We wondered whether the above mentioned small charge accumulation close to the cysteine cluster is exclusively found in rat SNAP25. Comparison of the cysteine-rich region of the SNAP25 protein (UniProtKB/Swiss-Prot-data bank) in a variety of species revealed that this region carries a net positive charge in all species except Drosophila (Figure 1—figure supplement 1). The distribution of charged amino acids adds up to +3 in all seven mammalian species, and also in chicken, zebrafish and goldfish (although for zebrafish and goldfish there is also an isoform with a charge of +1). The cysteine-rich region has four cysteines for palmitoylation in the centre, which are flanked by four lysines on each side (Figure 1a). The sequence around the cysteines also contains five acidic amino acids: four upstream and one downstream of the cysteines. Hence, eight basic and five acidic amino acids yield a net charge of +3 located downstream of the cysteine cluster.

If positive charges close to the cysteine cluster (Figure 1a) constitute the main driving force for initial plasma membrane association, their elimination should diminish plasma membrane targeting. To test this hypothesis, we overexpressed GFP-tagged SNAP25 in neuroendocrine PC12 cells and analysed how plasma membrane targeting depends on these charges. Equatorial optical sections were imaged in live cells (Figure 1b). Linescans perpendicular to the plasma membrane reveal the GFP-SNAP25 distribution at the cell periphery (plasma membrane + cytosol that optically cannot be resolved) and the cytosol (Figure 1b and c). Relating these values to each other yields a ratio >1 if there is a plasma membrane-associated fraction.

A variety of SNAP25 mutants with increased or decreased charge in the cysteine-rich region were analysed. As control for non-targetable protein, cysteine palmitoylation sites were substituted by glycines (Figure 1d). As expected, SNAP25 proteins that lacked cysteines (construct SNAP25_(C-to-G)) did not locate to the plasma membrane (Figure 1d). We then reduced the charge from wt-SNAP25 (+3) to −1 by substituting four lysines with alanines (for construct details, see Figure 1a). In SNAP25-1_distal, the outer four lysines up- and downstream of the cysteine cluster are exchanged, and in SNAP25-1_proximal the four inner ones are exchanged. Both of these constructs showed strongly diminished plasma membrane targeting (Figure 1e). Further reducing positive charges to a net charge of −5, by substituting all eight lysines with alanines (SNAP25-5; see Figure 1a), abolishes targeting almost completely (Figure 1e).

We also tested a construct termed SNAP25-5_hydrophob which is identical to SNAP25-5 except that the lysines were not replaced by alanines but instead by more hydrophobic leucines (Figure 1a). The reasoning for increasing the hydrophobicity is a previous report suggesting that the hydrophobicity of this domain plays a role in initial membrane association (Greaves et al., 2009). Compared to SNAP25-5, SNAP25-5_hydrophob showed no increase in targeting; instead, targeting was further reduced and hardly detectable.

We next tested whether more positive charges would increase targeting efficiency. Here, the finding was ambiguous, as increasing the charge to +7 (SNAP25+7) or +10 (SNAP25+10; for constructs see Figure 1a) diminishes or promotes targeting, respectively (Figure 1e). Substituting the cysteines by glycines in the SNAP25+10 mutant (SNAP25+10_(C-to-G)) causes a cytosolic distribution (Figure 1d). Hence, an increase in positive charges cannot substitute for attachment by palmitoylation.

There is another small cluster of positive charges downstream of the cysteine-rich region in the C-terminal part of the SNAP25 linker. Elimination of these charges by introducing the mutations R191A, R198A and K201A has no effect on membrane targeting (Figure 1f). This indicates that the mere presence of positive charges is not sufficient for membrane targeting. Rather, the position of charged residues within the protein structure determines their effect.

SNAP23 is 60% identical to SNAP25. Like SNAP25, it carries three positive charges in the cysteine-rich region (Figure 2a). Reduction of the positive charges by six units in SNAP23-3 diminishes membrane association (Figure 2). As observed for SNAP25, increase of positive charges can promote (see SNAP23+10 in Figure 2 and SNAP23+16 in Figure 2—figure supplement 1) or diminish (see SNAP23+11a and SNAP23+11b in Figure 2—figure supplement 1) membrane targeting. Finally, exchange of cysteines for glycines (SNAP23_(C-to-G)) yields a cytosolic distribution (Figure 2), showing that palmitoylation is also required for stable attachment of SNAP23.

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In conclusion, the analysis of SNAP25/SNAP23 constructs shows that membrane targeting is dependent on positive charges located close to the cysteine cluster. Charges > +3 can increase or decrease targeting, suggesting that primary structure is not the only determinant for stronger targeting and that secondary structural elements are also important. Structural features may define whether the charges can be exposed to the membrane environment. Alternatively, they may determine the geometry of the electrostatic contact (and hence the accessibility of the cysteines) that plays a role in the palmitoylation reaction.

In addition to immunofluorescence imaging, we employed an independent method to analyse the subcellular distribution. SNAP25 constructs that showed strong effects in live cells and the constructs without palmitoylation sites were studied by cell fractionation (Figure 3). In contrast to the imaging analysis, the biochemical cell fractionation experiment makes it possible to relate the absolute amounts of the membrane and cytosol fraction to each other. In line with the imaging experiments, we observed that SNAP25-5 has a weaker and SNAP25+10 a stronger membrane association than wt-SNAP25 (+3). In the imaging experiment, no membrane-associated fraction of SNAP25_(C-to-G) or SNAP25+10_(C-to-G) was visible. This is different in the membrane fractionation assay where we detected a small membrane-associated fraction for SNAP25+10_(C-to-G) (Figure 3). This fraction might have been overlooked by confocal microscopy, which is unable to resolve small plasma membrane-associated pools in the presence of a strong cytosolic background. For a better microscopic analysis, it was necessary to eliminate the cytosolic background. We therefore turned to ‘unroofed cells’ (Heuser, 2000), also called plasma membrane sheets. To achieve this, cells were exposed to a brief ultrasound pulse that exerts a shearing force to the upper cellular parts, leaving behind the basal plasma membranes. Then, the membrane sheets were imaged to quantify the recruited protein in the absence of the cytosolic background. Wt-SNAP25 (+3), SNAP25-5 and SNAP25+10 were readily detected on the membrane sheets (Figure 4) in amounts that would be expected from the confocal microscopic subcellular distribution analysis. SNAP25_(C-to-G) was not detectable, either because it is present at undetectable amounts after membrane sheet generation (see also the hardly detectable membrane fraction of SNAP25_(C-to-G) in the cell fractionation assay in Figure 3), or because it binds weakly and washes off during sample mounting and imaging (which takes up to ≈ 35 min in total). By contrast, SNAP25 +10_(C-to-G) was clearly present, despite the absence of palmitoylation sites, documenting a higher plasma membrane affinity when compared to SNAP25_(C-to-G).

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Figure 4

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Cell fractionation and membrane sheet experiments both show that an increase in positive charge produces a membrane-associated fraction even in the absence of palmitoylation. This poses the question of whether the small increase in membrane targeting of SNAP25+10 is, in part, achieved independently from stable membrane-attachment through palmitoylation.

To clarify this issue, we examined whether the extent of plasma membrane targeting correlates with the degree of palmitoylation for wt-SNAP25 (+3), SNAP25-5 and SNAP25+10, using SNAP25_(C-to-G) as a negative control. Freshly transfected cells were incubated with a palmitate analogue carrying an alkyne-group to allow click-labelling with a fluorophore. Hence, the newly synthesized SNAP25 constructs should be palmitoylated by the clickable palmitate. Cells were lysed and the constructs were immunoprecipitated using their GFP-tags. A Cy5-fluorophore was covalently attached to the alkyne group through click-chemistry and the immunoprecipitate was subjected to western blot analysis. The membrane was immunostained for GFP, and the palmitate-Cy5 fluorescence was related to the GFP signal. As expected, the data show that SNAP25-5 is much less palmitoylated than wt-SNAP25 (+3), and SNAP25_(C-to-G) shows no detectable traces of clickable-palmitates (Figure 5). SNAP25+10 is not significantly more palmitoylated than wt-SNAP25 (+3) (Figure 5).

Figure 5

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The outcome of this experiment is well in line with (i) the linescan analysis (Figure 1), (ii) the biochemical cell fractionation assay (Figure 3) and (iii) the membrane sheet association assay (Figure 4). The four different test systems indicate that SNAP25-5 is less targeted to the plasma membrane and less palmitoylated than wt-SNAP25 (+3). Moreover, SNAP25_(C-to-G) cannot associate with the membrane. Finally, SNAP25+10 shows a trend towards increased targeting. However, the data suggest that SNAP25+10 associates with the plasma membrane not only through palmitoylation but also by pure protein electrostatics.

Initial plasma membrane association via protein electrostatics may not be the only mechanism affected by the introduced mutations. A disturbed interaction with palmitoyl transferases is unlikely as all of the substitutions are located distal to the QPARV motif which is important for the interaction of SNAP25 and DHHC palmitoyl transferases (Greaves et al., 2010). However, some mutations map to the N-terminal SNARE motif of SNAP25 that interacts with plasmalemmal syntaxin 1A. Early studies on SNAP25 plasma membrane targeting proposed syntaxin as the receptor for initial contact establishment (Vogel et al., 2000; Washbourne et al., 2001), although a subsequent study provided compelling evidence against this hypothesis (Loranger and Linder, 2002).

To test whether our mutations affect the formation of a complex with syntaxin, we employed a fluorescence recovery after photobleaching (FRAP) assay. This assay is capable of probing interactions between SNAP25 and syntaxin by measuring the mobility of SNAP25 (Halemani et al., 2010). Mobility diminishes the stronger SNAP25 binds to syntaxin and the more syntaxin is present. The slow-down is dependent on a syntaxin–SNAP25 interaction, as it requires the N-terminal SNARE motif of SNAP25. Moreover, a mutant carrying introduced prolines in the N-terminal SNARE-motif of SNAP25 (prolines interfere with the alpha-helix formation, which in turn is required for SNARE-complex formation) moves almost independently of the syntaxin concentration (Halemani et al., 2010). We tested our constructs on membrane sheets with or without co-expressed syntaxin 1A-RFP. Syntaxin 1A-RFP slows down the mobility of wt-SNAP25 (+3), SNAP25-5 and SNAP25+10 in a similar fashion, arguing against an altered capability of complex formation caused by the introduced mutations (Figure 6). Therefore, we conclude that the differences in initial membrane association are not due to an altered affinity of binding to syntaxin.

Figure 6

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Next, we tested whether membrane association of SNAP25 is directly mediated by negatively charged lipids. We used reconstituted liposomes containing distinct lipid compositions but lacking any proteins (thus eliminating the role of potential SNAP25 binding partners such as syntaxin 1) (Figure 7). This assay also clarifies whether the few amino acids located in the relatively small SNAP25 segment do indeed affect lipid binding. GST-tagged wt-SNAP25 (+3), SNAP25-5 and SNAP25+10 were expressed in bacteria and thus lack the palmitoylation modification. The constructs were purified, immobilized on glutathione beads and incubated with Atto647N-PE-labelled liposomes containing either PC/PS or PC/PS/PE/PI/cholesterol in the absence or the presence of distinct phosphoinositides. SNAP25-bound liposomes were quantified by their Atto647N-PE fluorescence and the results were corrected for unspecific binding to GST-beads. All SNAP25 constructs show only weak liposome binding in the absence of phosphoinositides, which excludes phosphatidylserine (PS) as a major/sole binding partner (Figure 7).

Figure 7

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The presence of 4% PI(4,5)P₂ significantly enhanced SNAP25 binding in the PC samples and in the complex lipid mixture. As in the cellular assays, the reduction or addition of positive charges reduced or increased the SNAP25 interaction, respectively. Replacing PI(4,5)P₂ by PI(3,4,5)P₃ profoundly increased binding, but largely diminished the differences between SNAP25 constructs. This suggests that the presence of additional negative charges creates additional binding contacts, which are probably outside of the cysteine-rich region. In order to keep the number of negative charges comparable to those provided by 4% PI(4,5)P₂, PI(3,4,5)P₃ was reduced to 2.8%. This condition yielded a stronger difference in binding in the range between the cell fractionation (Figure 3) and the membrane sheet assay (Figure 4). Although the magnitudes of the effects are difficult to compare because of the different assay systems (the complex composition of the plasma membrane versus the simple lipid mix in liposomes, concentrations of binding partners, and the presence of SNAP25-palmitoylation in the cellular experiments may modulate the outcome), the observations point to primary interactions that occur in the cysteine-rich region.

Since PS is not capable of recruiting SNAP25, and as PI(3,4,5)P₃ is much less abundant in the cellular membrane than PI(4,5)P₂ (Balla, 2013), these findings point to PI(4,5)P₂ as the most likely intracellular binding partner. However, the effect of PI(3,4,5)P₃ on wt-SNAP25 (+3) binding was very prominent. Thus, membrane microdomains that are locally enriched in PI(3,4,5)P₃ at the plasma membrane or in an endosomal compartment (Wang and Richards, 2012) could also be preferential sites for SNAP25 targeting. The liposome binding assay thus identifies phosphoinositides as primary membrane-targeting factors interacting with positive charges in the vicinity of the cysteine cluster. However, we cannot exclude the possibility that other sites in SNAP25 contribute to phosphoinositide-dependent binding.

Plasmids for the expression of GFP-SNAP25 (Halemani et al., 2010) and GFP-SNAP23 are based on the expression vector pEGFP-C1 (GenBank accession No. U55763, Clontech, Mountain View, CA), which contains a monomeric variant of mEGFP fused N-terminally to the sequence of full-length rat SNAP25B (NP_112253.1) or SNAP23 (NP_073180). All fusion proteins contain a linker of five amino acids between mEGFP and the N-terminus of SNAP25B or SNAP23. Mutations in the SNAP25 or SNAP23 coding sequence were introduced via fusion PCR with purchased oligonucleotides (Eurofins Genomics), followed by insertion into the above-mentioned expression vector using the SacI and BamHI sites, or in the case of SNAP25_(C-to-G), the XhoI and KpnI restriction sites. The construct for expression of C-terminally RFP-tagged rat Syntaxin 1A is based on the expression vector pEGFP-N1 (GenBank accession No. U55762, Clontech, Mountain View, CA), into which Syntaxin-RFP is inserted using the XhoI and the NotI restriction sites. A twelve amino acid linker connects Syntaxin 1A (NP_446240) to a monomeric RFP (Campbell et al., 2002) lacking the first amino acid.

For expression of GST-tagged constructs, the coding sequences for wt-SNAP25 (+3), SNAP25-5 and SNAP25+10 were amplified with primers carrying restriction sites for BamHI (forward primer) and EcoRI (reverse primer). The sequences were first subcloned into the pGEM-T easy vector system (catalogue no. A1360, Promega) by TA cloning, and in a second step subcloned into the expression vector pGEX-6P1 (GE Healthcare Life Sciences) via the BamHI and EcoRI restriction sites.

All constructs were verified by sequencing.

PC12 cells (a gift from Rolf Heumann, Bochum, Germany; similar to clone 251 (Heumann et al., 1983)) were cultured in DMEM with high (4.5 g/l) glucose (PAN biotech) supplemented with 10% horse serum (Biochrom), 5% fetal calf serum (Biochrom) and 100 U/ml penicillin/100 ng/ml streptomycin (PAN biotech). During the course of the project, characteristic features of the cell line — such as morphology, expression of neuronal proteins, and their responsiveness to NGF — were regularly confirmed. Cells were maintained at 37°C and 5% CO₂ in a sterile incubator and tested negative for mycoplasmic infections (GATC Biotech, Konstanz, Germany).

Cells were transfected with the Neon Transfection System (Thermo Fisher Scientific, Waltham, MA, USA). The tip (100 µl) was loaded with 10 µg plasmid DNA of each construct. Cells were transfected by applying a pulse at 1410 V and 30 ms pulse width. Cells were plated onto poly-L-lysine (PLL) (Sigma, Cat. No: P-1524) coated coverslips (25 mm diameter, Menzel Gläser, Braunschweig, Germany)) and maintained for at least 48 hr before imaging.

For membrane sheet generation, cells were subjected to a brief ultrasound pulse in ice-cold sonication buffer (120 mM KGlu, 20 mM KAc, 20 mM HEPES-KOH, 10 mM EGTA; pH 7.2).

PC12 cells were imaged at 37°C in Ringer solution (130 mM NaCl, 4 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 48 mM D(+)αGlucose, 10 mM HEPES; pH 7.4) using a FluoView1000 confocal laser scanning microscope (Olympus) and a UPLSAPO 60x oil objective (NA 1.35). Focussing on the glass–cell interface, all green cells were imaged provided they displayed a clear cell–glass contact area. This excludes dead or dying cells that are in the process of rounding up and detaching. In addition, we avoided large clumps of cells because for such cells it is difficult to identify a longer section of cell membrane required for analysis. For measuring membrane-to-cytosol ratios, PC12 cells expressing GFP-tagged constructs were scanned in a 256 pixel x 256 pixel field (12 bit image) at a pixel size of 137 nm using for excitation 488 nm. The equatorial plane of the cell was imaged. To facilitate subsequent linescan analysis, the scanned field was rotated to allow for each cell placing a horizontal linescan (length 100 pixel; five pixel width, averaged) perpendicular to the plasma membrane that records the averaged fluorescence intensities along the 100 pixel linescan (including background fluorescence). Fluorescence intensities were background corrected and normalized to the peak intensity at the plasma membrane. Traces from several cells imaged that day (ranging from 14–40 per condition) were aligned with reference to the peak intensity at the plasma membrane. The cytosolic fluorescence level was averaged over five pixels starting at a 10 pixel distance from the peak intensity at the plasma membrane, and the cell periphery/cytosol ratio was calculated subsequently.

For mobility measurements by FRAP, the laser intensities of the 488 nm laser (for GFP) and the 543 nm (for RFP) were reduced to a minimum to prevent bleaching effects during the recording. Membrane sheets were analysed by scanning a 100 pixel x 100 pixel field with a pixel size of 0.414 µm. Recordings started with a pre-bleaching phase of three images, followed by a 500 ms bleaching step and the recording of the recovery phase. For bleaching, a region of interest (ROI) with a size of 7 pixels x 7 pixels (2.9 μm x 2.9 μm) was bleached using a 488 nm laser in combination with a 405 nm laser (both set to their maximum intensity). After bleaching, image sequences were taken at 1.2 Hz for 113 s with the scanning speed set to 40 µs per pixel. Recovery traces were background-subtracted and normalized to the average of the pre-bleach values. For one experiment, several normalized recovery traces were averaged (7–15 membrane sheets per condition). A hyperbolic curve y(t) = offset + maximal recovery x t/(t + t_1/2) was fitted to the averaged recovery trace, yielding the half time (t_1/2) of recovery.

To measure the association of SNAP25 constructs with the plasma membrane on plasma membrane sheets, a Zeiss Axio Observer D1 epifluorescence microscope equipped with a Plan-Apochromat 100x/NA 1.4 oil immersion objective and a 12 bit CCD camera (1376 × 1040 pixel) was used, yielding a pixel size of 64.5 nm x 64.5 nm. Freshly prepared membrane sheets were imaged in sonication buffer supplemented with TMA-DPH (1-(4-tri-methyl-ammonium-phenyl)−6-phenyl-1,3,5-hexatriene-p-toluenesulfonate; Thermo Fisher Scientific), up to ≈ 35 min after membrane-sheet generation. TMA-DPH staining was applied for visualization of the shape and integrity of the membrane sheets. Pictures were taken using filter sets F11-000 (AHF Analysentechnik, Tübingen, Germany) for TMA-DPH (blue channel) and F36-525 (AHF Analysentechnik, Tübingen, Germany) for GFP (green channel). From individual membrane sheets, the fluorescence intensity was measured in 30 pixel x 30 pixel ROIs and background subtracted. For each experiment and condition, the values of 21–96 membrane sheets were averaged.

About 48 hr after transfection, 9 × 10⁶ PC12 cells were detached from the substrate by trypsin treatment for 2 min (0.05% Trypsin and 0.02% EDTA in PBS, PAN Biotech, Cat# P10-0231SP) and trypsin was inactivated by adding medium. Cells were pelleted by centrifugation at 1000 x g at room temperature for 3 min and the pellet was washed with PBS. Cell pellets were resuspended in 750 µl ice-cold homogenization buffer (300 mM sucrose, 5 mM Tris-HCl, 0.1 mM EDTA, 1 mM PMSF freshly added; pH 7.4). Using a Potter-Elvehjem homogenizer, cells were kept on ice and homogenized in a volume of 0.75 ml by applying 100 strokes. The homogenate was centrifuged for 8 min at 800 x g at 4°C, yielding pellet P1 (containing non-homogenized cell debris) and supernatant S1. S1 was transferred into a new tube for a second centrifugation step for 120 min at 20,000 x g and 4°C, yielding pellet P2 (containing the enriched membrane fraction, which was resuspended in 750 µl homogenization buffer) and the supernatant S2 with the cytosolic fraction. Protein concentrations of P2/S2 were adjusted to the lowest concentration in the series, before adding respective amounts of 4x Laemmli buffer followed by incubation at 95°C for 10 min. Samples of 10 µg per lane were subjected to SDS-PAGE analysis using a 12% polyacrylamide gel. Proteins were then transferred onto a Roti-NC nitrocellulose membrane (Carl Roth, Germany) applying semi-dry blotting. Nitrocellulose membranes were blocked with PBS-T (0.05% Tween20 in PBS) containing 5% milk powder for one hour, and incubated with primary antibody against GFP diluted in blocking solution overnight at 4°C. Membranes were washed for 20 min with PBS-T three times. For detection a second antibody tagged with HRP (RRID:AB_631747, Cat# sc-2030, Santa Cruz Biotechnology, USA) was applied for 1 hr and, after washing three times with PBS-T, chemiluminescence was detected with Luminol Reagent (sc-2048, Santa Cruz Biotechnology, USA) using autoradiographic films. Films were scanned and band intensities were quantified from the digital images.

Ten million PC12 cells were transfected with 15 µg GFP fusion constructs of wt-SNAP25 (+3), SNAP25-5, SNAP25+10 or SNAP25_(C-to-G). After one hour, the medium was replaced with DMEM containing 15% delipidized FCS (PAN biotech) and 100 µM palmitate-alkyne (a kind gift from the Thiele lab, LIMES Institute, Bonn). After 15 hr of feeding, cells were harvested via trypsinization and scraping, washed once with PBS, and resuspended in lysis buffer (1% Triton, 1x cOmplete protease inhibitor cocktail, 150 mM NaCl, 5 mM MgCl₂, 25 mM HEPES; pH 7.2). Lysis was promoted by vortexing and sonication. Samples were then centrifuged for 10 min at 14,000 x g, and the supernatant was bound to a GFP-trap (Chromotek) for 2 hr at 4°C to immunoprecipitate the GFP-SNAP25 fusion constructs. After several washing steps, incorporated palmitate alkyne was clicked to a Cy5-labelled azide (Sigma, c_final = 100 µM) in 100 mM HEPES, pH 7.2, containing 500 µM tetrakis(acetonitrile)copper(I)tetrafluoroborate (Sigma) for 1 hr at 37°C. The samples were then washed to remove non-bound Cy5, and the GFP-constructs were eluted by boiling in Laemmli buffer, and loaded onto an SDS-PA gel. Proteins were then wet blotted to a nitrocellulose membrane. The membrane was blocked with a 1:1 mixture of Odyssey blocking buffer (Li-Cor) and PBS, and then incubated with a rabbit anti-GFP antibody (RRID: AB_303395; abcam, catalog no. ab-290, diluted 1:1000 in blocking solution containing 0.1% Tween-20). After washing, the membrane was incubated with an IRDye 800CW-coupled goat-anti rabbit secondary antibody (Li-Cor, catalog no. 9263221, diluted 1:10,000 in blocking solution containing 0.1% Tween-20). The Cy5 fluorescence of the palmitate and the IRDye 800CW fluorescence of the GFP were imaged with an Odyssey infrared imaging system (Li-Cor) at 700 nm and 800 nm, respectively. The fluorescent bands were quantified using ImageJ’s Gel Analyser.

Atto647N-DPPE (Att647N-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine) was purchased from Atto-Tec. All other lipids were from Avanti Polar Lipids. The complex lipid mixture (5 µmol total amount of lipid) contains 34.5 mol % 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 15 mol % 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS), 20 mol % 1-hexadecanoyl-2-octadecenoyl-sn-glycero-3-phosphoethanolamine (POPE), 25 mol % cholesterol (from ovine wool), 5 mol % liver L-α-phosphatidylinositol (PI, from liver) and 0.5 mol % Atto647N-DPPE. For the lipid mixes containing brain L-α-phosphatidylinositol-4,5-bisphosphate (PI(4,5)P₂) or 1-stearoyl-2-arachidonoyl-sn-glycero-3-phospho-(1'-myo-inositol-3',4',5'-trisphosphate) (PI(3,4,5)P₃) the amount of PI was reduced accordingly. The lipids were dissolved in chloroform or chloroform/methanol (3:1 ratio, for PI(4,5)P₂ and PI(3,4,5)P₃), mixed and dried under a flow of nitrogen. The remaining chloroform was removed by vacuum for 4 hr. The lipids were dissolved in 1 ml reconstitution buffer (25 mM HEPES/KOH pH 7.4, 200 mM KCl, 1% (w/v) OG (n-Octyl-β-D glucopyranoside), 1 mM DTT (1,4-dithiothreitol)) by 30 min shaking. To form liposomes, OG was diluted below the critical micelle concentration by the addition of 2 ml buffer (25 mM HEPES/KOH; pH 7.4, 200 mM KCl, 1 mM DTT). The residual OG was removed by flow dialyses with 4 L 25 mM HEPES/KOH pH 7.4, 135 mM KCl, 1 mM DTT overnight. Subsequently, a Nycodenz gradient centrifugation was performed to isolate the liposomes. Therefore, the dialyzed samples were mixed with an equal volume of 80% (w/v) Nycodenz and transferred into two SW60-tubes (Beckman Coulter). Layers of 750 µl 35% (w/v) Nycodenz, 150 µl 11.6% (w/v) Nycodenz and 100 µl fusion buffer were added on top of the 40% (w/v) Nycodenz/liposome solution. The gradient was spun at 55,000 rpm for 3 hr 40 min at 4°C. The liposomes were isolated, followed by a buffer exchange (25 mM HEPES/KOH pH 7.4, 135 mM KCl, 1 mM DTT, 0.1 mM EGTA, 0.5 mM MgCl₂, 1 mM DDT) using a PD MiniTrap G-25 (GE Healthcare Life Sciences). The amounts of lipids were quantified by measurement of Atto647N fluorescence (excitation 639 nm, emission 669 nm) in a Fluoroskan Ascent FL Microplate Fluorometer (Thermo Scientific).

Wt-SNAP25 (+3), SNAP25-5 and SNAP25+10 pGEX-6P1 constructs were transformed into Escherichia coli (Rosetta (DE3)pLysS) and expression of the GST fusion proteins was induced with 1 mM IPTG. After harvesting, cells were lysed for 60 min at 4°C in 150 mM NaCl, 50 mM Tris-HCl, pH 7.4 and 1 mM EDTA containing Roche cOmplete protease inhibitor, 1 mM DTT, 100 μg/ml lysozyme and two units/ml DNAse I. The suspension was sonicated, centrifuged for 30 min at 20,000 x g, frozen in liquid nitrogen and thawed for binding of the constructs directly to glutathione beads.

To monitor SNAP25 interaction with liposomes, 42 µg GST-SNAP25 constructs or a equimolar amount of GST (glutathione S-transferase) were bound to 20 µl glutathione (GSH) sepharose four fast flow beads (GE Healthcare Life Sciences) prewashed 3 x with ddH₂O and 3 x with fusion buffer (25 mM HEPES/KOH pH 7.4, 135 mM KCl, 1 mM DTT, 0.1 mM EGTA, 0.5 mM MgCl₂, 1 mM DDT). 160 nmol liposomes in fusion buffer were added to the beads and incubated 1 hr at 4°C on a rotation wheel. The beads were washed once with 1 ml fusion buffer and resuspended in 80 µl fusion buffer. The bound liposomes were detected by measuring the Atto647N fluorescence. The amounts of liposomes specifically bound to the different GST-SNAP25 constructs were calculated by subtracting the values derived from the GST controls.

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

The authors would like to thank Michael Pankratz and Nora Karnowski for comments on the manuscript. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (TRR83 to THS and TL).

1. Received: July 5, 2016
2. Accepted: February 26, 2017
3. Accepted Manuscript published: February 27, 2017 (version 1)
4. Accepted Manuscript updated: February 28, 2017 (version 2)
5. Version of Record published: March 22, 2017 (version 3)

© 2017, Weber et al.

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