant flies are shown in (B). *, <i>p</i> < 0.05.</p>
<p>(C and D) Western blots show the SNARE complexes, tubulin, syntaxin 1A, SNAP-25, and N-Syb from fly-head extract inputs, immunoprecipitates (pellets), and SDS head extracts. The head extracts used for immunoprecipitation were obtained from the wild-type (+/+) and the <i>syx<sup>3â69</sup></i> mutant <i>(syx)</i> flies and incubated with Sepharose beadâcoupled SNAP-25 antibodies. Inputs and precipitates were boiled in SDS sample buffer before loading. One of the SDS head-extract samples was not boiled to preserve the SDS-resistant SNARE complexes. The Western blot was sequentially probed with a syntaxin 1A antibody for SNARE complexes and syntaxin 1A monomers, a different SNAP-25 antibody for SNAP-25 (which also recognizes SNAP-24), an N-Syb antibody for N-Syb, and a tubulin antibody for tubulin. Note that tubulin is absent from pellet and that the SNARE complexes are only present in the unboiled SDS head extracts. SNAP-24 is present only in the input lanes and the SDS extracts; it is absent from the pellets because the IP antibody is specific for SNAP-25. Compared to the wild-type lanes, there are slightly more N-Syb, syntaxin 1A, and SNAP-25 in the precipitates from the <i>syx<sup>3â69</sup></i> mutant <i>(syx)</i> mutant flies.</p></div
The T254I Mutation Exerts Dominant Positive Effects on Both Constitutive and Ca<sup>2+</sup>-Triggered Vesicle Fusion in <i>syx<sup>3â69</sup></i> Heterozygotes
<div><p>(A) Models of multimeric SNARE complexes found in the wild type (+/+), the homozygous <i>syx<sup>3â69</sup></i> mutant <i>(syx/syx),</i> and the heterozygous <i>syx<sup>3â69</sup></i> mutant (<i>syx/</i>+). Oligomerization of a mixture of the wild-type and the mutant SNARE complex predicts that the T254I mutant syntaxin 1A has dominant positive effects on vesicle fusion. The wild-type syntaxin 1A is illustrated in red, whereas the T254I mutant syntaxin 1A is in blue. For simplicity, SNAP-25 is omitted from these models. PM, plasma membrane; SV, synaptic vesicle.</p>
<p>(BâD) Representative traces of minis and evoked EPSPs in the heterozygous larvae are shown in (B) and (C), respectively. The average mini frequency and quantal content are shown in (D).</p>
<p>(E) The histogram shows that the normalized mini frequency in the heterozygote is significantly higher than that in the wild type, but much lower than that in the homozygous mutant. ***, <i>p</i> < 0.001.</p>
<p>(F) A histogram of the average EPSP amplitude recorded from the wild type (+/+), the heterozygote (<i>syx/</i>+), and the homozygote <i>(syx/syx)</i> at three different Ca<sup>2+</sup> concentrations: 0.4 mM, 0.8 mM, and 1 mM. At these Ca<sup>2+</sup> concentrations, the amplitude of EPSPs in the wild type is consistently lower than those in the heterozygote and the homozygote. Note that the difference between the wild type and mutants (the heterozygote and the homozygote) appears more dramatic at lower Ca<sup>2+</sup> concentrations. At higher Ca<sup>2+</sup> concentrations, this difference becomes smaller because EPSPs reach the âceilingâ set by the reversal potential. The amplitude of EPSPs is similar between the heterozygote and the homozygote. **, <i>p</i> < 0.01; ***, <i>p</i> < 0.001.</p></div
The SNARE Complexes Remain in <i>syx<sup>3â69</sup></i> Mutant Flies at the Restrictive Temperature
<div><p>(A and B) The SDS-resistant complex is not obviously affected in homozygous <i>syx<sup>3â69</sup></i> mutant flies at restrictive temperatures. A representative Western blot shows the syntaxin 1A monomer, the 7S SNARE complex, and the multimeric complex obtained from heads of the wild type (+/+) and the <i>syx<sup>3â69</sup></i> mutant (A). A control for total protein loaded is illustrated by the intensity of tubulin (bottom). Histograms of ratios of the 7S and multimeric complexes to monomer in wild-type (+/+) and <i>syx<sup>3â69</sup></i> mutant flies are shown in (B). Unless specifically noted, the complex-to-monomer ratio is normalized to that of the wild type at room temperature in this and other SNARE complex histograms. The SNARE complex was extracted from flies either at room temperature (âŒ22 °C) or after exposure to 38 °C for 20 min, as described above [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050072#pbio-0050072-b021" target="_blank">21</a>].</p>
<p>(C) An example of Western blots showing the SNARE complex in the wild-type (+/+), <i>comatose (comt<sup>tp7</sup>),</i> and <i>syx<sup>3â69</sup></i> mutant flies. The SNARE complex accumulates in the <i>comt<sup>tp7</sup></i> mutant, likely as a result of a block of the NSF ATPase activity at the restrictive temperature. Note that even though relatively less protein was loaded in the <i>syx<sup>3â69</sup></i> lanes (as judged by the intensity of the syntaxin band), the 7S complex remains in paralyzed <i>syx<sup>3â69</sup></i> flies.</p></div
Structural Modeling Suggests That the T254I Mutation in Syntaxin 1A Increases Direct Molecular Interactions within the +7 Layer
<div><p>(A) The core complex layers of the synaptic SNARE complex (1SFFC), consisting of two α-helical bundles from SNAP-25 (SN1 and SN2) and one bundle each from syntaxin 1A (Syx) and synaptobrevin (Syb), are shown (adapted from [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050072#pbio-0050072-b006" target="_blank">6</a>]). Although initially obtained as <i>cis</i> complexes with truncated SNAREs [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050072#pbio-0050072-b006" target="_blank">6</a>], these layers of the core complex are most likely found in pre-fusion <i>trans</i> SNARE complexes.</p>
<p>(B) Crystal structures of +1 and +7 layers of the synaptic core complex (1SFC [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050072#pbio-0050072-b006" target="_blank">6</a>]) show tightly and loosely packed bundles, respectively. Note the void space within the +7 layer. Our structural modeling shows that the mutation of the hydrophilic threonine at position 251 (which is equivalent to position 254 in <i>Drosophila</i> syntaxin 1A) to a hydrophobic isoleucine results in a relatively tightly packed +7 layer. This may allow direct molecular interactions between syntaxin 1A with its neighboring bundles from SNAP-25 and synaptobrevin. It is hypothesized that the T254I mutation in <i>syx<sup>3â69</sup></i> stimulates vesicle fusion by lowering the energy barrier for zippering of the SNARE complex.</p>
<p>(C) Representative +7 layer abstracted from the crystal structure of the endosomal SNARE (1GL2 [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050072#pbio-0050072-b020" target="_blank">20</a>]). Note that this layer is tightly packed and similar to the T251I mutant layer. Given the evolutionary conservation of hydrophobic residues at the +7 layer among âconstitutiveâ syntaxin orthologs (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050072#pbio-0050072-g001" target="_blank">Figure 1</a>B), this structural resemblance suggests that the T254I mutant syntaxin 1A may function as a constitutive syntaxin to promote vesicle fusion.</p></div
Behavioral and Electrophysiological Analyses Reveal That Synaptic Transmission Is Not Blocked in the <i>syx<sup>3â69</sup></i> Mutant Fly at Restrictive Temperatures
<div><p>(A and B) Temperature-sensitive paralysis and recovery of the <i>syx<sup>3â69</sup></i> mutant fly. (A) shows the still image of both wild type (+/+) and the <i>syx<sup>3â69</sup></i> mutant before, during, and after exposure to the restrictive temperature (38 °C). Although the wild-type flies are not paralyzed at 38 °C, the <i>syx<sup>3â69</sup></i> mutant flies are. However, the <i>syx<sup>3â69</sup></i> flies recover rapidly to standing position within 2â3 min once returned to the permissive temperature. The quantification of the recovery kinetics is shown in (B). Error bars in this and all other figures indicate the standard errors.</p>
<p>(C) The paralyzed <i>syx<sup>3â69</sup></i> mutant flies remain capable of responding to stimuli via the polysynaptic giant fiber (GF) pathway. The flies are anchored on a glass slide upside down with modeling clay while a stimulating electrode is inserted into one of the compound eyes (arrows). The <i>syx<sup>3â69</sup></i> fly constantly shakes its legs, head, and abdomen while paralyzed at 38 °C. In response to electrical stimulation of the giant fiber neuron, the mutant fly extends its legs phase-locked with each stimulus. However, the <i>Shi<sup>ts1</sup></i> fly is completely paralyzed and does not respond to the stimuli at the same restrictive temperature. The right-most panels summarize the cumulative spontaneous and electrical stimulationâevoked movements of legs in <i>syx<sup>3â69</sup></i> flies and the lack of leg movement in <i>Shi<sup>ts1</sup></i> flies. These behavioral observations strongly indicate that exposing the <i>syx<sup>3â69</sup></i> fly to 38 °C does not block synaptic transmission. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050072#pbio-0050072-sv004" target="_blank">Videos S4</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050072#pbio-0050072-sv005" target="_blank">S5</a>.</p>
<p>(D) Recordings from indirect flight muscles confirm that synaptic transmission is not blocked in <i>syx<sup>3â69</sup></i> flies at the restrictive temperature. Action potentials in DLMs driven by polysynaptic stimuli along the giant fiber pathway remain the same in the <i>syx<sup>3â69</sup></i> mutant fly as in the wild-type control fly before, during, and after exposure to the restrictive temperature. Synaptic-induced high-frequency action potentials are often observed in both the wild type and the <i>syx<sup>3â69</sup></i> mutant (unpublished data). These high-frequency action potentials also occur spontaneously in the mutant. (An example is shown in the inset box.)</p></div
ERG Recordings Show That Synaptic Transmission Is Not Blocked in the <i>syx<sup>3â69</sup></i> Mutant Fly at Restrictive Temperatures
<div><p>(A) ERGs are obtained from the wild-type fly at the permissive temperature (20 °C), during the restrictive temperature (38 °C), and during recovery at 20 °C following a brief white-light stimulation of the compound eye. The spikes before and following the sustained photoreceptor potential are called âonâ and âoffâ transient potentials (arrows), respectively. They are thought to reflect synaptic transmission from the photoreceptor to downstream interneurons. Note that these transient potentials are not significantly affected at 38 °C. The extracellular recording electrode also detects light-induced high-frequency action potentials (arrowheads), which normally result in startle escape.</p>
<p>(B) The âonâ and âoffâ transient potentials are absent in <i>Shi<sup>ts1</sup></i> flies exposed at 33 °C, consistent with a conditional block of vesicle recycling.</p>
<p>(C) Under the same experimental conditions, the âonâ and âoffâ transient potentials in the <i>syx<sup>3â69</sup></i> mutant fly remain essentially similar to those observed in the wild-type fly. Even though the amplitude of photoreceptor potentials is reduced and the duration of recovery is prolonged, synaptic transmission is not blocked at 38 °C in both the wild-type and the mutant fly. Additionally, the mutant fly also displays light-induced high-frequency action potentials (arrowheads), even though it is paralyzed.</p></div
Conservation and Divergence of Threonine 254 among Different Syntaxin Orthologs
<div><p>(A) Proposed model of SNARE complex assembly and disassembly in a synaptic vesicle cycle (adapted from [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050072#pbio-0050072-b005" target="_blank">5</a>]). (1) Synaptobrevin forms a partial <i>trans</i> SNARE complex with syntaxin 1A and SNAP-25. (2) By zippering in an N- to C-termini direction, the SNARE proteins form a <i>trans</i> complex and bring the synaptic vesicle close to the plasma membrane. SNARE-mediated synaptic vesicle exocytosis occurs either spontaneously (3) or evoked by Ca<sup>2+</sup> (4). (5) <i>cis</i> SNARE complexes are thought to be disassembled by NSF ATPase prior to vesicle recycling. ER, endoplasmic reticulum; PM, plasma membrane; SV, synaptic vesicle.</p>
<p>(B) Alignment of amino acids (aa) around position T254 in the <i>Drosophila</i> syntaxin 1A or equivalent residues in syntaxin orthologs from a variety of animals, yeast, and the plant <i>Arabidopsis</i>. The top panel shows a cartoon of syntaxin 1A and the region of the alignment. Syntaxins are organized as âplasma membraneâ or âintracellular compartmentsâ according to their cellular distributions. With the exception of syntaxin 4, most plasma membrane syntaxins are known to function in presynaptic terminals or neurosecretory cells for Ca<sup>2+</sup>-regulated exocytosis. Note that T254 is highly conserved among âpresynapticâ syntaxin 1A, 2, and 3A molecules. We call all other syntaxin orthologs shown here âconstitutiveâ syntaxins because they are used for constitutive secretion on the plasma membrane (PM) and intracellular compartments, such as the endosome and the lysosome, the <i>cis</i> and <i>trans</i> Golgi network (Golgi network), and endoplasmic reticulum (ER) [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050072#pbio-0050072-b001" target="_blank">1</a>]. The yeast plasma membrane syntaxin orthologs SSO1 and SSO2, and syntaxins 4 and 131 from <i>Arabidopsis</i> are also shown here. (A more complete alignment can be see in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050072#pbio-0050072-sg001" target="_blank">Figure S1</a>.) Unlike the synaptic syntaxins, syntaxin 4 and most syntaxin 11s have a valine (V) at the 254 equivalent position, syntaxins 6, 7, 12, 16, and 17 a leucine (L), and syntaxin 5 an isoleucine (I). The isoleucine found in the <i>syx<sup>3â69</sup></i> mutant resembles some of the wild-type syntaxin orthologs used for constitutive secretion. The core complex layers from 0 to +8 are identified at the bottom. The aa sequence was obtained from the NIH's National Center for Biotechnology Information (NCBI; <a href="http://www.ncbi.nlm.nih.gov" target="_blank">http://www.ncbi.nlm.nih.gov</a>) and aligned using the software DNAStar.</p></div