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The SNAP-25 linker supports fusion intermediates by local lipid interactions

  1. Ahmed Shaaban
  2. Madhurima Dhara
  3. Walentina Frisch
  4. Ali Harb
  5. Ali H Shaib
  6. Ute Becherer
  7. Dieter Bruns
  8. Ralf Mohrmann  Is a corresponding author
  1. Saarland University, Germany
  2. Max Planck Institute for Experimental Medicine, Germany
  3. Otto-von-Guericke University, Germany
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Cite this article as: eLife 2019;8:e41720 doi: 10.7554/eLife.41720

Abstract

SNAP-25 is an essential component of SNARE complexes driving fast Ca2+-dependent exocytosis. Yet, the functional implications of the tandem-like structure of SNAP-25 are unclear. Here, we have investigated the mechanistic role of the acylated “linker” domain that concatenates the two SNARE motifs within SNAP-25. Refuting older concepts of an inert connector, our detailed structure-function analysis in murine chromaffin cells demonstrates that linker motifs play a crucial role in vesicle priming, triggering, and fusion pore expansion. Mechanistically, we identify two synergistic functions of the SNAP-25 linker: First, linker motifs support t-SNARE interactions and accelerate ternary complex assembly. Second, the acylated N-terminal linker segment engages in local lipid interactions that facilitate fusion triggering and pore evolution, putatively establishing a favorable membrane configuration by shielding phospholipid headgroups and affecting curvature. Hence, the linker is a functional part of the fusion complex that promotes secretion by SNARE interactions as well as concerted lipid interplay.

https://doi.org/10.7554/eLife.41720.001

Introduction

Ca2+-triggered exocytosis is essential for the vesicular release of transmitters, peptides, and hormones. On the molecular level, assembly of membrane-bridging SNARE complexes is believed to induce membrane fusion by forcing vesicle and plasma membrane into close apposition (Jahn and Fasshauer, 2012; Südhof and Rothman, 2009). SNARE complexes are helix bundles formed by four cognate SNARE motifs, Qa, Qb, Qc, and R, which have been categorized based on sequence homology and the identity of characteristic polar residues in the central interaction layer (Bock et al., 2001; Fasshauer et al., 1998b). While prototypical SNARE complexes that drive intracellular fusion generally employ four single-pass transmembrane proteins with a single SNARE motif, exocytosis relies on ternary complexes out of one R-SNARE, one Qa-SNARE, and a specialized Qbc-SNARE with concatenated Qb and Qc motifs (Weimbs et al., 1998). In neurons and neuroendocrine cells, the set of exocytotic SNAREs typically comprises synaptobrevin (syb)−2 (R), syntaxin (stx)−1A (Qa), and SNAP-25 (Qbc).

The two SNARE motifs in Qbc proteins are connected by a ‘linker’ peptide, whose length varies between different isoforms. Although all Qbc SNAREs lack a transmembrane domain, several isoforms are membrane-anchored by acylated cysteine residues in the N-terminal linker region. In SNAP-25a and b, the principal isoforms in neurons and neuroendocrine cells, a cluster of 4 cysteines forms a palmitoylation site (Hess et al., 1992; Lane and Liu, 1997; Veit et al., 1996). While SNAP-25 is efficiently acylated by members of the DHHC acyltransferase family (Greaves et al., 2009; Greaves et al., 2010), not all cysteines seem to be modified in vivo (Foley et al., 2012). Interestingly, membrane association is facilitated by stx-1A interactions, which likely promotes lipidation by membrane-bound acyltransferases (Gonelle-Gispert et al., 2000; Vogel et al., 2000). Yet, a SNAP-25 linker fragment (aa 85–120) lacking both SNARE motifs can still be effectively acylated (Gonzalo et al., 1999), likely owing to transient membrane interactions via hydrophobic and positively charged residues (Greaves et al., 2009; Weber et al., 2017). Mutation of one or more cysteine residues results in a correlated reduction in plasma membrane localization of SNAP-25 (Gonelle-Gispert et al., 2000; Nagy et al., 2008; Washbourne et al., 2001). Acylation-deficient SNAP-25 mutants were inconsistently reported to be dysfunctional (Washbourne et al., 2001), to partially drive exocytosis (Gonelle-Gispert et al., 2000), or to primarily change release kinetics (Nagy et al., 2008).

Site-directed spin-labelling experiments indicated that the SNAP-25 linker is mostly unstructured (Margittai et al., 2001) and thus may act as a simple connector. This view was fostered by data that the linker is only loosely associated with the SNARE complex, from which the N-terminal linker segment can be proteolytically cleaved (Fasshauer et al., 1998a). Moreover, the linker domain is dispensable for liposome fusion in vitro (Parlati et al., 1999) and the physical linkage of SNARE motifs seems inconsequential for secretion in cracked Botulinum toxin (BoTn) /E treated PC12 cells, as infusion of a toxin-resistant Qc motif can reinstate release (Chen et al., 1999). However, rescue efficiency of complementing SNAP-25 fragments in cellular assays seems to strictly depend on experimental conditions, as another study reported a recovery of secretion only in permeabilized cells after initial rundown of release (Wang et al., 2008). Contradicting the notion of a dispensable linker, functional differences between the Qbc isoforms SNAP-25 and SNAP-23 have recently been attributed to a short sequence variation within the linker (Nagy et al., 2008).

Here, we have studied the role of the SNAP-25 linker domain in a systematic structure-function analysis. Starkly objecting the prevalent notion of an inert connector, our new results establish that the linker peptide is a vital component of SNARE complexes that serves multi-tiered mechanistic functions during fast Ca2+-dependent secretion. In particular, we present first evidence that the linker establishes local lipid:SNARE contacts, which may critically support lipidic fusion intermediates in late mechanistic stages of the exocytotic process.

Results

The SNAP-25 linker is a functional component of the fusion machinery

Earlier work suggested that the physical linkage of the two SNARE motifs in SNAP-25 is not a strict prerequisite of transmitter release (Chen et al., 1999; Wang et al., 2008). As former studies exclusively relied on population-based release assays with limited time resolution, we wondered whether expression of unlinked SNAP-25 fragments could fully reestablish the kinetic properties of fast secretion. To investigate potential kinetic deficits, we setup a reconstitution system based on Snap25-/- (KO) chromaffin cells, wherein two complementing SNAP-25 fragments (each containing one SNARE motif) were co-expressed using a bicistronic Semliki Forest (SF) virus. Ca2+-dependent secretion in infected KO cells was analyzed by capacitance measurements and amperometry in combination with Ca2+-uncaging (Mohrmann et al., 2013), allowing for the analysis of secretion kinetics at the millisecond scale. Due to an unforeseen instability of isolated SN2 fragments in chromaffin cells (Figure 1—figure supplement 1A), we used a mCherry (mCh)-SN2 fusion protein in all rescue paradigms. That said, the N-terminal mCh-tag is not expected to interfere with the function of SN2, since SNAP-25-based FRET probes carrying a fluorophore in a similar position exhibited normal interactions with cognate SNAREs (An and Almers, 2004; Wang et al., 2008). Strikingly, co-expression of a fragment covering SN1 and the full linker (SN1-L; aa 1–141) together with mCh-SN2 (aa 138–206) only resulted in a poor reconstitution of the secretory response to a brief increase of [Ca2+]i (~30 µM) in KO cells (Figure 1Aa,b), albeit both fragments were overexpressed at high levels according to our Western Blot analysis (Figure 1—figure supplement 1B). Moreover, release mediated by isolated SNAP-25 fragments under these conditions was kinetically delayed compared to controls expressing wildtype (WT) SNAP-25, as seen from a significant increase in the time constant of monoexponential fits (Figure 1Ab) of ΔCM. To better compare our rescue data with the original results of Chen et al. (1999), we also expressed SN25Δ26 (aa 1–180) together with mCh-SN2 in KO chromaffin cells (Figure 1—figure supplement 1C). Expression of SN25Δ26 instead of the shorter SN1-L fragment did however not enhance but clearly suppress secretion (Figure 1—figure supplement 1Ca,b), replicating a known dominant-negative action of SN25Δ26 (Sørensen et al., 2006). In sum, our data thus point to a yet unacknowledged mechanistic requirement for SNAP-25 linker continuity in secretion.

Figure 1 with 3 supplements see all
Integrity and primary structure of the SNAP-25 linker are important for Ca2+-dependent secretion.

(A) Cartoon (top) illustrates the structure of the two co-expressed SNAP-25 fragments: SN1-L (aa 1–141) comprises SN1 and the full linker, while mCherry (mCh)-SN2 represents SN2 (aa 138–206) N-terminally fused to mCherry. (a) Electrophysiological measurements of secretion in KO chromaffin cells co-expressing mCh-SN2 and SN1-L showed a poor rescue of secretion. Averaged traces for [Ca2+]i (top), capacitance measurements (middle) and amperometric recordings (bottom) in KO cells (gray) and in KO cells expressing the fragments (light green) or WT protein (black) are shown. Arrow indicates UV-flash. (b) Quantification of the data presented in (a). Average capacitance changes after 5 s (total ΔCM) and 1 s (fast burst) are depicted. Sustained release represents mean ΔCM/s occurring between 1 s and 5 s. Kinetics of the secretory burst was approximated by a monoexponential function, yielding τburst. (B) Cartoon (top) illustrates the substitution of the whole linker (aa 83–141) by a flexible G/S peptide in SN25 LGGS. (a) Characterization of GFP-SN25 LGGS-mediated secretion in KO chromaffin cells by Ca2+-uncaging. Depicted are averaged traces for [Ca2+]i (top), capacitance measurements (middle) and amperometric recordings (bottom) in KO cells (gray) and KO cells expressing GFP-SN25 LGGS (pastel blue) or WT protein (black). (b) Quantitative analysis of the average capacitance changes after 5 s (total ΔCM) and 1 s (fast burst). Sustained release represents mean ΔCM/s occurring between 1 s and 5 s. Data are shown as mean ± SEM. Statistical analysis was done with ANOVA and Tukey’s test.

https://doi.org/10.7554/eLife.41720.002

To evaluate the prevalent view of the linker as a functionally dispensable connector, we next generated a mutant SNAP-25 variant (denoted SN25 LGGS), in which the entire linker was substituted by a flexible, equally-sized peptide. Following well-established design principles for inert ‘linker’ peptides in fusion proteins (Argos, 1990; Chen et al., 2013), the artificial SNAP-25 linker was exclusively composed of glycine and serine residues (ratio of 2:1, varied sequence) in order to prevent interactions with the adjoining SNARE motifs (Figure 1B, cartoon). SN25 LGGS or WT SNAP-25 was expressed in KO chromaffin cells by means of a SF virus, and secretion was assayed by membrane capacitance measurements and amperometric recordings. While expression of WT SNAP-25 reinstated the full secretory response upon stimulation by Ca2+-uncaging, no rescue of transmitter release was detected in KO cells expressing SN25 LGGS (Figure 1Ba,b). Reasoning that the linker mutant might have weakened stimulus-secretion coupling or otherwise delayed secretion, we performed membrane capacitance measurements for extended time intervals using strong Ca2+-stimuli. Intriguingly, we found that even infusion of a 19 µM Ca2+-containing solution via the patch pipette for up to 5 min did not result in a recovery of secretion in cells expressing SN25 LGGS (Figure 1—figure supplement 2A). Thus, the linker substitution mutant SN25 LGGS is dysfunctional, when expressed in KO cells. Nevertheless, SN25 LGGS exerted a mild dominant-negative effect in WT chromaffin cells, suggesting that the mutant still interacts with cognate SNARE partners via its intact SNARE motifs but fails to support exocytosis (Figure 1—figure supplement 2B). In order to visualize and quantify protein expression in our experiments, mutant and WT SNAP-25 carried an N-terminal GFP-tag, which was previously shown not to interfere with protein function (Delgado-Martínez et al., 2007). Since total GFP fluorescence intensity of cells expressing SN25 LGGS was indistinguishable from WT controls (Figure 1—figure supplement 3A), the observed secretion phenotype of SN25 LGGS cannot be simply due to insufficient expression of the mutant protein. Noteworthy, it has been reported before (Nagy et al., 2008) and is also demonstrated here (Figure 3A) that an acylation-deficient SNAP-25 mutant (SNAP-25 C84S, C85S, C90S, C92S; SN25 LN4CS) can still mediate substantial amounts of release, which excludes the possibility that the loss of acylation and the resulting mislocalization of SN25 LGGS (Figure 1—figure supplement 2C) primarily account for the severe secretion deficits. Hence, our data suggest a mechanistic requirement for specific biophysical properties and/or special motifs of the SNAP-25 linker peptide during fusion.

The SNAP-25 linker is critical for t-SNARE interaction and complex assembly

Substitution of the linker domain by an unrelated flexible peptide in SN25 LGGS may result in abnormal SNAP-25 folding and potentially could even affect the integrity of SNARE motifs. To investigate whether SN25 LGGS can still engage in ternary core complex formation, we mixed bacterially expressed SN25 LGGS or WT protein with syb-2 (GST-fusion protein) and stx-1A in equimolar concentrations (3 µM) and quantified the amount of assembled complexes by SDS-PAGE and Coomassie staining after defined time intervals (Figure 2A). Note that we did not work with an excess of SNAP-25 to suppress unproductive 2:1 stx-1A:SNAP-25 complexes (Fasshauer and Margittai, 2004), since this might have diminished potential kinetic differences between mutant and WT SNAP-25. As a ‘negative’ control, we also employed a 1:1 mixture of isolated SN1 (aa 1–82) and SN2 (aa 142–206) fragments.

Figure 2 with 1 supplement see all
SNAP-25 linker motifs promote t-SNARE interactions and ternary complex assembly.

(A) Kinetics of SNARE complex assembly was assayed by mixing equimolar amounts of purified GST-syb-2 (aa 1–116) and stx-1A (aa 1–262) with (a) SN25 WT, (b) SN25 LGGS, or (c) isolated SN1/SN2 fragments. SNARE complex formation was analyzed by SDS PAGE after different times (indicated above lanes). (d) Quantitative analysis of complex formation. Biexponential fits of the kinetic profiles (left panel) demonstrated a significant decrease of the initial rate constant for SN25 LGGS and SN1/SN2 fragments (right panel). (B) Binding of SN25 LGGS or SNAP-25 fragments to immobilized stx-1A was tested in pulldown assays. Depicted are representative Coomassie-stained gels for different experimental conditions: (a) pulldown of SN25 WT, (b) SN25 LGGS (‘*” marks a contaminating degradation product), (c) SN1 and SN2, (d) SN1-L and SN2, (e) SN1 and L-SN2, and (f) L-SN2. (g) GST-GFP was used as a negative control. Each gel shows load (‘L’), GST-stx-1A containing beads after binding reaction (‘B’), and supernatant (‘S’). (h) To quantify the binding to immobilized stx-1A, we calculated the average retention ratio nprey/nstx-1A for each protein/fragment. Depicted errors are SEM. n is indicated in the corresponding panels. Statistical analysis was done with ANOVA and Tukey’s test.

https://doi.org/10.7554/eLife.41720.010

Interestingly, our assay demonstrated that SN25 LGGS was still able to engage in SNARE complex formation under these conditions, which largely excludes a gross misfolding of the SNARE motifs (Figure 2Ab). The assembly kinetics of SNARE complexes containing SN25 LGGS was however clearly decelerated in comparison to WT controls (Figure 2Ad), which might be in part due to the high glycine content and the deviating biophysical properties of the artificial linker. Yet, complex formation in experiments with SN25 LGGS was considerably faster than the kinetics observed for separated SN1 and SN2 motifs (Figure 2Ab-d), emphasizing the importance of a physical linkage of both motifs for reaction speed. Fitting the kinetic profiles with a biexponential function, we found that the rate constant for the initial rapid phase of SNARE assembly was significantly decreased for SN25 LGGS in comparison to controls (Figure 2Ad). In case of linker-less SNAP-25 fragments, our analysis also revealed a strong decrease in the fast rate constant, which was accompanied by a reduction in the total amount of SNARE complexes assembled within 24 hr (2.19*105 ± 0.17*105 a.u., WT: 4.37*105 ± 0.52*105 a.u.; p<0.01).

SNARE complexes formed by SN25 LGGS or separated SN1 and SN2 motifs exhibited a characteristic size shift (~180 kDa; Figure 2Ab-c), which possibly suggests the formation of SNARE complex dimers. Indeed, previous work by Fdez et al. (2008) has shown that two fully formed ternary complexes containing linker-less SN1/SN2 fragments can assemble into dimeric structures with attached C-terminal tips. Thus, the primary structure of the SNAP-25 linker peptide likely affects the biochemical properties of SNAREpins and their propensity to dimerize.

The SN25 LGGS–induced slowdown of complex assembly may hint at a role of the linker in providing conformational support for SNARE complex nucleation. Therefore, we investigated whether linker substitution also affects the interplay of SNAP-25 and stx-1A. To evaluate linker:stx-1A interactions, we immobilized GST-stx-1A (aa 1–265) on glutathione-sepharose beads and quantified the ‘pulldown’ of SN25 LGGS or WT SNAP-25 (Figure 2B). Stx-1A-loaded beads were incubated with each prey protein (5 µM) for 20 hr at 4°C in order to approach binding equilibrium. Intriguingly, we found that SN25 LGGS bound to stx-1A at a ~ 50% lower molar ratio than WT protein (Figure 2Ba,b,h). Earlier biochemical work demonstrated that stx-1A only interacts with truncated SNAP-25 fragments that contain the unperturbed SN1 motif (Chapman et al., 1994), derogating the role of the linker and SN2 motif in directly binding to stx-1A H3. Given the low retention of SN25 LGGS, we wondered whether linker motifs assist the recruitment of SN2 into helical assemblies, thereby intensifying stx-1A interactions. Thus, we tested whether inclusion of the linker in SNAP-25 fragments could enable pulldown of SN2. When an equimolar mix of isolated SN1 and SN2 fragments was incubated with stx-1A, SN1 was almost exclusively retained on beads (Figure 2Bc,h), as expected (Chapman et al., 1994). Adding the linker back to SN1 (aa 1–142; SN1-L) did not increase fragment binding, nor did it facilitate the retention of SN2 (Figure 2Bd,h). In contrast, a continuous linker-SN2 fragment (aa 83–206; L-SN2) bound to immobilized GST-stx-1A in the presence of isolated SN1 motifs (Figure 2Be,h) but not in their absence (Figure 2Bf,h). The amount of retained SN1 also significantly increased in the presence of L-SN2, suggesting that linker-mediated recruitment of SN2 also stabilizes the whole assembly. Thus, SN2 and linker form a functional unit that likely binds to SN1:stx-1A complexes.

As the requirement for a continuous linker-SN2 segment in t-SNARE interactions might limit ternary complex nucleation, we also compared SNARE complex assembly in mixtures containing either SN1-L and SN2, SN1 and L-SN2, or linker-less SN1 and SN2 together with stx-1A and GST-syb-2 (Figure 2—figure supplement 1A). We observed that the presence of the linker in either fragment combination significantly facilitated SNARE assembly over linker-less fragments, as judged by the time constants of monoexponential fits of assembly profiles (Figure 2—figure supplement 1B). Intriguingly, the combination of SN1/L-SN2-fragments was slightly more efficient than SN1-L/SN2 in promoting complex formation, indicating that linker interactions during initial t-SNARE assembly might indeed determine overall assembly kinetics. That said, our biochemical assays likely underestimate the effect of t-SNARE interactions, as fragments can easily interact with each other in solution without steric hindrance.

In summary, motifs within the native SNAP-25 linker are required for normal t-SNARE interactions as well as the fast assembly of ternary SNARE complexes. Accordingly, the severe secretory phenotype of SN25 LGGS is probably caused by ineffective SNARE interactions that hinder complex initiation.

N- and C-terminal linker regions mechanistically support Ca2+-dependent release

To learn more about the fusion-promoting function of the linker, we generated two partial substitution mutants, in which the N-terminal region (aa 83–118; SN25 LNGGS) or the C-terminal linker portion (aa 119–141; SN25 LCGGS) was exchanged by a corresponding flexible G/S peptide of equal length (Figure 3A,B). We based this subdivision on earlier reports that the N-terminal linker segment could be proteolytically excised (cuts at aa 84/115/120/125) from fully assembled complexes (Fasshauer et al., 1998a) and that a C-terminal motif (aa 120–129) determines isoform-specific release properties (Nagy et al., 2008). Expression of either mutant in KO chromaffin cells produced only small secretory responses in uncaging experiments (Figure 3Aa,Ba). Yet, secretion levels for both mutants clearly exceeded residual release in KO cells, indicating a partial functionality of the mutant proteins.

Figure 3 with 2 supplements see all
Motifs in N- and C-terminal linker regions promote secretion.

(A) Characterization of secretion deficits induced by GFP-SN25 LNGGS or GFP-SN25 LN4CS. Cartoon illustrates the structure of the tested mutants. (a) Averaged traces for [Ca2+]i (top panel), capacitance measurements (middle) and amperometric recordings (bottom) in KO cells expressing GFP-SN25 LNGGS (cyan), GFP-SN25 LN4CS (orange), or WT protein (black) are shown. Arrow indicates UV flash. (b) Quantitative analysis of the kinetic properties of the secretory burst. The mean amplitude of RRP and SRP as well as the corresponding time constants τfast and τslow are depicted. Sustained release rate reflects the slope of the linear secretion component. The mean exocytotic delay indicates the average latency of secretion after the flash. (B) Characterization of secretion in KO cells expressing GFP-SN25 LCGGS or GFP-SN25 LC4G. Cartoon illustrates the structure of the C-terminal linker substitutions mutants. (a) Averaged recordings for the GFP-SN25 LCGGS mutant (blue), GFP-SN25 LC4G (red) or wildtype controls (black) are shown. Panel organization as in (Aa). (b) Quantitative analysis of kinetic parameters. All data are presented as mean ± SEM; n is indicated in the corresponding panels. Statistical comparisons were performed with ANOVA and Tukey’s post-hoc test.

https://doi.org/10.7554/eLife.41720.014

Fitting capacitance traces by exponential functions typically delivers detailed information about the different secretory components, especially about release from the readily releasable pool (RRP) and slowly releasable pool (SRP) (Voets et al., 1999). In case of SN25 LNGGS, a detailed kinetic analysis showed that secretion is not only severely decreased but also markedly slowed-down in comparison to controls (Figure 3Ab). In particular, we observed a complete loss of the RRP component, a profound reduction in SRP size, a substantially prolonged time constant for release of SRP vesicles, a moderate decrease in sustained release, and a delay in the onset of secretion (Figure 3Ab). Hence, substitution of the N-terminal linker segment generated a ‘complex’ phenotype, simultaneously altering several mechanistic aspects of the fusion process including priming, primed vesicle stability, and fusion triggering. Noteworthy, SN25 LNGGS lacks the palmitoylation motif and consequently exhibits a dramatically reduced plasma membrane targeting (Figure 3—figure supplement 1A) despite a normal overall expression (Figure 1—figure supplement 3A). In comparison, the palmitoylation-deficient mutant SN25 LN4CS, which is also not targeted to the plasma membrane (Figure 3—figure supplement 1A), showed much milder but clearly related release deficits (Figure 3Aa,b), indicating that the loss of membrane association is not the sole cause of diminished transmitter release.

The phenotype of the C-terminal substitution mutant SN25 LCGGS appeared almost identical to the one observed for SN25 LNGGS (Figure 3Ba; see normalized plots in Figure 3—figure supplement 1B). Our kinetic analyses showed that SN25 LCGGS strongly reduced the size of both primed vesicle pools and simultaneously increased the time constants for RRP- and SRP-mediated release in comparison to controls (Figure 3Bb). Additionally, secretion onset was prolonged, and sustained release was decreased for SN25 LCGGS (Figure 3Bb). Thus, C-terminal linker substitution also caused defects in priming, maintenance of primed vesicles, and triggering – very much like SN25 LNGGS.

As structural data suggested an interaction of nonpolar side-chains in the very C-terminal linker segment with a hydrophobic groove on the core SNARE complex (Sutton et al., 1998), we further characterized a mutant, in which the four corresponding amino acids were substituted by glycine residues (F133G, I134G, V137G, A141G; denoted SN25 LN4G; Figure 3B). SN25 LC4G produced a substantially smaller and slower secretory response than in controls, as indicated by a strong reduction in RRP size and an increased time constant of RRP release (Figure 3Bb). Hence, even this small substitution induced a complex release phenotype that is reminiscent of the defects seen in SN25 LCGGS. The phenotypic similarity to SN25 LCGGS became even stronger, when more residues in the very C-terminal linker segment were mutated, as demonstrated by replacing the last nine amino acids by glycine residue in SN25 LC9G (Figure 3—figure supplement 2). Clearly, the potential core complex interaction site and the adjoining linker sequence are important for a fusion intermediate that determines the fate of the primed vesicle. Note that all C-terminal linker mutants were normally targeted to the plasma membrane and exhibited a similar overall expression level like the WT protein in controls (Figure 1—figure supplement 3A,Bb), excluding the possibility that the observed secretion deficits are due to reduced protein availability.

Ternary SNARE complex assembly appeared only slightly decelerated in biochemical experiments with SN25 LNGGS and SN25 LCGGS (Figure 3—figure supplement 1Ca–d). Biexponential fits of the kinetic profiles still revealed a significantly decreased initial rate constant for SN25 LNGGS and SN25 LCGGS in comparison to controls (Figure 3—figure supplement 1Ce,f). Thus, N- and C-terminal linker regions each must contain motifs that provide additive support for SNARE assembly. Noteworthy, SNARE complexes formed by SN25 LNGGS (but not SN25 LCGGS) also exhibited a characteristic shift towards higher molecular mass in this assay. Furthermore, we tested both linker substitution mutants in pulldown experiments with immobilized stx-1A (Figure 3—figure supplement 1D) and found that exclusively the retention of SN25 LCGGS was significantly reduced compared to WT SNAP-25 (Figure 1—figure supplement 1Dd). This finding highlights that only the C-terminal linker motif substantially stabilizes t-SNARE dimers, while the contribution of the N-terminal linker region is negligible.

In summary, our data suggest that motifs in the N-terminal as well as in the C-terminal linker segment are critical for fast, Ca2+-dependent vesicle fusion. Both linker segments likely stabilize late fusion intermediates in a cooperative fashion, preventing unpriming and promoting Ca2+-dependent fusion triggering.

The acylated N-terminal linker segment promotes triggering

While priming-related release deficits of linker substitution mutants can be readily explained by perturbed SNARE assembly, the observed kinetic slow-down of secretion also suggests a distinctive function of linker motifs in late fusion steps. As mutation of the four acylated cysteine residues in the N-terminal linker segment (SN25 LN4CS) suffices to decelerate release kinetics and decrease apparent RRP size (Figure 3Aa,b; cf. Nagy et al., 2008), we wondered about a specific mechanistic contribution of the acylated linker in fusion triggering. Interestingly, the palmitoylation motif is located directly downstream of SN1 in all known orthologs of SNAP-25, thus mediating membrane contacts near the C-terminal end of the assembling SNARE complex. To distinguish the effects of local lipid interactions from potential deficits due to a general protein mislocalisation, we attached the minimal palmitoylation motif of SNAP-25 (aa 83–120) (Gonzalo et al., 1999) to the N-terminus of GFP-tagged SN25 LN4CS (denoted P-GFP-SN25 LN4CS), thereby providing a secondary acylation point for re-targeting to the plasma membrane (Figure 4A, cartoon). Indeed, P-GFP-SN25 LN4CS was almost exclusively localized at the plasma membrane of infected KO cells (Figure 4A). WT SNAP-25 carrying the same modification (P-GFP-SN25 WT) showed a similar membrane expression, only slightly deviating from the distribution pattern of P-GFP-SN25 LN4CS by a stronger intracellular protein accumulation (Figure 4A, example image), which likely represents a Golgi/recycling endosomal SNAP-25 pool (Aikawa et al., 2006). P-GFP-SN25 WT efficiently rescued secretion in KO chromaffin cells and even reached a slightly higher release level than typically seen in other control recordings (Figure 4B). This increase in total secretion might be attributed to a more pronounced membrane targeting compared to the unmodified WT protein. In contrast, the retargeted P-GFP-SN25 LN4CS mutant still caused secretion deficits similar to the original acylation mutant despite its high membrane expression (Figure 4Ba,b; cf. Figure 3Aa,b). Restoration of membrane localization clearly did not abate the primary deficits in fusion triggering and pool stability. Hence, we conclude that linker acylation at a defined intramolecular position is required for efficient membrane fusion, pointing to a mechanistic role for local linker-mediated membrane contacts.

Intramolecular acylation of SNAP-25 is required for efficient triggering and stability of primed vesicles.

(A) Cartoon illustrates the construction of SNAP-25 variants carrying a secondary palmitoylation motif fused to the GFP-tag. The cellular localization of SNAP-25 variants was analyzed by confocal microscopy. Exemplary pictures of KO cells expressing P-GFP-SN25 LN4CS or double-acylated WT protein are shown. Scale bar is 5 µm. Mean radial line scans for P-GFP-SN25 LN4CS (red) and P-GFP-SN25 WT (black) demonstrate similar plasma membrane localization. Inset depicts the average fluorescence near the plasma membrane. (B) Characterization of secretion properties in KO cells expressing P-GFP-SN25 LN4CS (red) or P-GFP-SN25 WT (black). (a) Shown are averaged Ca2+-imaging results (top), capacitance measurements (middle) and amperometric recordings (bottom). Arrow indicates UV flash. (b) Quantitative analysis of RRP and SRP size, time constants of release components, sustained release rate, and exocytotic delay. Data are depicted as mean ± SEM; n is indicated in the panels. Statistical comparisons were done using Student’s t-test.

https://doi.org/10.7554/eLife.41720.020

In a plausible scenario, the linker-based membrane anchor might facilitate fusion by relaying SNARE-generated force onto the plasma membrane. To further test this idea, we constructed linker mutants, in which the distance between the acylation site and SN1 was increased by insertion of flexible spacer peptides (6, 10, or 14 residues; mutants denoted SN25 shift6aa/10aa/14aaL) in order to mechanically ‘uncouple’ the acyl anchors from the core complex (Figure 5A). We especially relied on G/S-containing spacers, as the high rotational freedom of the included glycine residues should substantially lessen conformational strain and thereby further hamper force transfer. Deviating from the secretion phenotype of SN25 LN4CS, none of these spacer insertions caused a detectable reduction in the size of the secretory burst (exemplified by SN25 shift14aaL in Figure 5Ba). However, all mutants consistently induced a moderate slow-down of release, as seen in scaled plots of ΔCM traces (Figure 5Bc) and reflected by increased time constants of RRP and SRP-mediated secretion in the kinetic analysis (Figure 5Bb). Moreover, we observed a significantly prolonged latency of the secretory burst in all mutants that further confirmed deficits in fusion triggering (Figure 5Bb). Since total expression and membrane localization of the three mutants were indistinguishable from WT controls (Figure 1—figure supplement 3A,Bc), the kinetic changes cannot be due to a shortage of mutant protein on the plasma membrane. Intriguingly, the phenotype is also not caused by increased linker length per se, as the initial linker segment can be extended by seven amino acids without functional deficits using natural insertions found in the longer N-terminal linker region of the related SNAP-23 isoform (Figure 5—figure supplement 1A,B). Similarly, a net extension of the linker by 14 amino acids using an additional spacer after A118 was well tolerated (Figure 5—figure supplement 1A,C).

Figure 5 with 2 supplements see all
The acylated N-terminal linker motif does not play an essential role in force transfer.

(A) Structure of the three mutants SN25 shift6/10/14aaL, which contain ‘uncoupling’ spacers, and the double mutant SN25 shift14aaLN4CS. (B) The secretion phenotype of GFP-SN25 shift14aaL (olive; WT in black) is shown as example for the functional deficits induced by spacer insertions. (a) Averaged [Ca2+]i measurements (top), capacitance traces (middle) and amperometric recordings (bottom) are shown. UV flash is indicated by arrow. (b) Analysis of kinetic parameters for all three tested mutants: GFP-SN25 shift6aaL (beige), GFP-SN25 shift10aaL (gray), and GFP-SN25 shift14aaL (olive). Data for the kinetic parameters were normalized to the corresponding WT control values. Statistical analyses were performed between mutant and control measurements (Student’s t-test). (c) Normalized plots of averaged capacitance traces highlight the kinetic deceleration caused by spacer insertions. All three pairs of WT/mutant traces are equally scaled. (C) Comparative electrophysiological analysis of secretion in GFP-SN25 LN4CS (orange), GFP-SN25 shift14aaLN4CS (red-brown), and WT protein-expressing KO cells (black). (a) Panel organization as in (Ba). (b) Quantitative analysis of kinetic parameters. (c) Normalized plot of the secretory burst component highlighting the changed kinetics of the double mutant. All data are mean ± SEM; n is indicated in the corresponding panel. Statistical comparisons were performed using ANOVA and Tukey’s test.

https://doi.org/10.7554/eLife.41720.022

In order to directly test the idea of linker-mediated force transfer, we next introduced the longest spacer peptide into SN25 LN4CS, generating a double mutation denoted SN25 shift14aaLN4CS (Figure 5A). The rationale behind this experiment was that elimination of SNAP-25 acylation should break up the most relevant mechanical coupling between linker and membrane, as the binding free energy of four lipid-embedded palmitoyl groups (Pool and Thompson, 1998) should by far exceed the energetic contributions of other linker:membrane interactions. In comparison, the free energy gain for electrostatic membrane interactions of lysine residues (five in the N-terminal linker) is very small (Kim et al., 1991), and unstructured peptide strands are generally expected to exhibit a low binding affinity to the membrane interface (Almeida, 2014; White and Wimley, 1999). Without a significant mechanical coupling between linker and plasma membrane, no exacerbation of the phenotype would be expected for SN25 shift14aaLN4CS in comparison to SN25 LN4CS. Contrary to our expectation, however, the secretory burst in SN25 shift14aaLN4CS –expressing cells was further decelerated (Figure 5Ca), as can be well appreciated in normalized plots of ΔCM (Figure 5Cc) and is evident from a significant decrease in RRP/SRP fusion rates and the prolonged exocytotic delay in kinetic analyses (Figure 5Cb). The substantially aggravated phenotype of SN25 shift14aaLN4CS in combination with the generally mild phenotypes of uncoupling spacer insertion mutants clearly argue against a prominent contribution of the linker to SNARE force transfer. Still, given the pronounced kinetic phenotype of the non-palmitoylated SN25 LN4CS mutant and its retargeted P-GFP-SN25 LN4CS variant, the membrane interactions of the acyl anchors near the C-terminal end of the core complex must be mechanistically important for efficient fusion initiation and likely promote a favorable membrane configuration for merger.

The acerbated release deficits of SN25 shift14aaLN4CS also highlight acylation-independent functions of the N-terminal linker in fusion triggering, possibly involving additional lipid or protein interactions. Work by Weber et al. (2017) recently suggested that the N-terminal linker motif mediates transient membrane contacts via electrostatic interactions between lysine side-chains and phospholipid head groups. To test whether these weak lipid interactions are functionally important for secretion, we mutated five lysine residues in the N-terminal linker region of SN25 LN4CS (K83A, K94A, K96A, K102A, K103A; denoted SN25 LN5KA,4CS; Figure 5—figure supplement 2A, cartoon), and analyzed potential effects on secretion in Ca2+-uncaging experiments. Substitution of the lysine residues very slightly decelerated secretion in comparison to SN25 LN4CS, as indicated by an increased time constant of RRP release (Figure 5—figure supplement 2A–C). Since no difference in overall expression or membrane targeting of SN25 LN5KA,4CS compared to SN25 LN4CS was noticeable (Figure 1—figure supplement 3A,Ba), we conclude that the lysine residues contribute to the linker:lipid interactions that facilitate Ca2+-dependent fusion triggering. Noteworthy, the role of the lysine residues in the linker:membrane interface is probably more pronounced within the intact acylated linker but would be difficult to study due to an involvement in the acylation process (Weber et al., 2017).

In sum, our data suggest that the N-terminal linker region engages in different interactions with the plasma membrane that are important for late mechanistic steps of the fusion process. While these linker:membrane contacts may only play a limited role in force transfer, our results hint at a mechanistic scenario, in which acyl anchors and local electrostatic membrane interactions cooperatively establish a suitable membrane configuration for fusion triggering.

The N-terminal linker section controls fusion pore expansion

To investigate whether the linker is also required for normal fusion pore evolution, we elicited secretion by infusion of an intracellular solution containing 19 µM Ca2+ and studied the kinetics of amperometric spikes in KO cells expressing different linker mutants (Figure 6). In accord with Nagy et al., 2008, we found that SN25 LN4CS significantly prolonged the pre-spike signal (PS) (Figure 6C) but left the main spike signal unperturbed (Figure 6B). Interestingly, the N-terminal substitution mutant SN25 LNGGS affected the PS as well as the kinetics of the main spike: Similar to SN25 LN4CS, the PS was significantly prolonged (Figure 6Cc), exhibiting reduced amplitudes (Figure 6Ca,b) and diminished current fluctuations (Figure 6Ce,f). Additionally, rise time and half width of the main spike signal were significantly increased (Figure 6Bc,d), while the total charge remained unchanged (Figure 6Ba). The effects on main spike waveform induced by SN25 LNGGS clearly indicate that the N-terminal linker region surrounding the cysteine cluster participates in the control of late fusion pore expansion. Confirming this notion, removal of the flanking lysine residues in SN25 LN5KA,4CS was sufficient to delay main spike kinetics, establishing a congruent phenotype to SN25 LNGGS (Figure 6B–C). Moreover, similar deficits in early and late fusion pore behavior were observed for SN25 shift14aaL (Figure 6B–C), demonstrating that a displacement of the palmitoylation motif effectively disturbs the linker-mediated support of pore dilation. The lack of kinetic alterations in cells expressing the C-terminal substitution mutant SN25 LCGGS (Figure 6B–C) finally suggests that fusion pore regulation is selectively tied to the N-terminal linker region. Hence, our data suggest that lipid interplay of the N-terminal linker segment with the neck of the fusion pore governs the behavior of the pore from its initial opening to the final stages of membrane merger.

The N-terminal linker segment controls fusion pore expansion.

(A) Exemplary amperometric spikes recorded in KO cells expressing different N- and C-terminal linker mutants. The kinetic parameters determined during analysis are marked for a spike recorded in control experiments with WT SNAP-25. (B) Quantitative analysis of main spike properties. Data were collected for the following conditions (# of events/ # of cells): WT protein (black, 12633/100), GFP-SN25 LN4CS (orange, 1419/22), GFP-SN25 LNGGS (cyan, 1986/27), GFP-SN25 LN5KA,4CS (grey, 1391/16), GFP-SN25 shift14aaL (olive, 2377/22) and GFP-SN25 LCGGS (blue, 1014/16). Cumulative frequency distributions are displayed for (a) spike charge, (b) amplitude, (c) 50–90% rise time, and (d) half width. Bar graphs depict means of the median determined from the frequency distribution for each cell. (C) Analysis of PS waveform. Cumulative frequency distributions for (a) PS amplitude, (b) initial amplitude, (c) duration, (d) charge, (e) fluctuation frequency, and (f) root mean square (rms) noise of the current derivative are shown. As in (B), bar graphs show mean of cell medians for each parameter. Errors are SEM. Statistical comparisons were performed by ANOVA using Tukey’s test.

https://doi.org/10.7554/eLife.41720.028

Rescue of linker-induced secretion deficits by membrane-active agents

If N-terminal linker:membrane interactions critically modulate lipidic fusion intermediates, application of membrane-active reagents might be able to ‘rescue’ release deficits. The underlying idea is that linker:membrane interplay might mechanistically help to facilitate spontaneous lipid stalk formation and membrane merger, and thus might serve a function that could be mimicked by application of membrane-perturbing compounds. While such treatments generally promote fusion (in mutants as well as in controls), the relative effect should be disproportionally strong in KO cells expressing SN25 LNGGS, if the action of the compound can lessen the impact of the mechanistic ‘bottleneck’ imposed by the mutation.

Application of short-chain alcohols was reported to facilitate hemifusion/fusion of liposomes in reduced protein-free systems by altering membrane properties (Chanturiya et al., 1999; Paxman et al., 2017). Here, we introduced low concentrations (2%) of methanol (MeOH) into cells via the patch pipette and characterized potential changes of secretion properties in Ca2+-uncaging experiments. Indeed, MeOH infusion into SNAP-25 WT-expressing KO cells mildly increased total secretion (+25%) by strengthening the SRP component (Figure 7). The very same treatment resulted in a profound recovery of the secretory burst in SN25 LNGGS–expressing KO cells, basically doubling total secretion (Figure 7B). The restoration of release suggests that the alcohol-induced changes in lipid properties can partially compensate for the loss of linker:membrane interactions. Interestingly, MeOH treatment of SN25 LNGGS–expressing KO cells selectively reconstituted primed vesicle pools and the sustained release component, leaving release rates unchanged (Figure 7C). Thus, our data primarily point to a MeOH-induced recovery of priming and priming stability. As short-chain alcohols likely affect hydration repulsion (Ly and Longo, 2004; Paxman et al., 2017), it stands to reason that MeOH compensates for an insufficient local shielding of the plasma membrane surface caused by elimination of local linker:lipid interactions.

Intracellular application of methanol (MeOH) compensates for release deficits of GFP-SN25 LNGGS.

(A) The effect of low amounts of MeOH (2%) on secretion was tested in KO cells expressing either GFP-SN25 WT (dark green; untreated: black) or GFP-SN25 LNGGS (bright green; untreated: cyan) using Ca2+-uncaging. Averaged [Ca2+]i measurements (top), capacitance traces (middle) and amperometric recordings (bottom) are shown. UV flash is indicated by arrow. (B) Total ΔCM was quantified 5 s after flash application (left) and the relative gain in secretion in the presence of MeOH was calculated. Note the significantly stronger effect of MeOH on GFP-SN25 LNGGS-mediated release. (C) Analysis of kinetic parameters for secretion in presence and absence of MeOH. RRP and SRP size, time constants of release components, sustained release rate, and exocytotic delay were quantified. Data are shown as mean ± SEM; n is indicated at the bottom. Statistical comparisons between treated and untreated conditions were done using Student’s t-test.

https://doi.org/10.7554/eLife.41720.030

Curvature-inducing lipids are known to either facilitate or inhibit membrane merger in the context of various model systems, including liposome fusion and virus-mediated membrane fusion (Chernomordik and Kozlov, 2003; Melia et al., 2006). As it was reported that cone-shaped lipids support the highly-bent stalk intermediate during fast Ca2+-triggered fusion of secretory vesicles (Churchward et al., 2008), we investigated whether induction of negative curvature by infusion of oleic acid (OA) can abate the phenotype of SN25 LNGGS. In control experiments, we infused SNAP-25 WT–expressing KO cells with intracellular solution containing 19 µM Ca2+ and 5 µM OA and characterized secretion by membrane capacitance measurements as well as amperometric recordings. OA infusion in these control experiments induced a ~ 50% gain in total capacitance over 2 min compared to vehicle-treated cells (Figure 8A). In stark contrast, infusion of SN25 LNGGS–expressing KO with OA almost tripled the membrane capacitance increase seen within 2 min (Figure 8A), indicating that the facilitating action of OA can in part accommodate for the loss of N-terminal linker interactions. Hence, we propose that the membrane anchor and flanking linker sequences support a critical lipidic fusion intermediate that is sensitive to curvature-inducing agents.

Intracellular infusion of oleic acid (OA) partially rescues exocytosis and reverses the kinetic delay of amperometric spikes induced by GFP-SN25 LNGGS.

(A) Characterization of secretion from KO cells expressing SN25 LNGGS or WT protein during infusion of solution containing 19 µM free Ca2+ together with either OA (5 µM) or vehicle (DMSO). (a) Depicted is the mean capacitance change (∆CM) over 120 s for each condition. (b) Total ∆CM after 120 s was averaged from the indicated number of cells. (c) The relative gain in ∆CM with OA over vehicle infusion was significantly higher in SN25 LNGGS expressing cells compared to WT controls. (B) Properties of the main amperometric spike, displayed as cumulative frequency distribution (left panel) and as cell weighted averages for the indicated parameters (right panel, a–d). (C) Properties of the prespike foot signal (PS) displayed as cumulative frequency distribution (left panel) and as cell weighted averages (right panel) for the indicated parameters (a–e). Values are given as mean of median determined from the parameter’s frequency distribution for each cell. Data are represented as mean ± SEM and were collected for the following conditions (# of events/ # of cells): WT protein (black, 1969/20), WT + OA (dark green, 2640/21), GFP-SN25 LNGGS (cyan, 968/26) and GFP-SN25 LNGGS+ OA (pastel pink, 2296/25). Statistical testing was done by ANOVA and Tukey’s post-hoc test.

https://doi.org/10.7554/eLife.41720.032

As curvature-perturbing lipids have been shown to affect the life-time of the nascent fusion pore (Zhang and Jackson, 2010), we also investigated whether OA application could reverse the kinetic slow-down of amperometric spikes in SN25 LNGGS-expressing cells (Figure 8B,C). In control experiments with SNAP-25 WT-expressing cells, application of OA resulted in a clear acceleration of PS and main spike waveform, as reflected by a decreased rise time and half width (Figure 8Bc,d), an increased spike amplitude (Figure 8Bb), and a shortening of PSs (Figure 8Ca). A similar kinetic speed-up of spike kinetics was also observed, when OA was introduced into SN25 LNGGS–expressing KO cells (Figure 8B,C), in result compensating for all decelerating effects of the linker mutation on PS and main spike signals. The full reversal of the kinetic deficits of SN25 LNGGS suggests that linker-mediated membrane interactions and OA both influence the same mechanistic determinant of pore evolution. Thus, our data suggest the possibility that the N-terminal lipid contacts could directly regulate pore expansion by altering membrane curvature near the fusion pore neck.

Discussion

Despite the fundamental mechanistic role of SNARE proteins in fast Ca2+-triggered exocytosis, the functional implications of the tandem-like structural organization of SNAP-25a have not been sufficiently understood. Here, we studied the mechanistic role of the linker domain of SNAP-25, uncovering two unrecognized mechanistic functions: [1] Linker motifs govern SNARE protein interactions and facilitate the formation of binary and ternary complexes. [2] The N-terminal linker segment engages in local membrane contacts that support critical lipidic fusion intermediates.

The SNAP-25 Linker is a functional component of the fusion machinery

Earlier reconstitution experiments with two complementing SNAP-25 fragments indicated that a physical linkage of SNARE domains in SNAP-25 is not a prerequisite of Ca2+-dependent exocytosis (Chen et al., 1999; Wang et al., 2008). While we confirmed here that co-expression of separate SN1- and SN2-containing fragments was in principle sufficient to reinstate Ca2+-dependent vesicle fusion in KO chromaffin cells, an elaborated analysis showed that secretory responses were dramatically diminished and kinetically distorted in spite of an abundance of expressed fragments (Figure 1A, Figure 1—figure supplement 1B). Hence, our results suggest that the structural integrity of the linker domain is indeed important for fast Ca2+-triggered exocytosis in neuroendocrine cells, refuting the prevalent notion of a functionally dispensable linker domain. Previous work might have underestimated the relevance of the linker due to technical limitations of the employed BoTn /E based rescue paradigms and population-based secretion assays (Chen et al., 1999). Moreover, secretion in permeabilized or cracked cells might not follow canonic release pathways (Wang et al., 2008). To remedy such experimental shortcomings, we have performed all rescue experiments in Snap25-/- cells using electrophysiological techniques that allowed us to study secretion kinetics on the single cell level.

Corroborating the idea of a mechanistically relevant linker, we found that mutation of linker motifs severely impaired secretion. In fact, complete substitution of the linker domain by a flexible peptide produced a fully dysfunctional SNAP-25 mutant (Figure 1B), while partial substitutions still dramatically reduced primed pool size and fusion rate (Figure 3A,B). Albeit large linker substitutions could potentially result in a globally misfolded protein causing unspecific secretory defects, several lines of evidence argue against such scenario: [1] All linker mutants formed SNARE complexes in vitro, showing that both SNARE motifs remain functional (Figure 2A, Figure 3—figure supplement 1C). [2] The phenotypes of the large substitutions SN25 LNGGS and SN25 LCGGS qualitatively reflect the deficits of corresponding ‘small’ mutants SN25 LN4CS and SN25 LC4G/LC9G (Figure 3; Figure 3—figure supplement 2), implying incremental changes in specific mechanistic processes. [3] Intracellular application of membrane-modifying agents rescued key phenotypical features in SN25 LNGGS–expressing cells (Figures 7 and 8). If the mutations would just unspecifically interfere with complex nucleation and initial priming, the resulting phenotype should not be highly sensitive to membrane-active compounds that primarily reduce the energy-demand of membrane merger in final fusion steps.

As especially noticeable for the N- and C-terminal mutants SN25 LNGGS and SN25 LCGGS (Figure 3A–B), large linker substitutions caused very similar phenotypes despite targeting non-overlapping regions. While several findings indeed suggest a differential mechanistic function of the N- and C-terminal linker (see discussion below), the apparent convergence of phenotypes likely reflects a structural interdependence between certain motifs due to the relative short length of the linker peptide. Given that the linker motifs are part of the same stretched-out peptide chain that lines the core complex, a potential linker dislocation at one end may well result in an altered configuration at the other end, in this way possibly affecting positioning and function of distant linker sections. The view that the linker is indeed not completely randomly oriented along the SNARE core but at least in sections engages in local interactions with the SNARE complex is supported by recent work of White et al. (2018), showing a diffuse density for the SNAP-25 linker at the N-terminal and C-terminal ends of the EM structure of the fully formed complex.

Taken together, the native linker provides critical functional support for the fusion process, in particular in late mechanistic steps, wherein its motifs stabilize primed vesicles and facilitate fusion triggering. While previous experiments with SNAP-23/25 chimeric proteins only hinted at a regulatory role of a singular C-terminal linker motif (Nagy et al., 2008), our new mutational analysis ultimately identifies the SNAP-25 linker as a vital component of the fusion machinery.

The SNAP-25 Linker is required for productive SNARE interactions

How does the linker domain mechanistically promote secretion? Our biochemical experiments demonstrated that normal SNAP-25:stx-1A interactions critically depend on motifs within the linker (Figure 2B), uncovering a putative key aspect of linker function. Since earlier interaction studies could only detect binding between SN1 and stx-1A (Chapman et al., 1994; Hayashi et al., 1994), the mechanism, by which SN2 enters t-SNARE acceptor complexes, has remained unclear. Here, we provide new insight into initial t-SNARE interplay by showing that a continuous linker-SN2 fragment (but not isolated SN2 motifs) binds to immobilized stx-1A in the presence of free SN1, even increasing the relative retention of SN1 (Figure 2Be,h). In reverse, attachment of the linker to SN1 does not allow for binding of SN2 to stx-1A (Figure 2Bd), which suggests that only the continuous linker-SN2 fragment acts as a functional unit. We therefore conclude that motifs within the linker peptide are essential for induction and/or stabilization of interactions between SN2 and independently forming SN1:stx-1A complexes. Our experiments with partial substitution mutants moreover revealed that specifically the C-terminal linker segment promotes stx-1A interactions (Figure 3—figure supplement 1D). An attractive interpretation would be that local interactions of the C-terminal linker region with the initial segment of SN2 increase its helicity, enabling the association of SN2 with the bi-helical SN1:stx-1A H3 complex (Figure 9). As stx-1A:SNAP-25 complexes seem to dynamically alternate between conformational states with different engagement of SN1 and SN2 (Weninger et al., 2008), the observed linker-mediated recruitment of SN2 should critically determine conformational dynamics and lifetime of binary complexes. In accord with this conclusion, experiments with SNAP-25-based intramolecular FRET sensors indicated that the two SNARE motifs of SNAP-25 would only tightly associate with stx-1a on the plasma membrane, if physically connected by the linker (Wang et al., 2008).

Mechanistic model of the role of the SNAP-25 linker domain in exocytosis.

Overview of the putative mechanistic functions of the SNAP-25 linker (cartoon, left side) with reference to the phenotypic features of linker mutants analyzed in this work (right side, box). The cartoon depicts key assembly states of a SNARE complex and potential correlated interactions of the SNAP-25 linker with core complex and/or plasma membrane. Syb-2 is shown in blue, stx-1A in red, and SNAP-25 in green. The acylated N-terminal (NT) linker segment is indicated by the cyan region; the blue section represents the backfolding C-terminal (CT) loop of SNAP-25. Helical segments are shown as boxes. Yellow circles indicate cysteine residues that were modified by acylation (black wavy lines). Positively charged lysine residues are indicated by red circles in the magnified view (for simplicity, only the SNARE motifs of syb-2 and stx-1A are shown in the close-up). Box contains original data (taken from Figure 2B, Figure 3A, Figure 5B/C, and Figure 6) in simplified form.

https://doi.org/10.7554/eLife.41720.034

On a related note, it is interesting that the retention of SN25 LGGS on immobilized stx-1A closely matched the relative molar binding of isolated SN1 fragments, rendering it highly probable that SN25 LGGS only interacts with stx-1A via its SN1 motif. This provides a ready explanation for the dramatic secretion phenotype of SN25 LGGS–expressing KO cells, as binary acceptor complexes, which putatively serve as platforms for syb-2 interactions and vesicle priming (Fasshauer and Margittai, 2004; Pobbati et al., 2006), would be severely diminished. Our finding that SN25 LGGS decelerates ternary complex assembly in biochemical experiments with free SNARE proteins (Figure 2A) would also be consistent with a scenario, in which complex nucleation is compromised by altered stx-1A interactions of SN25 LGGS. That said, we cannot exclude that formation of competing unproductive intermediates, e.g. complexes containing only the SN1 motifs together with stx-1A H3 (cf. Misura et al., 2001), may contribute to the slowdown of assembly kinetic in vitro and the severe secretion phenotype of SN25 LGGS in cells. Moreover, linker interactions with the core complex might also facilitate assembly after the initial nucleation stage, as even the N-terminal substitution mutant SN25 LNGGS exhibited a mild slow-down of ternary complex assembly in spite of normal binding to stx-1A (Figure 3—figure supplement 1C,D). In line with this idea, MD simulations have indeed suggested potential stabilizing interactions of the linker with the SNARE core complex (Shi et al., 2017).

N-terminal linker:membrane interplay accelerates Ca2+-triggered fusion

Attenuated SNARE interactions of linker mutants might indirectly decelerate secretion kinetics by causing a shortage of trans-SNARE complexes and an altered stoichiometry of SNARE supercomplexes at the vesicular contact site (Acuna et al., 2014; Mohrmann and Sørensen, 2012; Mostafavi et al., 2017). That said, our structure-function analysis also provided strong evidence for a direct mechanistic involvement of the N-terminal linker region in fusion triggering. While Nagy et al. (2008) found that a palmitoylation-deficient SNAP-25 mutant induced a slow-down of secretion, vaguely hinting at a mechanistic requirement for linker acylation, it has remained unclear to what extent the phenotype was affected by the apparent mislocalization of the mutant protein. Here, we have demonstrated that retargeting of the acylation-deficient mutant SN25 LN4CS to the plasma membrane via a secondary palmitoylation site (fused to the GFP-tag) did not mitigate the primary release defects of the mutant (Figure 4B), thus excluding the possibility that a reduced membrane expression of SN25 LN4CS per se is responsible for defective fusion triggering. Rather, our data emphasize the requirement of a correctly positioned membrane anchor within the linker of SNAP-25. In this respect, it is highly interesting that the palmitoylation motif directly adjoins the C-terminal end of the SN1 motif throughout all known SNAP-25 orthologs of the animal kingdom, which places the membrane anchor invariantly near the prospective fusion site at the C-terminal end of the assembled SNARE complex, where it might interact with membrane matrices during fusion.

An attractive explanation for the triggering phenotype of SN25 LN4CS would be that the acyl anchors of the linker relay mechanical force from the core complex onto the plasma membrane, thereby increasing local membrane straining. To test this idea, we produced a set of linker mutants, in which the mechanical coupling between SN1 and the palmitoylation motif was loosened by interjacent insertion of flexible spacer peptides. While similar-sized extensions of the JMD of stx or syb-2 dramatically diminished vesicle fusion (Deák et al., 2006; Kesavan et al., 2007; Wang et al., 2001), spacer insertion at the N-terminal end of the linker in SNAP-25 only resulted in a moderate triggering defect with reduced release rates and an increased exocytotic delay (Figure 5B), which is clearly distinct from the phenotype observed for the non-acylated SN25 LN4CS mutant. Raising even more doubt about a potential role of the acylated linker in force transfer, we found that insertion of the largest spacer peptide into the palmitoylation-deficient mutant SNAP-25 LN4CS further reduced release rates (Figure 5C). Since the linker acyl anchors serve as the primary mechanical coupling point to the plasma membrane, their loss should have vastly precluded any effect of upstream spacer insertion. The considerably exacerbated phenotype of SN25 LN shift14aaLN4CS however indicates that spacer insertion at least in part disrupts an acylation-independent fusion-facilitating function of the N-terminal linker region. In line with this idea, we observed that substitution of several lysine residues, which are thought to mediate very weak electrostatic membrane contacts of the N-terminal linker segment (Weber et al., 2017), further decelerated release kinetics in SN25 LN5KA,4CS (Figure 5—figure supplement 2). Considering the very moderate defects of spacer insertion mutants and the substantial contribution of acylation-independent effects, it stands to reason that force transfer is not the primary function of linker-mediated membrane interactions. The profound secretory deficits of the non-palmitoylated SN25 LN4CS variant and the observed acylation-independent function of the N-terminal linker segment would rather point to a scenario, in which the palmitoylation motif and the surrounding linker region establish an interface between the SNARE complex and the plasma membrane that directly facilitates triggering by local linker:lipid contacts.

Mechanistic importance of linker:lipid interactions in membrane fusion

Membrane merger is putatively initiated by spontaneous lipid tail protrusions (lipid splay) near apposition points with reduced hydration or within curved membrane segments (Smirnova et al., 2010; Stevens et al., 2003; Tahir et al., 2016), nucleating an expanding stalk that transforms into a lipidic fusion pore (Kawamoto and Shinoda, 2014; Risselada et al., 2014; Risselada et al., 2011). Intriguingly, simulations have recently indicated an increased propensity for lipid splay near membrane-embedded viral fusion peptides (Apellániz et al., 2014; Kasson et al., 2010; Larsson and Kasson, 2013), suggesting that local protein:lipid interactions are crucial for membrane merger during viral fusion. Moreover, transmembrane domains (TMDs) of SNARE proteins have also been suspected to promote spontaneous stalk formation by disruption of lipid packing in adjoining membrane sections (Dhara et al., 2016; Risselada et al., 2011). Given these ideas about the mechanistic role of protein:lipid interfaces in fusion, it might be speculated that N-terminal linker:membrane contacts could also be involved in initiation of membrane merger. To test this notion we tried to 'rescue' the secretory phenotype of SN25 LNGGS by membrane-active compounds that putatively facilitate lipid stalk formation. While it is expected that such treatments also facilitate release in controls, a functional compensation should manifest in a disproportionally strong recovery of release in KO cells expressing the linker mutant. Indeed, we found that the secretion deficits of SN25 LNGGS can be partially ‘rescued’ by intracellular application of MeOH or OA, which are both known to alter bilayer properties:

  1. Infusion of small amounts of MeOH into KO cells expressing SN25 LNGGS produced an overly strong increase of total release in comparison to WT controls (Figure 7B), indicating a partial recovery of secretion. Short-chain alcohols were previously shown to exert a facilitating effect on the hemifusion/fusion of liposomes (Chanturiya et al., 1999; Paxman et al., 2017). Although the underlying mechanism is not yet fully understood, it has been argued that alcohol partitioning at the water:lipid interface alters the properties of lipid bilayers, resulting in a functionally relevant reduction in hydration repulsive pressure and interfacial tension (Ly and Longo, 2004; Paxman et al., 2017; Stetter and Hugel, 2013). In our experiments with SN25 LNGGS, MeOH application conspicuously increased the size of both primed pool components and sustained release, but was unable to elevate release rates of RRP/SRP vesicles. Given the current mechanistic ideas about alcohol-induced effects, a reduced hydration repulsion may ease the convergence of membranes and alleviate the priming defect induced by SN25 LNGGS. This interpretation implies that the linker:lipid interactions contribute to the dehydration and shielding of phospholipid head groups and thereby energetically promote priming. Indeed, several positively charged residues framing the palmitoylation motif might contribute to this mechanistic function.

  2. Infusion of the curvature-inducing lipid OA also overly boosted total secretion in SN25 LNGGS-expressing cells, indicating a partial compensation of release deficits. Curvature-perturbing lipids are well known to either inhibit or facilitate liposome fusion, viral membrane fusion, and Ca2+-dependent exocytosis (reviewed in Chernomordik and Kozlov, 2003; Zhang and Jackson, 2010). The bidirectional actions of curvature-inducing agents have been largely attributed to the de-/stabilization of curvature-sensitive lipidic fusion intermediates, in accord with recent simulations that report a strong curvature dependence of the energy landscape of membrane fusion (Kawamoto et al., 2015). Given the observed recovery of secretion in SN25 LNGGS-expressing cells after cis-application of OA, it seems natural to assume that the linker:lipid interface could also be required for the establishment and/or maintenance of curved membrane segments in critical fusion intermediates. Interestingly, recent studies on S-acylated proteins have suggested that protein palmitoylation does not only allow for curvature-dependent sorting (Hatzakis et al., 2009; Larsen et al., 2015) but may itself induce membrane curvature (Chlanda et al., 2017; Weber et al., 2017). In the same line, MD simulations indicated that the palmitoyl lipid anchors of SNAP-25 may sequester cone-shaped cholesterol and stabilize local undulations of the plasma membrane near t-SNARE assemblies (Sharma and Lindau, 2017). Hence, it might be speculated that the acylated N-terminal linker supports highly-bent bilayers segments in plasma membrane protrusions or lipid stalks (Figure 9).

While the compensatory actions of membrane-active compounds hint at a specific role of linker:lipid interactions in fusion initiation, it should be noted that the available evidence for this scenario is still circumstantial, and other mechanistic interpretations may also apply. In particular, a supporting function of the N-terminal linker in the putative ‘zippering’ of the JMDs of stx-1A and syb-2 (Stein et al., 2009) may represent an alternative explanation, as the linker would be closely involved in inducing membrane merger without directly affecting bilayer properties.

A postfusional role of linker:lipid interactions in controlling pore dilation

After initial membrane merger, cis-SNARE complexes putatively line the neck of the fusion pore, allowing for a tight contact between the palmitoylation motif and the curved flanks of the lipidic pore (Figure 9). Here, we have demonstrated that substitution of the N-terminal linker region (but not the C-terminal segment) delayed the discharge kinetics of catecholamines from single vesicles, suggesting that linker:membrane interactions actively control the speed of fusion pore dilation (Figure 6). While mutation of the cysteine residues in the palmitoylation motif selectively increased the length of the PS (Nagy et al., 2008), we found that perturbations of the whole palmitoylation motif and surrounding sequences (either by direct substitution in SN25 LNGGS or by a positional shift in SN25 shift14aa) additionally decelerated the kinetics of the main spike signal. Interestingly, we could demonstrate that this slow-down of the main spike signal is primarily caused by the loss of several lysine residues surrounding the central cysteine group, as substitution of these amino acids in SN25 LN5KA,4CS fully replicated the phenotype of the full substitution mutant SN25 LNGGS (Figure 6). Hence, our data indicate that different linker:lipid interactions are required for subsequent phases of pore expansion: transition from a semi-stable nascent pore to a dilating pore depends on the membrane-embedded acyl anchors of the linker, whereas phospholipid interplay with positively charged residues in the linker is needed for the late phase of pore expansion. Possibly, the changing relevance of different linker:lipid interactions is caused by the shifting geometry of the evolving pore.

It is conspicuous that the phenotypic alterations with respect to the overall secretory response and the transmitter discharge kinetics from single vesicles appear uncorrelated in N- and C-terminal linker mutants. Despite sharing the same kinetic phenotype in Ca2+-uncaging experiments, SN25 LNGGS and SN25 LCGGS strictly differ in fusion pore behavior. As mentioned before, we attribute the convergence of secretion phenotypes in linker mutants to a structural interdependence of different linker motifs during SNARE complex formation. Fusion pore evolution, however, constitutes the final step of the fusion process, when SNARE assembly is almost complete, and indirect effects of the C-terminal linker should be largely diminished. Accordingly, only the direct dependence of pore dilation on N-terminal linker interactions with the plasma membrane should remain relevant.

The decelerating effect of SN25 LNGGS on spike kinetics was completely reversed by cis-application of cone-shaped OA (Figure 8B–C), identifying membrane curvature as a critical factor for linker-mediated regulation of fusion pore expansion. Indeed, theoretical considerations of membrane bending energies explained different pore dilation states by unequal extension mobilities and suggested a strict dependence of pore evolution on membrane tension as well as spontaneous curvature (Chizmadzhev et al., 1995). Using a related theoretical approach to deduce the energy state of a metastable fusion pore, Zhang and Jackson (2010) also identified membrane curvature as an important determinant of nascent pore stability. Interestingly, based on the assumed relationship between energy landscape and pore life time, a changed PS duration in cells expressing syb-2 TMD mutants was attributed to TMD-induced local curvature changes (Chang and Jackson, 2015). Given these ideas about curvature and pore behavior, it seems plausible that the N-terminal acyl anchors of the linker could regulate pore dilation by locally inducing/stabilizing negative membrane curvature. Moreover, we speculate that interactions of the lysine residues in the N-terminal linker region could lower the energetic toll of phospholipid head group crowding in pore segments with highly negative curvature, which would deliver a ready explanation for the additional deceleration of main spike kinetic in cells expressing SN25 LN5KA,4CS.

Conclusion

In summary, our data establish a radically changed view of the mechanistic role of the SNAP-25 linker in fast Ca2+-triggered exocytosis, acknowledging facilitating functions of linker motifs in multiple mechanistic steps en route to fusion (Figure 9). In particular, our structure-function analysis revealed two intertwined mechanistic roles of the linker domain: First, the linker serves as an organizer for early t-SNARE interactions and SNARE complex assembly, thus promoting priming and fusion by protein:protein interplay. Second, the acylated N-terminal linker segment engages in membrane contacts that are critical for the maintenance of primed fusion intermediates and fusion triggering. Strikingly, we deliver first clues that linker:lipid contacts facilitate fusion triggering by promoting a favorable membrane configuration for membrane merger. In addition, we demonstrate that linker:lipid contacts are crucial for fast fusion pore evolution, implying that interactions between the linker and highly-curved membrane segments of the pore may facilitate its expansion. In effect, the multi-tiered facilitating functions of the SNAP-25 linker enhance the performance of the exocytotic machinery, enabling it to meet the speed and fidelity demands of synaptic transmission and neuroendocrine secretion.

Materials and methods

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Strain, strain
background
(Mus Musculus)
C57BL/6
Genetic reagent
(Mus Musculus)
Snap25 null alleleWashbourne et al., 2001,
Genetic ablation of the
t-SNARE SNAP-25
distinguishes mechanisms
of neuroexocytosis.
Nat Neurosci. 5 (1):19–26.
PMID:11753414
Snap25tm1Mcw, MGI:2180178
cDNA
(Rattus Norvegicus)
Snap25a WTSorensen et al., 2002
,
The SNARE
protein SNAP-25 is linked to fast
calcium triggering of
exocytosis.Proc Natl Acad Sci U
S A 99(9):6449. PMID: 11830673
amino acid sequence
identical to murine
Snap25a
Antibodymouse anti-SNAP-25,
SySy 71.1
Synaptic SystemsCat# 111 011WB 1:1000
AntibodyHRP-conjugated
goat-anti mouse
Bio-Rad LaboratoriesCat# 170–5047WB 1:5000
Antibodyrabbit anti-SNAP-25,
AB1762
Sigma AdrichAB1762WB 1:1000
AntibodyHRP-conjugated
goat-anti rabbit
Bio-Rad LaboratoriesCat# 170–5046WB 1:5000
Transfected
construct
(Mus Musculus)
pSFV1 GFP-SNAP-25
WT
Sorensen et al., 2002,
The SNARE
protein SNAP-25 is linked to fast
calcium triggering of
exocytosis.Proc Natl Acad Sci
U S A 99(9):6449.
PMID: 11830673
contains rat Snap25a
WT cDNA; also abbreviated
as pSFV1 GFP-SN25 WT
Transfected
construct
(Mus Musculus)
pSFV1 GFP-SN25 LGGSthis paperderived from pSFV1
GFP-SNAP-25 WT,
substitution of the linker
by varied G/S sequence
Transfected
construct
(Mus Musculus)
pSFV1 GFP-SN25 LNGGSthis paperderived from pSFV1
GFP-SNAP-25 WT, substitution
of the NT linker (aa 83–118)
by varied G/S sequence
Transfected
construct
(Mus Musculus)
pSFV1 GFP-SN25 LCGGSthis paperderived from pSFV1
GFP-SNAP-25 WT, substitution
of the CT linker (aa 119–141)
by varied G/S sequence
Transfected
construct
(Mus Musculus)
pSFV1 GFP-SN25 LC4Gthis paperderived from pSFV1
GFP-SNAP-25 WT, CT
substitution F133G, I134G,
V137G, A141G
Transfected
construct
(Mus Musculus)
pSFV1 GFP-SN25 LC9Gthis paperderived from pSFV1
GFP-SNAP-25 WT,
substitution of F133-A141
by glycine residues
Transfected
construct
(Mus Musculus)
pSFV1 GFP-SN25 LN4CSthis paperderived from pSFV1
GFP-SNAP-25 WT,
substitution of 4x cysteine
by serine
Transfected
construct
(Mus Musculus)
pSFV1 P-GFP-SN25
WT
this paperderived from pSFV1 GFP-SNAP-25
WT, insertion of the minimal
SNAP-25 palmitoylation sequence
(aa 83–120) NT of GFP
Transfected
construct
(Mus Musculus)
pSFV1 P-GFP-SN25
LN4CS
this paperderived from pSFV1 GFP-SN25
LN4CS, insertion of the minimal
SNAP-25 palmitoylation
sequence (aa 83–120) NT of
GFP
Transfected
construct
(Mus Musculus)
pSFV1 GFP-SN25
shift6aaL
this paperderived from pSFV1
GFP-SNAP-25 WT, insertion of
a six aa G/S spacer after G82
Transfected
construct
(Mus Musculus)
pSFV1 GFP-SN25
shift10aaL
this paperderived from pSFV1
GFP-SNAP-25 WT, insertion
of a 10 aa G/S spacer after G82
Transfected
construct
(Mus Musculus)
pSFV1 GFP-SN25
shift14aaL
this paperderived from pSFV1
GFP-SNAP-25 WT, insertion
of a 14 aa G/S spacer after G82
Transfected
construct
(Mus Musculus)
pSFV1 GFP-SN25
Lext7aa
this paperderived from pSFV1
GFP-SNAP-25 WT, NT
insertion of 7 aa analogous
to linker of SNAP-23
Transfected
construct
(Mus Musculus)
pSFV1 GFP-SN25
Lext14aa
this paperderived from pSFV1 GFP-SN25
Lext7aa, insertion of a 7aa G/S
spacer after A119
Transfected
construct
(Mus Musculus)
pSFV1 GFP-SN25
shift14aaLN4CS
this paperderived from pSFV1 GFP-SN25
shift14aaL, substitution of
4x cysteine by serine
Transfected
construct
(Mus Musculus)
pSFV1 GFP-SN25
LN5KA,4CS
this paperderived from pSFV1
GFP-SN25 LN4CS, substitution
K83A, K94A, K96A, K102A, K103A
Transfected
construct
(Mus Musculus)
pSFV1 GFP-SN1-L
IRES SN2
this paperbicistronic expression vector
(emcv IRES), newly cloned from
pSFV1 and Snap25 cDNA
fragments (SN1-L: aa 1–141;
SN2: aa 138–206)
Transfected
construct
(Mus Musculus)
pSFV1 mCherry-SN2
IRES SN1-L
this paperbicistronic expression vector
(emcv IRES), newly cloned from
pSFV1 and Snap25 cDNA
(SN1-L: aa 1–141; SN2: aa 138–206)
Transfected
construct
(Mus Musculus)
pSFV1 mCherry-SN2
IRES SN25Δ26
this paperbicistronic expression
vector (polio virus IRES), newly
cloned from pSFV1 and
Snap25 cDNA fragments
(SN25Δ26: aa 1–180; SN2:
aa 138–206)
Recombinant
DNA reagent
pET28 SN25WTthis paper6xHis-tagged SNAP-25a
WT for protein expression
Recombinant
DNA reagent
pET28 SN25 LGGSthis paper6xHis-tagged SN25 LGGS
for protein expression
Recombinant
DNA reagent
pET28 SN25 LNGGSthis paper6xHis-tagged SN25 LNGGS
for protein expression
Recombinant
DNA reagent
pET28 SN25 LCGGSthis paper6xHis-tagged SN25 LCGGS
for protein expression
Recombinant
DNA reagent
pET28 SN1this paper6xHis-tagged SN1 (aa 1–82)
fragment for protein
expression
Recombinant
DNA reagent
pET28 SN2this paper6xHis-tagged SN2 (aa 142–206)
fragment for protein expression
Recombinant
DNA reagent
pET28 SN1-Lthis paper6xHis-tagged SN1-L (aa 1–142)
fragment for protein expression
Recombinant
DNA reagent
pET28 L-SN2this paper6xHis-tagged L-SN2 (aa 83–206)
fragment for protein expression
Software,
algorithm
IgorProWaveMetrics
Software
Software,
algorithm
AutesPNPI electronics
Software,
algorithm
ImageJNIH software

Mutagenesis and viral expression

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Substitution or insertion mutations in SNAP-25a were generated by overlap extension polymerase chain reaction (PCR) using appropriate primers containing the desired non-homologous sequences. For expression of the mutant proteins in chromaffin cells, the produced PCR fragments were cloned into a Semliki Forest shuttle vector (pSFV1, Invitrogen) using NsiI or NsiI/BssHII sites for insertion. All SNAP-25 variants were N-terminally fused to GFP using a spacer of 25 amino acids in order to allow for identification of infected cells and quantification of expression levels. To generate bicistronic expression vectors an internal ribosomal entry site (IRES) and a second ORF were inserted into pSFV1 using a BssHII restriction site. In order to yield similar or disparate expression levels of both products, either an encephalomyocarditis virus IRES or a polio virus IRES were used to drive the second ORF. To recognize infected cells, the SN2 fragment was N-terminally tagged with mCherry using a spacer of 18 amino acids. SF viruses were produced using standard protocols. SNAP-25 fragments and linker mutants were also subcloned in pET28a vectors using NheI/BamHII restriction sites for the production of recombinant proteins.

Cell culture and electrophysiological recordings

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Adrenal glands from E18/19 mouse embryos of both genders were used for isolation of chromaffin cells following standard protocols. All mice were handled in compliance with the federal German animal welfare act and local regulations of the University of Saarland. Cultured chromaffin cells were infected with SF viruses on the second and third day in vitro, and electrophysiological recordings were performed 5–6 hr post infection. Cultures derived from a given Snap25-/- animal were equally committed to all experimental conditions. Recorded cells for each tested mutant were derived from ≥2 KO animals. Secretion was characterized by capacitance measurements and simultaneous amperometric recordings as described earlier (Mohrmann et al., 2013). Similar numbers of recordings were performed for all conditions during individual sessions. The order, in which different mutants and controls were measured within a session, was varied to avoid a bias in the age of the cultures. Catecholamine release was stimulated either by direct infusion with intracellular solution containing 19 µM free Ca2+ via the patch pipette or by photolytic Ca2+-uncaging after infusion of nitrophenyl-EGTA (Synaptic Systems). In uncaging experiments, photo-artifacts were removed from amperometric traces by subtraction of a reference measurement with only the carbon fiber electrode in the field of view. Ratiometric Ca2+-imaging with fura-4F and furaptra (Invitrogen) was performed as reported before (Mohrmann et al., 2013). [Ca2+]i was raised to 800–900 nM by transient illumination of the cell before a UV flash was applied. The intracellular solution for Ca2+-uncaging experiments was composed of (in mM): 100 Cs-glutamate, 32 Cs-HEPES, 8 NaCl, 4 CaCl2, 2 Mg-ATP, 0.3 GTP, 5 Nitrophenyl-EGTA, 0.4 fura-4F, 0.4 furaptra, and 1 Vitamin C, pH 7.2 (osmolarity adjusted to 290 mOsm). For Ca2+-infusion experiments a modified intracellular solution was used (in mM): 110 Cs-glutamate, 8 NaCl, 20 diethylene triamine penta-acetic acid, 5 CaCl2, 2 MgATP, 0.3 Na2GTP, and 40 Hepes-CsOH, pH 7.3 (osmolarity adjusted to 290 mOsm). The extracellular solution contained (in mM): 145 NaCl, 2.8 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, pH 7.2 (osmolarity adjusted to 300 mOsm). For intracellular infusion of oleic acid (OA), a 2.5 mM stock solution was prepared in DMSO, from which a final concentration of 5 µM OA (1:500 dilution) was prepared in 19 µM free Ca2+ containing intracellular solution. For vehicle controls, the same volume of DMSO (without OA) was added to the intracellular solution (0.2% final concentration of DMSO). Membrane capacitance and amperometric spikes were measured simultaneously for 120 s.

Electrophysiological data were analyzed using custom-written macros in IGOR Pro (v. 6.22A, Wavemetrics). Primed vesicle pools and the corresponding release rates were estimated by approximating capacitance traces by three exponential functions. Generally, the two fast exponential functions describe vesicle release from the readily-releasable poolRRP<50ms) and the slowly-releasable pool (50 ms<τSRP<500ms), while sustained release is represented by the third slow component. In case that the algorithm delivered a negative amplitude for one component or the fast time constants were not separated by a factor of at least two, the capacitance change was fitted by a biexponential function. Noteworthy, the different kinetic components cannot always be clearly distinguished, when pool sizes and release rates are simultaneously affected.

Single amperometric spikes were recorded using home-made carbon fiber (ø 5 µm, Amoco) electrodes. Digitized amperometric currents were filtered at 3 kHz prior to analysis. Only amperometric spikes with a peak amplitude >4 pA and within the charge range from 10 to 5000 fC were considered for frequency analysis. Amperometric events with a peak amplitude >7 pA were selected for the analysis of kinetic properties using custom macros (Autesp). To gain reliable data on current fluctuations in the pre-spike signal, we restricted our analysis to prespike currents with a total duration longer than 2 ms. For the analysis of prespike signal flickers, the current derivative was filtered at 1.2 kHz. Only deflections exceeding a threshold level of ±6 pA/ms (corresponding to 4*SD) were counted, and fluctuation frequency was calculated as the number of detected suprathreshold fluctuations divided by prespike signal length. Due to the large number of experimental conditions, different sets of mutants were tested in separate experiments. Data were pooled after confirmation that control experiments delivered statistically consistent results.

Localization and expression analyses

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The subcellular localization of GFP-tagged SNAP-25 mutants was investigated 5–6 hr post infection using a laser scanning microscope (LSM 710, Carl Zeiss Microimaging GmbH). Confocal images near the equatorial plane of live cells were acquired at 25°C using a 100x/1.4 oil-objective (Zeiss Plan-Apochromat) with 488 nm excitation, pinhole size of 1 airy unit, and fixed gain settings. Protein distribution near the membrane was quantified by radial line scans covering a distance of ~1.5 µm on both sides of the plasma membrane (ImageJ). Resulting fluorescence profiles were aligned using the maximum of the first derivative, that is the position of maximal fluorescence gain, as anchor point (custom macro, IGOR, Wavemetrics). The position of the plasma membrane was determined based on the clearly detectable fluorescence maximum of GFP-SN25 WT, and membrane expression of mutant proteins was quantified at the corresponding position.

For quantification of total protein expression, epifluorescence pictures of live cells were acquired at 25°C using a 100x/1.3 NA oil-objective (Zeiss) on an Axiovert 25-microscope equipped with a CCD camera (AxioCam MRm). GFP fluorescence throughout the cell body was quantified using ImageJ. Protein levels of SNAP-25 fragments expressed in Snap25-/- chromaffin cells by bicistronic SFV were quantified in Western Blot experiments. Primary antibodies specifically recognizing an N-terminal epitope (71.1, Synaptic Systems) or C-terminal epitope (AB1762, Millipore) were employed to quantify fragments using a standard chemoluminescence detection system (Pierce ECL substrate kit). To calculate expression ratios, the concentration of each fragment was determined by a three-point calibration curve, in which defined amounts of bacterially-purified full length protein were used as reference.

Biochemical analyses

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His-tagged SNAP-25 mutants were bacterially expressed (E. coli BL21DE3) and purified using a nickel-nitrilotriacetic acid column (Qiagen), as previously described (Mohrmann et al., 2013). To investigate ternary SNARE complex formation SNAP-25 variant, stx-1A (aa 1–262), and GST-syb-2 (aa 1–116) were first dialyzed into a solution containing (in mM): 100 NaCl, 20 Tris, 1 DTT, 1 EDTA, 0.5% Triton X-100, pH 7.4. Equal amounts (~3 µM) of SNAP-25/mutant variant, stx-1A, and GST-Syb-2 were mixed in a test volume of 80 µl and incubated for different time intervals at 25°C under slight agitation. SNARE assembly was stopped by addition of SDS-containing sample buffer, and (unboiled) samples were analyzed by SDS-PAGE and Coomassie blue staining of protein bands. Gels were scanned on a flat-bed scanner, and SNARE complex formation was quantified by densitometry using ImageJ.

For stx-1A binding assays with SNAP-25 fragments and linker mutants, GST-stx-1a (aa 1–262, 25 µM) was first immobilized on glutathione-sepharose beads (2 hr, 4°C). After extensive washing, beads were incubated for 20 hr at 4°C with test proteins (5 µM) in a binding buffer containing 100 mM NaCl, 20 mM Tris, 1 mM DTT, 1 mM EDTA, 0.5% Triton X-100 (pH 7.4). Subsequently, beads were washed four times, resuspended in fresh binding buffer, and protein retention was analyzed by SDS-PAGE and Coomassie blue staining. The amount of bound protein was quantified by densitometry using ImageJ. Assuming that Coomassie staining is directly proportional to protein size under our conditions, we calculated the molar ratio nbound/nstx1A accounting for the different molecular mass of test proteins.

Statistics

Electrophysiological measurements of secretion in individual cells were considered as independent biological replicates throughout this study. In all electrophysiological experiments, we aimed for a minimal sample size of >10 independent recordings. The actual number of replicates (n) is provided in the corresponding figure panels. Balanced numbers of recordings for all experimental conditions were acquired in each recording session. In biochemical assays, data from individual samples of mixed purified proteins were handled as independent biological replicates for statistical comparisons. Data were excluded from analysis, if common technical standards in individual experiments were violated (e.g. unstable electrophysiological recordings with high leak current). We did not use a statistical method to identify and eliminate ‘outliers’. Statistical tests were performed in Sigmaplot 12 (Systat Software). Following standard procedures, we generally used unpaired Student’s t-test for comparisons between two experimental conditions and ANOVA for experiments featuring multiple conditions. For specific comparisons between groups after ANOVA, we employed Tukey’s post-hoc test, if not stated otherwise. Extended statistical data are provided for each figure as Source Data. Significance levels: ‘*’ for p<0.05, ‘**’ for p<0.01, and ‘***’ for p<0.001.

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Decision letter

  1. Axel T Brunger
    Reviewing Editor; Stanford University, United States
  2. Eve Marder
    Senior Editor; Brandeis University, United States

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "The SNAP-25 linker supports fusion intermediates by local lipid interactions" for consideration by eLife. Your article has been reviewed by Randy Schekman as the Senior Editor, a Reviewing Editor, and three reviewers. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

In this manuscript, the authors use mutagenesis, secretion measurements from mouse adrenal chromaffin cells, and in vitro SNARE assembly and pulldown methods to re-investigate the role of the linker region, which connects the two SNARE-motifs in SNAP-25 (denoted SN1 and SN2). They identify two different functions for the N- and C-terminal end of the linker. The C-terminus of the linker is found to stabilize the syntaxin-SNAP-25 acceptor complex for VAMP/synaptobrevin, whereas the palmitoylated N-terminus of the linker supports fast triggering, priming and fusion pore expansion. Interestingly, the effect of replacing the N-terminus of the linker (including the palmitoylated cysteines) with a flexible GGS-motif is overcome by infusion of oleic acid, which promotes curvature due to its cone-shape.

The authors investigate SNARE complex formation in vitro using split SNAP25 constructs and show convincingly that syntaxin binds the constructs SN1 plus linker-SN2 and that the presence of the linker on SN2 is essential. These results point to a role of the linker in efficient SNARE complex assembly. Replacing the linker within unsplit SNAP25 by GGS repeats with the same total length also supports SNARE complex formation, although with only 50% efficiency compared to wt SNAP25. However, this construct does not support fusion in response to caged calcium stimulation as assessed by capacitance measurements and amperometry. Mutation of the 4 cysteins to serine reduces fusion amplitude and slows down the kinetics. Adding an extra palmitoylation site linked to the N terminus does provide plasma membrane localization but the 4CS mutation still reduces fusion amplitude and speed, suggesting that the location of the palmitoylation site matters. To address this question the authors performed experiments introducing GGS linkers of variable length between SN1 and the Palmitoylation sites. These mutations have rather small impact on fusion. The amplitudes are unchanged and only small changes in kinetics are observed.

There are several concerns and suggestions (detailed below) that should be addressed in a revised manuscript. Moreover, the presentation requires revision.

Essential revisions:

The author's statement "The aggravated phenotype of SN25 shift14aaLN4CS strongly argues against a significant contribution of the linker to force transfer but rather suggests that delayed fusion is caused by a misalignment of membrane and protein interactions of the N-terminal linker section." Is unclear and questionable. First, the impaired fusion kinetics of the LN-4CS, the shift6aaL, shift10aaL and shift14aaL render the opposite conclusion likely (that the force transfer IS impaired) – how else do the authors explain the phenotypes? Moreover, the rationale for this experiment is apparently that the 4CS-mutation has already eliminated the possibility for force transduction to the membrane, and therefore the exacerbation based on the 14 aa insertion must be due to something else. However, the authors themselves show in Figure 5—figure supplement 2 that removal of lysine residues on the background of the 4CS-mutation lead to similar changes (longer time constants of RRP release), and the authors conclude "the lysine residues contribute to the linker:lipid interactions that facilitates Ca2+-dependent fusion triggering". Since the lysines are still present in the shift14aaLN-4CS mutation this finding undermines the conclusion that the linker is not conferring force between the SNAREs and the lipid membrane. Another point is that the insertion of 14aa close to the nascent fusion pore could have dominant-negative effects, which might affect fusion. It is also not clear why "misalignment of membrane and protein interactions" is an alternative to impaired force transfer. Would such a misalignment not affect force transfer? Given that the authors did not do mutations to change the force released by the SNAREs, the conclusion about the transfer of force should be tempered.

What is the reason to use both methanol and oleic acid to rescue the LN-GGS? Methanol seems to have almost rescued the uncaging-induced secretion amplitude, but not the time constants of release, whereas oleic acid rescues the shape of amperometric spikes. Does this represent two different phenotypes, or only one? I.e. did methanol not rescue the amperometric spike shape, and was this the reason that oleic acid was then used? (And vice versa?)

Apparently, the authors conclude that the two ends of the SNAP-25 linker subserves two very different functions: the C-terminal part is involved in supporting t-SNARE interactions, whereas the N-terminal linker with its acyl side-chains 'engage in local lipid interactions'. However, why are the phenotypes of the LN-GGS and the LC-GGS then basically indistinguishable in uncaging experiments (Figure 2)? Does this not undermine the conclusion? Can the phenotype of the LC-GGS in uncaging experiments be rescued by methanol and oleic acid?

Mutation of the 4 cysteins to serine reduces fusion amplitude and slows down the kinetics but to a lesser degree than the full replacement with SGG repeats or SGG replacement in the N terminal residues 82-118 of the linker. Very similar results are obtained for corresponding mutations in the C terminal parts of the linker (residues 118-142), leaving the palmitoylation sites intact. It would be interesting to know if these constructs are actually palmitoylated in the cells or if there are convincing reasons to assume that they are. Maybe this is shown in Figure S4 Ai but needs to be stated specifically in the main text.

In Figure 4Bii it is impossible to tell which trace is which. There are (presumably) 3 black control traces with slightly different kinetics and 2 or 3 "shift" traces which are slightly slower but also very similar. As presented, it is unclear what can actually be concluded form this data. Also, the rationale of using a highly flexible linker rather than a rigid linker is not obvious because it raises the question how much the Palmitoylation sites are spatially displaced from their wt location.

Would it be possible to generate an LC4C construct where a palmitoylation site is introduced at the N terminal and of the linker next to SN2?

Surprisingly, Figure 4C shows that the effect of 14aaL is much more pronounced in the LN4CS mutant, which lacks the palmitoyl anchor. Doesn't this result suggest that the lipid anchors stabilize a reasonable SNARE complex structure, making it rather resistant to the introduced spacers and that in the absence of this stabilization the extra spacers have more detrimental impact on the SNARE complex?

In the Discussion section this leads to contradicting statements: "insertion at the N terminal end…… decrease release kinetics irrespective of membrane anchorage" and "…the phenotypes……reflect functional deficits originating from misalignment of the Palmitoylation site with other components of the fusion machinery".

The analysis of quantal events in Figure 5 is very interesting. However, it needs to be explained how PS amplitude and initial PS amplitude were defined. An example of spike analysis illustrating the different parameters would help. What is the significance of the observed changes in PS amplitude values? Do they suggest a change in fusion pore structure?

It is interesting that LC-GGS is indistinguishable from wild type, while there is very strong reduction in amplitude and kinetics of secretion in the flash experiment. This should be discussed more specifically.

For the results of Figure 6 and Figure 7, effects of methanol and oleic acid, the discussion is a bit misleading. The authors point to a rescue effect of the LN-GGS mutation, but it seems that these compounds similarly affect the results in normal wild type cells. It is not convincingly explained why these specifically relate to a rescue effect of the LN-GGS mutation.

The authors suggest that the linker domain of SNAP-25 prevents dimerization of the SNARE complexes "by masking the sticky surfaces". However, there is no direct proof for that except an apparent size shift in the SDS-PAGE. Inspecting the Figure 1C and Figure 3—figure supplement 1C carefully, one can see that also with WT SNAP-25 construct, a band with a similar size-shift becomes visible at 24hours. Additionally, even though it's apparent from the reduction of the monomeric SNAREs that the high molecular mass represents the SNARE complex, the identity of this high molecular mass band is not clear, because it's just Coomassie staining. Especially in the case of separated SN1 and SN2 fragments (Figure 1C), as there is no obvious reduction in the SN fragments and also as SN2 cannot even bind to Stx1 (Figure 1D), it cannot be concluded that high-size band represents dimerized SNARE complexes. This part should be toned down.

Using the fragments SN1+L-SN2 for SNARE complex assembly would also be informative, as in this case the interaction of SN2 with Stx1 is partially rescued and that in turn could rescue Syb2 binding as well. Last, it is not very clear what the star in Figure 1D stands for, and also why there are two fragments when SNAP-25 LGGS is used. The lower band seems at the size of SN1-L. Is the SNAP-25 LGGS construct somewhat unstable and degraded?

The expression experiments using the SNAP-25 linker mutants are not very clear (Figure 1—figure supplement 3A,B). The total expression of these mutants might be comparable to WT, but it is not informative for the plasma membrane localization (except Figure 3—figure supplement 1A) However, the interpretation of the functional output using the mislocalized proteins should be done more cautiously. Also, a test for proper localization regarding the 5KA,4CS mutant as in Figure 3—figure supplement 1A would be useful. Removal of 5K in the absence of palmitoylation could further affect the membrane targeting and localization of SNAP-25, which would explain the further reduction of the release (Figure 5A). Finally, the release properties of the cells expressing only the 5KA mutant should be examined because a mislocalization of this mutant is less likely due to proper palmitoylation of the cysteines. This would be useful for understanding whether the effects of 5KA and 4CS mutations are additive.

The authors claim in subsection “N- and C-terminal linker regions mechanistically support Ca2+-dependent release”, that SNAP-25 LC4G mutant shows phenotypical traits of SNAP-25 LCGGS. However, SNAP-25 LC4G shows only an intermediate phenotype. This claim should be therefore toned down. Comparing the functional output of mislocalized proteins creates ambiguity. The authors mention in subsection “A postfusional role of linker:lipid interactions in controlling pore dilation” that "this slow-down of the main spike signal is primarily caused by the loss of several lysine residues surrounding the central cysteine group, as substitution of these amino acids in SN25 LN5KA,4CS fully replicated the phenotype of the full substitution mutant SN25 LNGGS." However, the resemblance between the phenotypes of SNAP25-LNGGS and LN5KA,4CS might well be merely due to lack of membrane attachment and thus mislocalization. A membrane targeting strategy similar to what is done in Figure 3 would be more informative regarding the 4CS mutants combined with linker insertion (14aa; Figure 4C) and 5KA mutants (Figure 5—figure supplement 2).

Addition of another membrane targeting motif (Figure 3) could potentially change the conformation of SNAP-25. Besides, comparing the release parameters in Figure 3 and in the other figures, it seems that this P-GFP-SNAP25 WT has a better release efficacy. This is a very minor point, but could be mentioned in discussion.

The interpretation of MeOH and oleic acid experiments should be toned down. It is clear that MeOH and OA facilitate release, but this could be due to a mechanism independent from the function of the linker region. In other words, MeOH or OA already increases and accelerates the NT release from cells expressing WT SNAP-25, and as the cells expressing the linker mutant already has deficits in NT release, it is not surprising that they show a higher sensitivity to MeOH and OA application. Even though the data is largely convincing, the possibility of independent action of MeOH or OA from linker-lipid interaction cannot be ruled out.

In further support of a structural significance of the SNAP-25 linker (rather than being a "flexible linker"), a recent higher resolution EM structure of the NSF/aSNAP/SNARE complex revealed diffuse density for the SNAP-25 linker at both the N-terminal and C-terminal ends, suggesting that these parts of the linker are associated with the SNARE complex, albeit probably sampling multiple conformations. Thus, the linker is not entirely flexible, but it is not uniquely structured either (see Figure 7 in White et al., 2018). Perhaps this recent finding could be added to the Discussion section.

https://doi.org/10.7554/eLife.41720.038

Author response

Essential revisions:

1) The author's statement "The aggravated phenotype of SN25 shift14aaLN4CS strongly argues against a significant contribution of the linker to force transfer but rather suggests that delayed fusion is caused by a misalignment of membrane and protein interactions of the N-terminal linker section." Is unclear and questionable. First, the impaired fusion kinetics of the LN-4CS, the shift6aaL, shift10aaL and shift14aaL render the opposite conclusion likely (that the force transfer IS impaired) – how else do the authors explain the phenotypes? Moreover, the rationale for this experiment is apparently that the 4CS-mutation has already eliminated the possibility for force transduction to the membrane, and therefore the exacerbation based on the 14 aa insertion must be due to something else. However, the authors themselves show in Figure 5—figure supplement 2 that removal of lysine residues on the background of the 4CS-mutation lead to similar changes (longer time constants of RRP release), and the authors conclude "the lysine residues contribute to the linker:lipid interactions that facilitates Ca2+-dependent fusion triggering". Since the lysines are still present in the shift14aaLN-4CS mutation this finding undermines the conclusion that the linker is not conferring force between the SNAREs and the lipid membrane. Another point is that the insertion of 14aa close to the nascent fusion pore could have dominant-negative effects, which might affect fusion. It is also not clear why "misalignment of membrane and protein interactions" is an alternative to impaired force transfer. Would such a misalignment not affect force transfer? Given that the authors did not do mutations to change the force released by the SNAREs, the conclusion about the transfer of force should be tempered.

We thank the reviewers for pointing out that our conclusions with regard to the spacer insertion mutants were imprecisely formulated. Due to length concerns, we tried to keep explanations very concise, which may have hurt accessibility and clarity. While we acknowledge the reviewers’ doubts about the correct interpretation of the mutant phenotypes, we still think that our findings render it unlikely that the acylated linker plays a prominent role in force transduction. Earlier work has demonstrated that extension of the juxtamembrane domain of synaptobrevin or syntaxin critically interferes with liposome fusion, vacuole fusion in yeast, and Ca2+-triggered secretion due to impaired force transfer (Deak et al., 2006; Guzman et al., 2010; Kesavan et al., 2007; McNew et al., 1999; Wang et al., 2001). In particular, it has been established that insertion of flexible spacers >=12 aa (high Gly-content) abolishes membrane fusion in most model systems (Deak et al., 2006; Guzman et al., 2010; Kesavan et al., 2007). Assuming that an effective cooperation between SNAP-25 and its cognate SNARE partners requires a similarly tight coupling of all membrane anchoring structures to the core complex, it is expected that the insertion of 14 amino acids before the acylation site is sufficient to severely compromise linker-mediated force transfer and to induce secretion defects similar to the non-acylated SN25 LN4CS mutant. However, cells expressing SN25 shift14aaL exhibited unexpectedly weak secretory deficits (Figure 5B), thus questioning the idea that the acylated linker has a substantial contribution to force transfer.

The notion of linker-mediated force transduction is further challenged by our finding that insertion of the 14 aa-long spacer into SN25 LN4CS exacerbated its phenotype (Figure 5C). The rationale behind this experiment was that elimination of membrane anchorage should abolish force transfer and thus should preclude any additional effects of spacer insertions on force transduction. The more pronounced kinetic slowdown of secretion in the combined mutant however implies that spacer insertion interferes with mechanisms that do not depend on linker acylation. As insinuated by the reviewer, it might be argued that elimination of acylation in SN25 LN4CS does not abolish all lipid interactions, and that the residual membrane contacts still play a role in force transfer. While membrane binding energies for unstructured peptides are generally considered to be negligibly small (Almeida, 2014; White and Wimley, 1999), recent work by Weber et al., (2017) indeed suggested that an excess of positive charged lysine residues around the cysteine cluster in the N-terminal could mediate transient membrane contacts. However, the membrane free binding energy of short peptides containing 5 lysine residues (which is comparable to the 5 lysine residues in the N-terminal linker segment) has been estimated to be just around -6 kJ/mol (Kim et al., 1991). In comparison, a crude assessment of the Gibbs free energy gain for the membrane insertion of the four palmitate moieties of SNAP-25 yields an maximal energy gain of -150kJ/mol according to Tanford’s formula (Pool and Thompson, 1998; Tanford, 1980). MD simulations have even estimated binding free energies of ~ 250 kJ/mol for regular TMDs (Chetwynd et al., 2010; Lindau et al., 2012). From the perspective of binding energies, it is thus implausible how the very weak electrostatic membrane association of SNAP-25 could significantly contribute to force transfer, as mechanical tension would likely disrupt these interactions before a membrane deformation occurs. In line with the labile nature of the electrostatic linker:membrane contacts, previous studies also reported very low binding of non-palmitoylated SNAP-25 to membrane sheets (Nagy et al., 2008; Weber et al., 2017).

While the weak lipid interactions of the flanking lysine residues cannot relay pulling force on the plasma membrane, their elimination still caused a slight but noticeable slowdown of secretion kinetics in SN25 LN5KA,4CS (Figure 5—figure supplement 2) – similar to the effects seen in spacer insertion mutants. We think that this finding hints at a scenario, in which the acylation site and surrounding residues are part of a membrane-interacting structure that actively induces fusogenicity but does not primarily serve as an anchorage-point for SNARE force transfer to the plasma membrane. Thus, it seems straight forward to assume that the functional defects caused by spacer insertion result from a displacement of this membrane interacting linker structure, which was ambiguously referred to as a “misalignment” of the N-terminal linker segment in the original version of the manuscript. Given that treatment of cells with the membrane-perturbing agents oleic acid and methanol disproportionally facilitated release in cells expressing the N-terminal substitution mutant SN25 LNGGS (Figure 7, Figure 8), we speculate that local membrane interactions of the N-terminal linker structure could indeed directly alter membrane properties to similarly increase fusogenicity. Despite the compensatory action of oleic acid on fusion pore evolution in the mutant (Figure 8), we cannot exclude that the presence of the spacer could also sterically affect the fusion pore in its direct vicinity, as proposed by the reviewer. Nevertheless, such effect would not change the general interpretation of our results with regard to force transfer, as the flexible spacer should still loosen the mechanical coupling to the acylated linker. Hence, one would have expected to find a more pronounced phenotype for SN25 shift14aaL in such scenario, if linker-mediated force transfer were indeed highly relevant.

To accommodate the reviewers’ concerns, we have largely rewritten the corresponding passages in Results section and Discussion section. The relative weakness of the electrostatic membrane interactions of the lysine residues in the linker is now better discussed (subsection “The acylated N-terminal linker segment promotes triggering”), resulting in a clearer line of argument. Following the reviewers’ suggestion, we have also tempered our conclusion about force transfer and now modestly state in the Discussion section:

“Considering the very moderate defects of spacer insertion mutants and the substantial contribution of acylation-independent effects, it stands to reason that force transfer is not the primary function of linker-mediated membrane interactions.”

What is the reason to use both methanol and oleic acid to rescue the LN-GGS? Methanol seems to have almost rescued the uncaging-induced secretion amplitude, but not the time constants of release, whereas oleic acid rescues the shape of amperometric spikes. Does this represent two different phenotypes, or only one? I.e. did methanol not rescue the amperometric spike shape, and was this the reason that oleic acid was then used? (And vice versa?)

We used methanol (MeOH) and oleic acid (OA) in our rescue experiments, because both were reported to facilitate membrane fusion by altering lipid bilayer properties. Their effects on membranes are not congruent, as infusion of cells with MeOH putatively modifies the surface hydration and lipid packing of the exposed leaflet, while OA inclusion primarily induces negative membrane curvature. Earlier preparatory experiments have indicated to us that infusion of MeOH only exerts a strong effect on total secretion but leaves fusion pore evolution largely unaffected. In proof, we provide a dataset illustrating the persistence of spike properties in wildtype (WT) cells infused with 2% MeOH (Author response image 1). In contrast to MeOH treatment, intracellular application of OA was found to generally facilitate fusion rate and to simultaneously accelerate spike kinetics. As we expected that MeOH treatment would not alter spike properties in SN25 LNGGS-expressing cells, we did not perform high-resolution amperometric recordings.

Author response image 1
Effect of intracellular Methanol (MeOH, 2%) application on the properties of amperometric spikes elicited by infusion of 19 µM Ca2+.

(a) Representative amperometric spikes recorded in WT chromaffin cells in the absence (black) or presence (red) of MeOH. (b) Superimposition of averaged spike waveforms from two successively recorded cells, one untreated (113 spikes), the other infused with MeOH (138 spikes). (c) Detailed kinetic analysis of the prespike foot signals: Shown are mean foot amplitude, charge, and duration (WT: 2308 events, 20 cells; WT+MeOH: 3730 events, 25 cells). (d) Quantitative analysis of main spike parameters: Depicted are mean amplitude, charge, rise time (50-90%), and half-width. No significant differences were observed. Error bars represent SEM.

https://doi.org/10.7554/eLife.41720.037

The results of the rescue experiments with MeOH and OA suggest that linker:membrane contacts are likely important for initial fusogenicity/fusion pore opening and the subsequent postfusional dilatation of the pore. While methanol and OA treatment can both ameliorate total secretion, only the OA-induced curvature seems to be important for the fast expansion of the fusion pore. From this perspective, one can distinguish two phenotypes of SN25 LNGGS in consecutive stages, as suggested by the reviewers.

Apparently, the authors conclude that the two ends of the SNAP-25 linker subserves two very different functions: the C-terminal part is involved in supporting t-SNARE interactions, whereas the N-terminal linker with its acyl side-chains 'engage in local lipid interactions'. However, why are the phenotypes of the LN-GGS and the LC-GGS then basically indistinguishable in uncaging experiments (Figure 2)? Does this not undermine the conclusion? Can the phenotype of the LC-GGS in uncaging experiments be rescued by methanol and oleic acid?

Several of our findings indeed suggest a differential mechanistic function of N- and C-terminal linker motifs in secretion. However, it is also true that we observed phenotypic similarities between different N- and C-terminal linker mutants. While larger linker substitutions invariably induced a “complex” phenotype with priming and fusion triggering deficits of different intensity, only very restricted manipulations within the very N-terminal region (especially in case of the spacer insertion mutants SN25 shift6/10/14aaL) resulted in a mechanistically distinct fusion defect. We think that the occurrence of complex phenotypes most likely reflects a structural interdependence between different motifs due to the relative short length of the linker peptide. New structural data derived from high-resolution cryo-EM experiments have in fact indicated that the linker more closely lines the core complex near its ends (White et al., 2018), implying the existence of interaction sites in both terminal regions. Thus, the substitutions in SN25 LNGGS or SN25 LCGGS should at least interfere with one of these interaction regions. Given that the linker motifs are part of the same stretched-out peptide chain, a potential linker dislocation at one end of the linker may well result in an altered configuration at the other end, in this way possibly affecting the function of almost all linker motifs. To acknowledge this idea, we have added a statement concerning the structural interdependence of motifs to the Results section:

“As especially noticeable for the N- and C-terminal mutants SN25 LNGGS and SN25 LCGGS (Figure 3A-B), large linker substitutions caused very similar phenotypes despite targeting non-overlapping regions. While several findings indeed suggest a differential mechanistic function of the N- and C-terminal linker (see discussion below), the apparent convergence of phenotypes likely reflects a structural interdependence between certain motifs due to the relative short length of the linker peptide. Given that the linker motifs are part of the same stretched-out peptide chain that lines the core complex, a potential linker dislocation at one end of the linker may well result in an altered configuration at the other end, in this way possibly affecting positioning and function of distant linker sections.”

It is an interesting question whether the uncaging phenotype of SN25 LCGGS can be rescued with methanol or OA in a similar way as seen for SN25 LNGGS. If our above interpretation is correct that large substitutions can affect N- as well as C-terminal linker motifs, a similar rescue of total secretion for both mutants should be expected. However, due to time restrictions, an experimental confirmation of this aspect was unfortunately beyond the scope of this revision.

Mutation of the 4 cysteins to serine reduces fusion amplitude and slows down the kinetics but to a lesser degree than the full replacement with SGG repeats or SGG replacement in the N terminal residues 82-118 of the linker. Very similar results are obtained for corresponding mutations in the C terminal parts of the linker (residues 118-142), leaving the palmitoylation sites intact. It would be interesting to know if these constructs are actually palmitoylated in the cells or if there are convincing reasons to assume that they are. Maybe this is shown in Figure S4 Ai but needs to be stated specifically in the main text.

We investigated the subcellular distribution of the C-terminal substitution mutant GFP-SN25 LCGGS by confocal laser microscopy and did not find any significant change in its membrane localization compared to the WT protein (shown in Figure 3 —figure supplement 1A). These data clearly suggest an unaltered palmitoylation level of the mutant protein. In contrast to GFP-SN25 LCGGS, membrane targeting of the N-terminal substitution mutant GFP-SN25 LNGGS, whose palmitoylation motif was ablated, was diminished and mirrored the distribution pattern of the non-palmitolayted GFP-SN25 LN4CS mutant. To complement the existing dataset, we have now also performed a detailed confocal analysis of the other two C-terminal mutants, SN25 LC4G and SN25 LC9G, and reconfirmed their normal localization at the plasma membrane (Figure 1—figure supplement 3Bb). We have rewritten the corresponding passage to better convey the information (subsection “N- and C-terminal linker regions mechanistically support Ca2+-dependent release”):

“Note that all C-terminal linker mutants were normally targeted to the plasma membrane and exhibited a similar overall expression level like the WT protein in controls (Figure 1—figure supplement 3A,Bb), excluding the possibility that the observed secretion deficits are due to reduced expression.”

In Figure 4Bii it is impossible to tell which trace is which. There are (presumably) 3 black control traces with slightly different kinetics and 2 or 3 "shift" traces which are slightly slower but also very similar. As presented, it is unclear what can actually be concluded form this data.

We thank the reviewers for giving us feedback on the presentation of the normalized capacitance traces in original Figure 4Biii. The panel was indeed intended to highlight the kinetic slowdown in the secretory response of the three spacer insertion mutants, SN25 shif6aa/10aa/14aaL. For this purpose, we subtracted the linear component and normalized all traces to the corresponding ΔCM value 1s after the uncaging flash. As the depicted data was derived from three separate experimental series with independent control recordings, we included the corresponding wildtype rescue traces (black lines) for each dataset but unfortunately did not mark, which control trace referred to which mutant condition. To accommodate the reviewers’ criticism, we now display the three pairs of traces separately (Figure 5Bc).

Also, the rationale of using a highly flexible linker rather than a rigid linker is not obvious because it raises the question how much the Palmitoylation sites are spatially displaced from their wt location.

We intentionally used flexible spacer peptides with high glycine content to distance the SNARE motif from the acylation site, as the high rotational freedom of glycine residues is expected to result in a random coil configuration with reduced conformational strain, which should aggravate the effect of spacer insertion on force transfer. Indeed, previous studies (Deak et al., 2006; Kesavan et al., 2007; McNew et al., 1999), which investigated the role of mechanical coupling by extension of the juxtamembrane domains of synaptobrevin/syntaxin, have exclusively relied on insertion of highly flexible spacers. While a rigid linker may allow for a relatively controlled displacement of the acyl anchors, it should be less efficient in hampering the mechanical coupling to the acyl anchors. Moreover, there is a greater risk to induce unwanted structural issues with respect to other parts of the fusion machinery, as a helical spacer might interact with the preceding amphiphatic SN1 helix or sterically affect the putative “zippering” of the juxtamembrane domains of synaptobrevin/syntaxin in its direct vicinity (Stein et al., 2009). Thus, we are convinced that the used flexible spacers serve our purpose best.

We have slightly altered the corresponding passage in the Results section to better explain the experimental rationale behind the usage of flexible linkers:

“To further test this idea, we constructed linker mutants, in which the distance between the acylation site and SN1 was increased by insertion of flexible spacer peptides (6, 10, or 14 residues; mutants denoted SN25 shift6aa/10aa/14aaL) in order to mechanically “uncouple” the acyl anchors from the core complex (Figure 5A). We especially relied on G/S-containing spacers, as the high rotational freedom of the included Glycine residues should substantially lessen conformational strain and thereby further hamper force transfer.”

Would it be possible to generate an LC4C construct where a palmitoylation site is introduced at the N terminal and of the linker next to SN2?

In principle, a short sequence containing the cluster of cysteine residues (e.g. aa 85-93) could be easily inserted into the linker near the N-terminal end of the complex directly preceding SN2. However, it is not clear whether acylation of the inserted cysteine residues at this position would occur in an efficient manner. Previous truncation experiments in SNAP-25 have identified the N-terminal linker region including aa 85-120 as the minimal palmitoylation motif, thus indicating a requirement for an extended linker segment in acylation (Gonzalo et al., 1999). While the corresponding region is obviously still present in SN25 LN4CS and could even mediate transient plasma membrane contacts as proposed by Weber et al., (2017), it is a concern that the changed relative position of the cysteine cluster may obstruct an efficient interaction with acyltransferases. A further issue arises from our observation that the linker section preceding SN2 is putatively involved in functionally relevant core complex interactions (Figure 3B, Figure 3—figure supplement 1C-D). An insertion of a secondary acylation site in the C-terminal linker region is therefore expected to cause deficits that need to be distinguished from those functional alterations that may originate from altering the position of the acyl anchors.

Considering the potential pitfalls of the experimental approach, we think that such mutant is not ideal. Moreover, we already present data showing that shifting the acylation site to an N-terminal position relative to the complex (P-GFP-SN25 LN4CS; Figure 4) resulted in similar deficits as the non-acylated SN25 LN4CS mutant. Clearly, one would expect that the proposed switch of the acyl anchors to the opposite end of the linker would at best lead to a similar result.

Surprisingly, Figure 4C shows that the effect of 14aaL is much more pronounced in the LN4CS mutant, which lacks the palmitoyl anchor. Doesn't this result suggest that the lipid anchors stabilize a reasonable SNARE complex structure, making it rather resistant to the introduced spacers and that in the absence of this stabilization the extra spacers have more detrimental impact on the SNARE complex?

We consider it difficult to adequately compare the phenotypical deficits caused by spacer insertion in SN25 shift14aaL and SN25 shiftLN4CS. Consequently, we are skeptical about the reviewers’ conclusion that “the effect of 14aaL is much more pronounced in the LN4CS mutant”. In general, largely disproportional changes of secretion parameters in SN25 shift14aaLN4CS-expressing cells should be indicative of a potential synergistic aggravation of the phenotype, as insinuated by the reviewer. Looking at our kinetic data, we however noticed only a ~1.5x increase in τRRP after spacer insertion in SN25 shift14aaLN4CS (Figure 5Cb) while the corresponding change in τRRP was 3.5x for SN25 shift14aaL (in comparison to wildtype controls; Figure 5Bb). Moreover, the relative changes in other parameters were actually quite similar: The time constants for SRP-mediated release were extendedby almost the same factor (1.7x), and the exocytotic delay roughly doubled in both cases. Thus, in our opinion, our data do not support the notion that the spacer-induced relative changes in SN25 shift14aaLN4CS were dramatically more pronounced than in SN25 shift14aaL. Our results would rather fit a scenario, in which the spacer insertion more or less proportionally modulates the stronger phenotype of SN25 LN4CS, thus providing little support for a stabilizing effect of linker acylation.

In the Discussion section this leads to contradicting statements: "insertion at the N terminal end…… decrease release kinetics irrespective of membrane anchorage" and "…the phenotypes……reflect functional deficits originating from misalignment of the Palmitoylation site with other components of the fusion machinery".

We are sorry that our conclusions concerning the role of the N-terminal linker region seem inconsistent to the reviewer. As already elaborated in response to comment #1, the aggravated phenotype of SN25 shift14aaLN4CS suggests that the triggering defect after spacer insertion is in substantial part due to an acylation-independent mechanism. Given that the acyl anchors are by far the most important lipid-interacting element for mechanical straining of the membrane, we interpret the modest phenotype of SN25 shift14aaL and the enhanced slowdown of secretion in the SN25 shift14aaLN4CS mutant as evidence against a prominent role of the linker in force transfer. Therefore, it stands to reason that the functional deficits of spacer insertion mutants actually result from a displacement of the N-terminal linker region that disrupts its local protein and/or lipid interactions near the C-terminal end of the core complex. In line with this idea, substitution of several lysine residues – which previously have been associated with electrostatic linker:plasma membrane interactions (Weber et al., 2017) – slightly decelerated secretion in SN25 LN5KA,4CS mutant (Figure 5—figure supplement 2).

While the role of linker acylation for efficient fusion triggering is somewhat “downplayed” by the observation that spacer insertion aggravates the phenotype of non-palmitoylated SNAP-25, it should be noted that the profound kinetic phenotype of the non-palmitoylated SN25 LN4CS mutant still indicates that the acyl anchors serve a major function in fusion initiation. Unfortunately, the coexistence of acyl anchor-dependent and acylation-independent mechanisms was not clearly expressed in our original text, leading to ambiguous statements. To clarify this issue, we have now reworked the corresponding passage in the Discussion section. The central statement referred to by the reviewers now reads:

“Since the linker acyl anchors serve as the primary mechanical coupling point to the plasma membrane, their loss should have vastly precluded any effect of upstream spacer insertion. The considerably exacerbated phenotype of SN25 LN shift14aaLN4CS however indicates that spacer insertion at least in part disrupts an acylation-independent fusion-facilitating function of the N-terminal linker region. In line with this idea, we observed that substitution of several lysine residues, which are thought to mediate very weak electrostatic membrane contacts of the N-terminal linker segment (Weber et al., 2017), further decelerated release kinetics in SN25 LN5KA,4CS (Figure 5—figure supplement 2). Considering the very moderate defects of spacer insertion mutants and the substantial contribution of acylation-independent effects, it stands to reason that force transfer is not the primary function of linker-mediated membrane interactions. The profound secretory deficits of the non-palmitoylated SN25 LN4CS variant and the observed acylation-independent function of the N-terminal linker segment would rather point to a scenario, in which the palmitoylation motif and the surrounding linker region establish an interface between the SNARE complex and the plasma membrane that directly facilitates triggering by local linker:lipid contacts.”

The analysis of quantal events in Figure 5 is very interesting. However, it needs to be explained how PS amplitude and initial PS amplitude were defined. An example of spike analysis illustrating the different parameters would help. What is the significance of the observed changes in PS amplitude values? Do they suggest a change in fusion pore structure?

As suggested by the reviewer, we have incorporated an example for the spike analysis in Figure 6. For the sake of simplicity, we have highlighted PS duration, PS amplitude, spike amplitude, spike half width and rise time (50%-90%) in the example trace. The initial PS amplitude is determined at the initiation of the PS event, where the current amplitude is at least 4 times of the baseline noise. As such, the initial PS amplitude indicates the width of the fusion pore at the moment of opening. As shown in the example trace, the PS amplitude denotes the maximum current flow during the initial fusion pore state. A change in this parameter likely mirrors the maximum size of the narrow pore. Both parameters, initial PS amplitude and the PS amplitude, were reduced in cells expressing the GFP-SN25 LNGGS mutant, indicating that the size of the pore at the first moment of opening as well as the maximum size of the fusion pore are significantly reduced by the linker mutation.

It is interesting that LC-GGS is indistinguishable from wild type, while there is very strong reduction in amplitude and kinetics of secretion in the flash experiment. This should be discussed more specifically.

The reviewers obviously refer to our finding that SN25 LCGGS-expressing cells exhibit a dramatically reduced secretory response but do not show detectable changes in the kinetics of amperometric spikes. This is especially remarkable, since the N-terminal linker substitution mutant SN25 LNGGS possesses a virtually identical secretion phenotype in flash experiments, but does show a prominent slowdown of spike kinetics in contrast to SN25 LCGGS. We have attributed the delayed transmitter discharge in SN25 LNGGS–expressing cells to the loss of critical membrane interactions of the N-terminal linker segment, which is left unchanged in SN25 LCGGS. Thus, N- and C-terminal linker segments seem to be differentially required in early and late stages of the fusion mechanism.

As mentioned in response to reviewer comment #3, the similarity of phenotypes between N- and C-terminal linker substitution mutants in flash experiments potentially indicates a structural crosstalk of N- and C-terminal linker motifs in defining the exact configuration of the linker along the core complex. Such structural effects should be largely limited to the complex assembly phase, before the linker is fully stretched out. If SNARE assembly is almost complete, the influence of C-terminal linker mutations on the overall linker configuration should cease, while the membrane-anchored, N-terminal linker region should still be able to exert effects in postfusional stages. This would provide a satisfactory explanation for the observed phenotypical difference of both mutants. Following the reviewers’ recommendation, these ideas have been briefly mentioned in the Discussion section:

“It is conspicuous that the phenotypic alterations with respect to the overall secretory response and the transmitter discharge kinetics from single vesicles appear uncorrelated in N- and C-terminal linker mutants. Despite sharing the same kinetic phenotype in Ca2+-uncaging experiments, SN25 LNGGS and SN25 LCGGS strictly differ in fusion pore behavior. As mentioned before, we attribute the convergence of secretion phenotypes in linker mutants to a structural interdependence of different linker motifs during SNARE complex formation. Fusion pore evolution, however, constitutes the final step of the fusion process, when SNARE assembly is almost complete, and indirect effects of the C-terminal linker should be largely diminished. Accordingly, only the direct dependence of pore dilation on N-terminal linker interactions with the plasma membrane should remain relevant.”

For the results of Figures 6 and Figure 7, effects of methanol and oleic acid, the discussion is a bit misleading. The authors point to a rescue effect of the LN-GGS mutation, but it seems that these compounds similarly affect the results in normal wild type cells. It is not convincingly explained why these specifically relate to a rescue effect of the LN-GGS mutation.

We generally define a successful “rescue” as a functional compensation resulting from interventions that target and successfully bypass the compromised mechanistic step. While this definition does not require that treatments are functionally neutral in control experiments, we strictly demand that successful interventions induce a substantially stronger enhancement of release in SN25 LNGGS-expressing KO cells than in controls. Considering the fusion process as a simplistic linear sequence of mechanistic reaction steps, non-compensating treatments should yield (at best) similar relative effects for mutant and controls, if they target steps upstream or downstream of the compromised process, leaving the mechanistic “bottleneck” unaffected. In contrast, an over-proportional recovery is expected for interventions that can effectively compensate for the compromised function of mutant SNAP-25 in the same mechanistic context. Indeed, we found that intracellular application of methanol or oleic acid resulted in a significantly higher relative increase in total secretion of SN25 LNGGS-expressing cells than in control experiments, thus fulfilling our basic criteria for a successful “rescue”. While the observed “rescue” strongly suggests that the N-terminal linker is important for a fusion intermediate that is sensitive to membrane-perturbing agents, our data does not necessarily imply that the membrane effects of the N-terminal linker motif and the compounds are congruent. Nevertheless, we consider it the most likely scenario that the N-terminal linker increases fusogenicity by locally altering membrane properties (like e.g. lipid packing and/or curvature). To improve the clarity of our argumentation we have changed several passages, in particular the Results section:

“If N-terminal linker:membrane interactions critically modulate lipidic fusion intermediates, application of membrane-active reagents might be able to “rescue” release deficits. The underlying idea is that linker:membrane interplay might mechanistically help to facilitate spontaneous lipid stalk formation and membrane merger, and thus might serve a function that could be mimicked by application of membrane-perturbing compounds. While such treatments generally promote fusion (in mutants as well as in controls), the relative effect should be disproportionally strong in KO cells expressing SN25 LNGGS, if the action of the compound can lessen the impact of the mechanistic “bottleneck” imposed by the mutation.”

Additionally, we streamlined the corresponding section in the Discussion section and added the following statement to stress the possibility of alternative explanations:

“While the compensatory actions of membrane-active compounds hint at a specific role of linker:lipid interactions in fusion initiation, it should be noted that the available evidence for this scenario is still circumstantial, and other mechanistic explanations might also apply. In particular, a supporting function of the N-terminal linker in the putative “zippering” of the JMDs of stx-1A and syb-2 (Stein et al., 2009) may represent an alternative explanation, as the linker would be closely involved in inducing membrane merger without directly affecting bilayer properties.”

The authors suggest that the linker domain of SNAP-25 prevents dimerization of the SNARE complexes "by masking the sticky surfaces". However, there is no direct proof for that except an apparent size shift in the SDS-PAGE. Inspecting the Figure 1C and Figure 3—figure supplement 1C carefully, one can see that also with WT SNAP-25 construct, a band with a similar size-shift becomes visible at 24hours. Additionally, even though it's apparent from the reduction of the monomeric SNAREs that the high molecular mass represents the SNARE complex, the identity of this high molecular mass band is not clear, because it's just Coomassie staining. Especially in the case of separated SN1 and SN2 fragments (Figure 1C), as there is no obvious reduction in the SN fragments and also as SN2 cannot even bind to Stx1 (Figure 1D), it cannot be concluded that high-size band represents dimerized SNARE complexes. This part should be toned down.

The dimerization of SNARE complexes formed out of isolated SN1, SN2, Syntaxin, and Synaptobrevin has been investigated in detail by the workgroup of Prof. Sabine Hilfiker. This group used size-exclusion chromatography, MALLS, and SAXS to characterize SNARE complex dimers and found that two fully formed SNARE complexes can C-terminally attach to each other in a salt-dependent way, forming a wing-shaped dimer (Fdez et al., 2008). While our analysis is rudimentary, we are convinced that the observed size-shifted bands for SNARE complexes employing linker-less SNAP-25 fragments indeed represents the described complex dimers. Moreover, Fasshauer et al., (1998) and others have shown that separated, linker-less SN1 and SN2 assemble into a SDS-stable core complexes, even though SN2 cannot bind directly to stx1 (Chapman et al., 1994). As the increased formation of complex dimers in the presence of SNAP-25 linker mutants in vitro is however not central to this work and admittedly distracts from more important results, we have decided to shorten the corresponding passages in the interest of a streamlined manuscript. Only a basic statement concerning the shift in band size was retained (subsection “The SNAP-25 linker is critical for t-SNARE interaction and complex assembly”):

“SNARE complexes formed by SN25 LGGS or separated SN1 and SN2 motifs exhibited a characteristic size shift (~180 kDa; Figure 2Ab-c), which possibly suggests the formation of SNARE complex dimers. Indeed, previous work by Fdez et al., (2008) has shown that two fully formed ternary complexes containing linker-less SN1/SN2 fragments can assemble into dimeric structures with attached C-terminal tips. Thus, the primary structure of the SNAP-25 linker peptide likely affects the biochemical properties of SNAREpins and their propensity to dimerize.”

Using the fragments SN1+L-SN2 for SNARE complex assembly would also be informative, as in this case the interaction of SN2 with Stx1 is partially rescued and that in turn could rescue Syb2 binding as well.

We have followed the reviewers’ recommendation and tested SNARE complex formation for a combination of SN1 and L-SN2 fragments (Figure 2—figure supplement 1). Although we had trouble to correctly match the amount of the small fragments in all experiments, we noticed a significant acceleration of complex assembly in comparison to controls using linker-less SN1 and SN2 fragments. That said, it needs to be taken into account that the “inverse” setup using SN1-L and SN2 also positively affected SNARE complex formation in parallel experiments, indicating that linker:core complex interactions may additionally exert a promoting effect on complex assembly. Our biochemical assays presumably underestimate the effect of t-SNARE interactions on the assembly kinetics, as fragments can easily interact with each other without steric hindrance in solution. A corresponding passage describing our findings has been added to the Results section:

“As the requirement for a continuous linker-SN2 segment in t-SNARE interactions might limit ternary complex nucleation, we also compared SNARE complex assembly in mixtures containing either SN1-L and SN2, SN1 and L-SN2, or linker-less SN1 and SN2 together with stx-1A and GST-syb-2 (Figure 2 – —figure supplement 1A). We observed that the presence of the linker in either fragment combination significantly facilitated SNARE assembly over linker-less fragments, as judged by the time constants of mono-exponential fits of assembly profiles (Figure 2—figure supplement 1B). Intriguingly, the combination of SN1/L-SN2-fragments was slightly more efficient than SN1-L/SN2 in promoting complex formation, indicating that linker interactions during initial t-SNARE assembly might indeed determine overall assembly kinetics. That said, our biochemical assays likely underestimate the effect of t-SNARE interactions, as fragments can easily interact with each other in solution without steric hindrance.”

Last, it is not very clear what the star in Figure 1D stands for, and also why there are two fragments when SNAP-25 LGGS is used. The lower band seems at the size of SN1-L. Is the SNAP-25 LGGS construct somewhat unstable and degraded?

We noticed a contaminating protein (band marked by “*”) in the purified fraction of bacterially expressed His6-SN25 LGGS that likely represents a partially degraded expression product. Since only low levels of the contaminating protein were present, and further optimization of the purification procedure failed to completely eliminate the side product, we used the resulting protein fraction despite the presence of the contamination. We do not expect any effect of the contamination on our results, as we did neither see a participation of the protein in complex formation nor in stx-1A-binding. A corresponding statement has been added to the legend of Figure 2.

The expression experiments using the SNAP-25 linker mutants are not very clear (Figure 1—figure supplement 3A,B). The total expression of these mutants might be comparable to WT, but it is not informative for the plasma membrane localization (except Figure 3—figure supplement 1A) However, the interpretation of the functional output using the mislocalized proteins should be done more cautiously. Also, a test for proper localization regarding the 5KA,4CS mutant as in Figure S4A would be useful. Removal of 5K in the absence of palmitoylation could further affect the membrane targeting and localization of SNAP-25, which would explain the further reduction of the release (Figure 5A).

We thank the reviewers for reminding us of the importance of reliable expression data for a valid interpretation of mutant phenotypes. To eliminate remaining uncertainties concerning the membrane expression of SNAP-25 linker mutants, we performed additional confocal analyses to study the membrane localization of our GFP-tagged SNAP-25 variants. These new expression data has been added as Figure 1—figure supplement 2C and Figure 1—figure supplement 3Ba-c. Interestingly, we did not find any additional decrease in the membrane expression of the SN25 LN5KA,4CS mutant that could account for the slight exacerbation of its phenotype in comparison to SN25 LN4CS. Accordingly, it must be concluded that local phospholipid interaction of the lysine residues play a mechanistic role in fusion initiation. Moreover, we did not observe any effect of spacer insertions on the membrane localization of the corresponding mutants, confirming that the observed phenotypes are not due to a lowered availability of the mutant protein on the plasma membrane.

Finally, the release properties of the cells expressing only the 5KA mutant should be examined because a mislocalization of this mutant is less likely due to proper palmitoylation of the cysteines. This would be useful for understanding whether the effects of 5KA and 4CS mutations are additive.

We agree with the reviewers that the role of the lysine residues in the N-terminal linker deserves more attention, as we might underestimate their function, when only looking at the combined SN25 LN5KA,4CS mutant. However, Weber et al., (2017)recently showed that these residues mediate transient membrane interactions that are important for acylation of SNAP-25 by membrane-bound DHHC acyl-transferases. This implies that mutation of the lysine residues should always produce effects on the acylation state of SNAP-25. Due to this complication, we consider it not worthwhile at this point to investigate the 5KA-mutation in isolation.

The authors claim in subsection “N- and C-terminal linker regions mechanistically support Ca2+-dependent release”, that SNAP-25 LC4G mutant shows phenotypical traits of SNAP-25 LCGGS. However, SNAP-25 LC4G shows only an intermediate phenotype. This claim should be therefore toned down. Comparing the functional output of mislocalized proteins creates ambiguity.

We are sorry for the ambiguous formulation. The corresponding sentence was intended to stress that both phenotypes are related and share certain key features, but it should not express that both mutants exhibit identical release deficits. As correctly stated by the reviewer, SN25 LC4G (and also SN25 LC9G) shows an intermediate phenotype in comparison to the larger substitution mutant SN25 LCGGS. The passage has been accordingly altered to (subsection “N- and C-terminal linker regions mechanistically support Ca2+-dependent release”):

“Hence, this small substitution in SN25 LC4G already induced a complex release phenotype that is reminiscent of the defects seen in SN25 LCGGS.”

The authors mention in subsection “A postfusional role of linker:lipid interactions in controlling pore dilation” that "this slow-down of the main spike signal is primarily caused by the loss of several lysine residues surrounding the central cysteine group, as substitution of these amino acids in SN25 LN5KA,4CS fully replicated the phenotype of the full substitution mutant SN25 LNGGS." However, the resemblance between the phenotypes of SNAP25-LNGGS and LN5KA,4CS might well be merely due to lack of membrane attachment and thus mislocalization. A membrane targeting strategy similar to what is done in Figure 3 would be more informative regarding the 4CS mutants combined with linker insertion (14aa; Figure 4C) and 5KA mutants (Figure 5—figure supplement 2).

We agree with the reviewers that the membrane expression of the SNAP-25 linker mutants might critically affect release properties, especially with respect to fusion pore behavior, which has recently been proposed to depend on SNARE stoichiometry (e.g. (Shi et al., 2012; Wu et al., 2017)). To investigate whether different membrane expression levels may account for phenotypical differences between related linker mutants, we have now performed an extended analysis of protein distribution using confocal microscopy. In particular, we deliver better information on the membrane expression of SN25 LN5KA4CS in comparison to SN25 LN4CS. As illustrated in Figure 1—figure supplement 3Ba,we did not observe any differences in the distribution profiles of both mutants, which indicates that electrostatic interactions of the N-terminal linker with phospholipids are only of minor importance for the steady-state level of non-palmitoylated SNAP-25 at the membrane. Moreover, our older data on the localization of SN25 LNGGS demonstrated that the larger substitution within the N-terminal linker region did not result in a more pronounced mislocalization in comparison to SN25 LN4CS (Figure 3—figure supplement 1A). With all three N-terminal linker mutants sharing a similar low membrane expression, there is currently no indication that the observed phenotypic difference in spike kinetics (SN25 LN4CS exhibits only prespike signal alterations, while SN25 LN5KA4CS and SN25 LNGGS also show a marked delay in main spike kinetics) are the consequence of differential membrane targeting of the mutant proteins. While very small changes may have escaped our confocal analysis, it would be hard to explain by stoichiometric changes why a dramatic decrease in SNAP-25 abundance would only affect the prespike signal in SN25 LN4CS, and a subtle further drop should account for the dramatically delayed main spike kinetic in SN25 LN5KA,4CS. Thus, we think the phenotypic differences are indeed the consequence of a differential interference with linker:lipid interactions.

While it surely is an interesting idea to analyze the functional effects of lysine substitution (5KA) in front of the retargeted P-GFP-SN25 LN4CS variant, we think that our dataset on membrane expression already firmly excludes a prominent influence of membrane targeting on the phenotypic features in SN25 LN5KA,4CS (s. discussion above). Similarly, we also do not see how plasma membrane expression could influence the phenotypical features of spacer insertion (shift14L), as normal membrane targeting was seen in SN25 shift14aaL (Figure 1—figure supplement 3Bc), and residual membrane expression was unaffected after spacer insertion in SN25 LN4CS (Figure 1—figure supplement 3Ba). Due to the high work effort and the relatively low gain in information, we decided against testing the “5KA” and “shift14aa” mutations in P-GFP-SN25 LN4CS.

Addition of another membrane targeting motif (Figure 3) could potentially change the conformation of SNAP-25. Besides, comparing the release parameters in Figure 3 and in the other figures, it seems that this P-GFP-SNAP25 WT has a better release efficacy. This is a very minor point, but could be mentioned in discussion.

We think that the higher secretory responses are a consequence of the slightly higher expression of P-GFP-SNAP-25 WT at the plasma membrane. As mentioning this point would have disrupted the line of argument in the Discussion section, we have added a corresponding statement to the Results section instead:

“P-GFP-SN25 WT efficiently rescued secretion in KO chromaffin cells and even reached a slightly higher release level than typically seen in other control recordings (Figure 4B). This increase in total secretion might be attributed to a more pronounced membrane targeting compared to the unmodified WT protein.”

The interpretation of MeOH and oleic acid experiments should be toned down. It is clear that MeOH and OA facilitate release, but this could be due to a mechanism independent from the function of the linker region. In other words, MeOH or OA already increases and accelerates the NT release from cells expressing WT SNAP-25, and as the cells expressing the linker mutant already has deficits in NT release, it is not surprising that they show a higher sensitivity to MeOH and OA application. Even though the data is largely convincing, the possibility of independent action of MeOH or OA from linker-lipid interaction cannot be ruled out.

It is true that rescue experiments with membrane–perturbing compounds (OA and MeOH) can only deliver circumstantial evidence with regard to the nature and functional significance of linker-mediated membrane interactions. That said, we do not see compelling reasons to assume that any given mutation generically renders the SNARE machinery highly sensitive to OA or MeOH. We expect that a compound-driven facilitation of secretion should be similar or smaller than the corresponding relative changes in WT cells, if the treatment has no direct effect on the fusion intermediate that depends on the mutated linker region (see our response to comment #12). In this case, the process affected by the linker mutation should still limit secretion, as vesicle fusion putatively constitutes a series of sequential mechanistic steps. Only interventions that actually target the same step or fusion intermediate are expected to lift the blockade and produce a substantial disproportional compensation.

There are of course scenarios, in which the higher responsiveness to the treatments could be due to indirect actions of the linker on the OA/MeOH-sensitive intermediate, and thus might result in a misleading positive “rescue”. This could occur, if the N-terminal linker region tightly supports the force transfer by syb and/or stx (e.g. via facilitates C-terminal core complex assembly and zippering of JMDs) and hence acts as a modulator rather than a direct effector on the lipidic fusion intermediate. In this case, the apparent rescue would provide little information about the role of linker-mediated membrane interactions. As we have not explicitly discussed the possible limitations of the approach in the original manuscript version, we have now added a corresponding statements in the text, stressing that our results hint at but not necessarily prove the proposed role of linker:lipid interaction in membrane merger (subsection “A postfusional role of linker:lipid interactions in controlling pore dilation”):

“While the compensatory actions of membrane-active compounds hint at a specific role of linker:lipid interactions in fusion initiation, it should be noted that the available evidence for this scenario is still circumstantial, and other mechanistic explanations might also apply. In particular, a supporting function of the N-terminal linker in the putative “zippering” of the JMDs of stx-1A and syb-2 (Stein et al., 2009) may represent an alternative explanation, as the linker would be closely involved in inducing membrane merger without directly affecting bilayer properties.”

In further support of a structural significance of the SNAP-25 linker (rather than being a "flexible linker"), a recent higher resolution EM structure of the NSF/aSNAP/SNARE complex revealed diffuse density for the SNAP-25 linker at both the N-terminal and C-terminal ends, suggesting that these parts of the linker are associated with the SNARE complex, albeit probably sampling multiple conformations. Thus, the linker is not entirely flexible, but it is not uniquely structured either (see Figure 7 in White et al., 2018). Perhaps this recent finding could be added to the Discussion section.

We thank the reviewers for bringing this new publication to our attention. These findings have now been mentioned in the Discussion section:

“The view that the linker is indeed not completely randomly oriented along the SNARE core but at least in sections engages in local interactions with the SNARE complex is supported by recent work of White et al., (2018), showing a diffuse density for the SNAP-25 linker at the N-terminal and C-terminal ends of the EM structure of the fully formed complex.”

https://doi.org/10.7554/eLife.41720.039

Article and author information

Author details

  1. Ahmed Shaaban

    1. ZHMB, Saarland University, Homburg, Germany
    2. Department of Molecular Neurobiology, Max Planck Institute for Experimental Medicine, Göttingen, Germany
    Contribution
    Formal analysis, Validation, Investigation, Visualization, Writing—review and editing
    Competing interests
    No competing interests declared
  2. Madhurima Dhara

    Institute for Physiology, Center of Integrative Physiology and Molecular Medicine, Saarland University, Homburg, Germany
    Contribution
    Formal analysis, Validation, Investigation, Visualization, Writing—review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7745-472X
  3. Walentina Frisch

    Institute for Physiology, Center of Integrative Physiology and Molecular Medicine, Saarland University, Homburg, Germany
    Contribution
    Formal analysis, Investigation
    Competing interests
    No competing interests declared
  4. Ali Harb

    ZHMB, Saarland University, Homburg, Germany
    Contribution
    Formal analysis, Investigation
    Competing interests
    No competing interests declared
  5. Ali H Shaib

    Department of Molecular Neurobiology, Max Planck Institute for Experimental Medicine, Göttingen, Germany
    Contribution
    Formal analysis, Investigation
    Competing interests
    No competing interests declared
  6. Ute Becherer

    Institute for Physiology, Center of Integrative Physiology and Molecular Medicine, Saarland University, Homburg, Germany
    Contribution
    Conceptualization, Resources, Methodology, Writing—review and editing
    Competing interests
    No competing interests declared
  7. Dieter Bruns

    Institute for Physiology, Center of Integrative Physiology and Molecular Medicine, Saarland University, Homburg, Germany
    Contribution
    Conceptualization, Resources, Funding acquisition, Methodology, Writing—review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2497-1878
  8. Ralf Mohrmann

    1. ZHMB, Saarland University, Homburg, Germany
    2. Institute for Physiology, Otto-von-Guericke University, Magdeburg, Germany
    3. Center for Behavioral Brain Science, Otto-von-Guericke University, Magdeburg, Germany
    Contribution
    Conceptualization, Software, Formal analysis, Supervision, Funding acquisition, Investigation, Writing—original draft, Project administration, Writing—review and editing
    For correspondence
    ralf.mohrmann@med.ovgu.de
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9279-5071

Funding

Deutsche Forschungsgemeinschaft (MO2312/1-1)

  • Ralf Mohrmann

Deutsche Forschungsgemeinschaft (SFB 1027)

  • Dieter Bruns
  • Ralf Mohrmann

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

Acknowledgements

The authors are grateful to N Vogel, D Alansary, B Niemeyer, J Rettig, and N Brose for valuable discussions and support. We would like to thank M Wirth, B Bimperling, and V Schmitt for excellent technical assistance. The work was supported by grants from the Deutsche Forschungsgemeinschaft to RM (MO2312/1-1, SFB 1027) and DB (SFB 1027). The authors declare no conflict of interest.

Ethics

Animal experimentation: Breeding Snap25 KO mice was done in compliance with the German animal welfare act and all local regulations of the Saarland University and the Otto-von-Guericke-University Magdeburg. Animals were only used for post portem tissue extraction (no experimentation on alive animals).

Senior Editor

  1. Eve Marder, Brandeis University, United States

Reviewing Editor

  1. Axel T Brunger, Stanford University, United States

Publication history

  1. Received: September 4, 2018
  2. Accepted: March 5, 2019
  3. Version of Record published: March 18, 2019 (version 1)

Copyright

© 2019, Shaaban et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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