Munc18-Bound Syntaxin Readily Forms SNARE Complexes with Synaptobrevin in Native Plasma Membranes

Munc18–1, a protein essential for regulated exocytosis in neurons and neuroendocrine cells, belongs to the family of Sec1/Munc18-like (SM) proteins. In vitro, Munc18–1 forms a tight complex with the SNARE syntaxin 1, in which syntaxin is stabilized in a closed conformation. Since closed syntaxin is unable to interact with its partner SNAREs SNAP-25 and synaptobrevin as required for membrane fusion, it has hitherto not been possible to reconcile binding of Munc18–1 to syntaxin 1 with its biological function. We now show that in intact and exocytosis-competent lawns of plasma membrane, Munc18–1 forms a complex with syntaxin that allows formation of SNARE complexes. Munc18–1 associated with membrane-bound syntaxin 1 can be effectively displaced by adding recombinant synaptobrevin but not syntaxin 1 or SNAP-25. Displacement requires the presence of endogenous SNAP-25 since no displacement is observed when chromaffin cell membranes from SNAP-25–deficient mice are used. We conclude that Munc18–1 allows for the formation of a complex between syntaxin and SNAP-25 that serves as an acceptor for vesicle-bound synaptobrevin and that thus represents an intermediate in the pathway towards exocytosis

Speeding up Directed Evolution: Combining the Advantages of Solid-Phase Combinatorial Gene Synthesis with Statistically Guided Reduction of Screening Effort

Efficient and economic methods in directed evolution at the protein, metabolic, and genome level are needed for biocatalyst development and the success of synthetic biology. In contrast to random strategies, semirational approaches such as saturation mutagenesis explore the sequence space in a focused manner. Although several combinatorial libraries based on saturation mutagenesis have been reported using solid-phase gene synthesis, direct comparison with traditional PCR-based methods is currently lacking. In this work, we compare combinatorial protein libraries created in-house via PCR versus those generated by commercial solid-phase gene synthesis. Using descriptive statistics and probabilistic distributions on amino acid occurrence frequencies, the quality of the libraries was assessed and compared, revealing that the outsourced libraries are characterized by less bias and outliers than the PCR-based ones. Afterward, we screened all libraries following a traditional algorithm for almost complete library coverage and compared this approach with an emergent statistical concept suggesting screening a lower portion of the protein sequence space. Upon analyzing the biocatalytic landscapes and best hits of all combinatorial libraries, we show that the screening effort could have been reduced in all cases by more than 50%, while still finding at least one of the best mutants

Synaptobrevin 2 Releases Munc18–1 from Complexed Syntaxin

<div><p>(A) Membrane sheets were incubated for 10 min in the absence (set to 100%) or presence of 10 μM of recombinant SNARE proteins as indicated, and Munc18–1 was quantified by immunofluorescence. Each experiment was performed six to seven times, values are given as mean ± standard deviation of the mean (SDM). Paired <i>t</i>-test analysis none:synaptobrevin 2 <i>p</i> < 0.0005 (<i>n</i> = 7); <i>t</i>-test synaptobrevin:SNAP-25 <i>p</i> < 0.005 (<i>n</i> = 6).</p> <p>(B) Incubation of membrane sheets with or without 10 μM synaptobrevin 2 for varying times, as indicated, followed by immunostaining for Munc18–1. Immunostaining intensity of immediately fixed membrane sheets was set to 100%. For each data point, four to six independent experiments were performed. Values are given as mean ± SEM.</p> <p>(C) Experiment as in (A), R-SNAREs were added during incubation as indicated. Values are given as mean ± standard deviation of the mean (SDM). Paired <i>t</i>-test synaptobrevin 2:none <i>p</i> < 0.00005 (<i>n</i> = 10), synaptobrevin 2:endobrevin <i>p</i> < 0.0005 (<i>n</i> = 10), and non-paired <i>t</i>-test synaptobrevin:VAMP4 <i>p</i> < 0.005 (<i>n</i> = 10 and 6).</p></div

Docking of Secretory Granules to Plasmalemmal Domains Enriched in Munc18–1

<div><p>(A) Plasma membrane sheet generated from a PC12 cell expressing the secretory granule marker NPY-GFP. Left, immunostaining for Munc18–1 (red channel); right, plasma membrane–docked, GFP-filled secretory granules (green channel). Circle indicates a fluorescent bead visible in all channels acting as a spatial reference for vertical shifts occurring during filter change.</p> <p>(B) Left, overlay from images shown in (A); right, magnified view from overlay. Linescans were placed through the centers of individual secretory granules (174 granules from ten membrane sheets were analyzed; for example, see dotted line), and granules were rated to be associated with a Munc18–1–rich domain when both signals had a maximum to within two pixels. Random co-localization was determined on mirrored images and subtracted (for details see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040330#s4" target="_blank">Materials and Methods</a>), resulting in 70% specific co-localization of granules with Munc18–1 domains.</p></div

Munc18–1 Membrane Recruitment Requires the Closed Conformation of Syntaxin

<p>Co-overexpression of myc-tagged Munc18–1 together with GFP-tagged syntaxin 1 (A) and (B) or an open mutant of syntaxin 1 (C) and (D). Membrane-recruited overexpressd Munc18–1 was selectively visualized on the background of endogenous Munc18–1 by immunostaining for the myc-tag. For each condition, the level of overexpressed syntaxin and of overexpressed, recruited Munc18–1 were determined for individual membrane sheets, plotted against each other, and linearly fitted (B) and (D). The ratio of the slopes wild-type (wt) syntaxin:open syntaxin was determined to be 2.1. The graphs contain data points from 411 membrane sheets for wt syntaxin 1/Munc18–1 and 417 for open syntaxin 1/Munc18–1 obtained from ten independent experiments.</p

Model of Munc18–1 Function in the SNARE Assembly Pathway

<p>Munc18–1 binds to a partially closed conformation of syntaxin that is organized in clusters and that may (bottom branch) or may not (top branch) be associated with SNAP-25 at a 1:1 stoichiometry. After vesicle docking, synaptobrevin interacts with this complex, thereby displacing Munc18–1. Alternatively, SNAP-25 is not yet associated with the complex, and synaptobrevin binding is associated with the simultaneous recruitment of SNAP-25. As result, SNARE <i>trans</i>-complexes form, leading to exocytosis. It is possible that some syntaxin molecules that are freely diffusing in the membrane are capable of binding Munc18–1 with high affinity in a closed conformation (left) similar to that observed in solution, thereby preventing it from entering SNARE complexes. Such a pool could be reflected by Munc18–1 remaining membrane associated even after extensive washing periods.</p

Characterization of Munc18–1 Complexes in Native Plasma Membranes

<div><p>(A) Munc18–1 is concentrated in microdomains in inside-out plasma membrane sheets from PC12 cells. Membrane sheets were fixed with paraformaldehyde immediately after preparation, followed by immunostaining for Munc18–1 (right panel). Integrity of the membrane was confirmed using counterstaining by the lipophilic dye TMA-DPH (left panel).</p> <p>(B) and (C) Time-dependent dissociation of Munc18–1 from the membrane sheets. The experiments were carried out as in (A), but between preparation and fixation, the sheets were washed with buffer for varying time periods (as indicated), resulting in a gradual decrease of Munc18–1 immunostaining intensity over time (C). Values were related to the immediately fixed condition which was set to 100% (<i>n</i> = 3–6 independent experiments for each data point, values are given as mean ± standard error of the mean (SEM); for details see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040330#s4" target="_blank">Materials and Methods</a>).</p> <p>(D) Sensitivity of the Munc18–1 acceptor to SNARE-cleaving light chains of clostridial neurotoxins. Membrane sheets were incubated for 10 min (see also arrow in C) in the absence (value used for normalization) or presence of 10 μM light chains as indicated, followed by fixation and immunostaining for Munc18–1. BoNT/C1 cleaves syntaxin 1A (BotNT/C1-mut, a cleavage-inactive BoNT/C1-point mutant was used as a control), BoNT/E cleaves SNAP-25, and synaptobrevin 2 is sensitive to TeTx. Values are given as mean ± SEM. <i>n</i> = 8, paired <i>t</i>-test analysis none:BoNT/C1 <i>p</i> < 0.0005.</p> <p>(E) Cleavage efficiency of BoNT/C1. Membrane sheets were incubated for 10 min in the absence (set to 100%) or presence of 10 μM light chains of BoNT/C1 or BoNT/C1-mut, followed by fixation and immunostaining for syntaxin 1. Values are given as mean ± SEM. n = 3.</p></div

SNAP-25 Is Essential for Synaptobrevin 2–Triggered Munc18–1 Release

<div><p>Membrane sheets were prepared from mouse embryonal chromaffin cells (E16–E18), isolated from wild-type or <i>snap25</i> knockout animals. Membrane sheets were incubated for 10 min in the absence or presence of 10 μM synaptobrevin 2, fixed, and immunostained for Munc18–1. Syntaxin was also visualized by immunostaining in order to make sure that membrane sheets were generated from chromaffin cells and not from fibroblast cells, which are also present in the culture (for details see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040330#s4" target="_blank">Materials and Methods</a>).</p> <p>(A) and (B) Membrane sheets generated from cells isolated from wild-type (WT) (A) or knockout (B) animals. Left panels, syntaxin staining; right panels, Munc18–1 staining. Upper and lower panels in (A) and (B), absence or presence of synaptobrevin during incubation, respectively.</p> <p>(C) Quantification of Munc18–1 immunostaining intensities. For clarity, values are expressed as percentage relating to values obtained in the absence of synaptobrevin. For each condition, seven independent experiments were performed. Values are given as mean ± SEM. Statistical test. <i>n</i> = 7, paired <i>t</i>-test knockout:wt <i>p</i> < 0.005.</p></div

Ca2+ induces clustering of membrane proteins in the plasma membrane via electrostatic interactions

This study reveals that calcium influx through voltage-gated calcium channels triggers reversible redistribution and clustering of plasma membrane proteins